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WO1999040118A1 - Anticorps diriges contre le recepteur kdr humain du vegf - Google Patents

Anticorps diriges contre le recepteur kdr humain du vegf Download PDF

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Publication number
WO1999040118A1
WO1999040118A1 PCT/JP1999/000478 JP9900478W WO9940118A1 WO 1999040118 A1 WO1999040118 A1 WO 1999040118A1 JP 9900478 W JP9900478 W JP 9900478W WO 9940118 A1 WO9940118 A1 WO 9940118A1
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Prior art keywords
antibody
region
chain
monoclonal antibody
kdr
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PCT/JP1999/000478
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English (en)
Japanese (ja)
Inventor
Kenya Shitara
Mikito Ito
Nobuo Hanai
Akeo Shinkai
Hideharu Anazawa
Masabumi Shibuya
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU22990/99A priority Critical patent/AU2299099A/en
Publication of WO1999040118A1 publication Critical patent/WO1999040118A1/fr

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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention is useful for the diagnosis or treatment of diseases in which the disease state progresses due to abnormal angiogenesis, such as proliferation of solid tumors, metastasis, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness.
  • An antibody that specifically binds to human VEGF receptor KDR, a hybridoma or transformant producing the antibody, a method for immunologically detecting human VEGF receptor KDR using the antibody, and a method for detecting the antibody Diagnostic and therapeutic methods for solid tumors, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and freshness.
  • Angiogenesis plays an important role in the formation of a circulatory system and many tissues in the vertebrate embryo during the embryonic period, and also in the mature individual (female), luteinization during the estrous cycle, and transient endometrium. It is closely involved in the growth of the placenta and placenta formation. In addition, pathological conditions include solid tumor growth, metastasis, diabetic retinopathy, and pathogenesis and promotion of rheumatoid arthritis, in which angiogenesis is deeply involved. O. Folkman et al .; J. Biol. Chem. , 267, 10931, 1992).
  • Angiogenesis is triggered by the secretion of angiogenic factors, the secretion of proteases from the endothelial cells of nearby existing blood vessels, the destruction of the basal membrane and stroma, and the subsequent migration of vascular endothelial cells. It consists of a process in which proliferation begins and blood vessels are formed by the formation of a lumen (J. Folkman et al .; J. Biol. Chem., 267, 10931, 1992). As factors inducing angiogenesis, Vascular permeability factor (VPF) / Vascular endothelial growth factor (VEGF) is known as the most important factor in angiogenesis in the above-mentioned developmental stage and angiogenesis in a pathological state (.
  • VPF Vascular permeability factor
  • VEGF Vascular endothelial growth factor
  • VPF / VEGF is a protein consisting of homodimers with a molecular weight of about 40,000. In 1983, it was identified as a vascular permeability factor (VPF) (DRSenger et al .; Science 219, 983, 1983.), which was reported in 1989 as an independent molecule as Vascular endothelial growth factor (VEGF) (N. Ferrara et al .; Biochem. Biophys. Res. Comm., 161, 851, 1989.
  • VPF vascular permeability factor
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial cell growth promoting activity
  • VEGF has been reported to be a growth factor with extremely high specificity for vascular endothelial cells (N. Ferrara et al .; Biochem. Biophys. Res. Comm., 161, 851, 1989).
  • Alternative splicing reports that VEGF has four types of proteins (KAHouck et al .; J. Biol. Chem., 267, 26031, 1991.).
  • kidney cancer A. Takahashi et al .; Cancer Research,, 4233, 1994
  • breast cancer S. F. Brown et al .; Human Pathology, 26, 86, 1995
  • brain tumor RRBerkman et al .
  • J Many human tumor tissues such as Clinical Investigation, 91, 153, 1993
  • gastrointestinal cancer LBBrown et al .
  • ovarian cancer TEOlson et al .; Cancer Research, 54, 276, 1994.
  • VEGF vascular endothelial growth factor
  • an anti-VEGF monoclonal antibody can suppress cancer metastasis in a human tumor metastatic cancer model in nude mice (O. Meyk et al .; Cancer Research, 56, 921, 1996). Therefore, if VEGF activity can be suppressed, it is expected that tumor growth and metastasis in cancer patients can be suppressed.
  • high concentrations of VEGF were detected in human pleural effusion and ascites in humans, indicating that it may be a major factor in pleural effusion and ascites retention (S. Kondo et al .; Biochimica et Biophysica Acta, 1221). , 211, 1994.), Blocking VEGF is also expected to prevent the accumulation of cancerous pleural effusion and ascites.
  • angiogenesis in diabetic retinopathy is positively correlated with VEGF levels in the patient's eye.
  • angiogenesis is suppressed when VEGF activity is suppressed by intraocular administration of an anti-VEGF neutralizing monoclonal antibody (APAdamis et al .; Arch Opthalmol., 114, 66). , 1996.). Therefore, it is expected that the suppression of excessively produced VEGF activity can suppress vascular regeneration in diabetic retinopathy.
  • VEGF receptors As human VEGF receptors, a receptor tyrosine kinase belonging to the family of receptors, Ome-1 (fins-like tyrosine kinase) (Shibuya et al .; Oncogene, 5, 519, 1990; C. Vries et al .; Science, 255, 989, 1992.) and KDR (kinase insert domain-containing receptor) (BITerman et al .; W092 / 14748, Priolity Feb. 22, 1991; BITerman et al .; Biochem. Biophys. Res. Comm., 187, 1579, 1992).
  • Mouse-type homolog of the human VEGF receptor KDR is Flk-K W.
  • the extracellular domains of Fit-1 and KDR / Flk-1 consist of seven immu- noglobulin-like domains, and the intracellular domain consists of a membrane protein with a molecular weight of 180-200 kilodaltons containing the ostium synkinase domain.
  • VEGF specifically binds to Fit-1 and KDR / Flk-1 with KD values of 20 pM and 75 pM, respectively.
  • Fk-1 and KDR / Flk-1 have been reported to be specifically expressed in vascular endothelial cells (TPQuinn et al .; Proc. Natl. Acad. Sci. USA, 90, 7533, 1993; R ⁇ . Kendail et al; Proc. Natl. Acad. Sci. USA, 90, 8915, 1993).
  • the homology between the amino acid sequences of human-type receptor KDR and mouse-type receptor Flk-1 indicates that the N-terminal tyrosine kinase domain and the C-terminal tyrosine kinase domain in cells are extremely 95% and 97%, respectively.
  • the homology between extracellular domains is slightly lower at 80% (B, Barleon et al .; J. Cellular Biochemistry, 54, 56, 1994.).
  • KDR mRNA expression is observed by in situ hybridization in vascular endothelial cells of joints of patients with rheumatoid arthritis (RAFava et al .; J. Experimental Medicine, 180, 341, 1994 ⁇ ), and VEGF- VEGF Reception Evening I suggest the importance of the KDR system.
  • VEGF receptor KDR / Rk-1 expresses KDR in the vascular endothelial cells of the bush artery, and in response to VEGF, proliferates and migrates. It is reported to be involved in cell growth and migration (J. Waltenberger et al .; J. Biol. Chem., 269, 26988, 1994.). In addition, flk-1 knockout mice in which the mouse flk-1 gene was disrupted did not show any mature vascular endothelial cells, did not form yolk sac blood islands, and died in utero. KDR / flk-1 has also been reported to be involved in the proliferation and differentiation of vascular endothelial cells (F, Shalaby et al .; Nature, 376, 62, 1995.).
  • inactive Flk-1 mutant from which the intracellular tyrosine kinase domain of Flk-1 has been removed, we attempted to introduce the virus into endothelial cells using a viral vector.
  • Inactive Flk-1 mutant virus and tumor cells It has been reported that the growth of tumors is suppressed when they are mixed and transplanted into animal cells (B, Millauer et al .; Nature, 367, 576, 1994.). Tumors are inhibited by inhibiting Flk-1 signaling. It has been shown that proliferation is suppressed.
  • a rat monoclonal antibody has been reported as a monoclonal antibody against the mouse Flk-1 extracellular domain. Using the extracellular domain of mouse Flk-1 and the FLAG TM fusion protein as immunogens, hybridomas were prepared from rats and the three types of rat monoclonal antibodies 4H3, 3B6, and 3C8 were expressed on cells. It has been reported that the mouse Flk-1 can be immunoprecipitated, but the function inhibiting activity of VEGF has not been analyzed (A, Wilks et al .; WO 95/21865, Priolity, 10 Feb. 1994.).
  • DC101 prepared in a rat using the extracellular domain of mouse Flk-l and SEAPs (Secretory alkaline phosphatase) fusion protein as an immunogen was used for mouse Flk-1 / ftns on the cell surface. It has been reported that it reacts with chimeric receptor Yuichi (a chimeric molecule whose extracellular domain is Flk-1 and whose intracellular domain is fins).
  • DC101 stimulates VEGF-stimulated mouse Flk-1 / fVns chimera receptor ftns has the activity of inhibiting tyrosine kinase autophosphorylation.
  • DC101 exhibits an antitumor effect in a xenograft model experimental system in which a human tumor is subcutaneously implanted into nude mice (P, Rockwell et al .; WO 95/21868, Priolity, 10 Feb. 1994.). .
  • DC101 responds to mouse Flk-1 but not to human KDR.
  • Hybridoma (Hybridoma, 465-471, 1997.) contains two mouse monoclonal antibodies 2-7-9 and 2 that react specifically with the 6th and 7th specificities of the immunoglobulin-like domain of human KDR.
  • -10-1 has been reported, but it binds to the first to fifth antibodies of the immunoglobulin-like domain of the human-type VEGF receptor KDR, or binds to the human-type VEGF receptor KDR, and inhibits the action of KDR. No neutralizing antibody is known.
  • antibodies that bind to the human VEGF receptor KDR and neutralize the action of KDR are useful for the growth of solid tumors in humans, the formation of metastases, arthritis in rheumatoid arthritis, diabetic retinopathy, immature infants It is expected to be useful for the diagnosis and treatment of diseases in which the disease progresses due to abnormal angiogenesis, such as retinopathy and desiccation.
  • an anti-human VEGF receptor KDR monoclonal antibody that can detect human VEGF receptor KDR-expressing cells and neutralizes the action of VEGF has not been established so far.
  • the present inventors diagnose and treat diseases in which the disease progresses due to abnormal angiogenesis, such as growth of solid tumors, metastasis formation, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness. Binds to the human-type VEGF receptor KDR, which is useful for The present inventors have made intensive studies on the development of a monoclonal antibody that neutralizes the action of KDR, and have completed the present invention.
  • the present invention relates to the following (1) to (37).
  • the monoclonal antibody is The monoclonal antibody according to any one of (1) to (3), which is a monoclonal antibody recognizing an epitope in the region described in (1).
  • a monoclonal antibody is murine l g G l belong to a subclass or mouse IgG2b subclass, the (1) to (5) or monoclonal antibody according to one of.
  • hybridoma according to (8) wherein the hybridoma is KM1668 (FERM BP-6216), KM1992 (FERM BP-6217) or KM1995 (FERM BP-6218).
  • a human chimeric antibody comprising the antibody heavy chain (H chain) variable region (V region) and the antibody light chain (L chain) of the monoclonal antibody according to any one of (1) to (6) above.
  • the amino acid sequences of the H chain V region and the chain V region are selected from the monoclonal antibody KM1668 (FERM BP-6216), the monoclonal antibody KM1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM BP-6218)
  • the human chimeric antibody according to the above (13) which has the same amino acid sequence as the amino acid sequence of the H chain V region and L chain V region of the monoclonal antibody.
  • the CDR-grafted antibody is a monoclonal antibody according to any one of (1) to (6), wherein the H chain and L chain V region complementarity determining regions and the human antibody H chain and L chain C
  • the CDR-grafted antibody according to the above (1) which is an antibody comprising a region and a V region framework region.
  • the amino acid sequences of the H chain V region and the complementarity determining region of the chain V region are determined by the monoclonal antibody KM1668 (FERM BP-6216) and the monoclonal antibody KM1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM BP-6218) having the same amino acid sequence as the amino acid sequence of the complementarity determining regions of the H chain V region and L chain V region of the monoclonal antibody selected from (16).
  • the described CDR-grafted antibody is determined by the monoclonal antibody KM1668 (FERM BP-6216) and the monoclonal antibody KM1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM BP-6218) having the same amino acid sequence as the amino acid sequence of the complementarity determining regions of the H chain V region and L chain V region of the monoclonal antibody selected from (16).
  • the amino acid sequence of the complementarity determining region of the H chain V region and L chain V region of the single chain antibody is the H chain V region and the monoclonal antibody selected from one of (1) to (6);
  • the amino acid sequence of the H chain V region and the chain V region of the single chain antibody is the monoclonal antibody KM1668 (FERM BP-6216), the monoclonal antibody M1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM BP- The single-chain antibody according to (19), having the same amino acid sequence as the amino acid sequence of the H chain V region and L chain V region of the monoclonal antibody selected from 6218).
  • the amino acid sequence of the H chain V region and L chain V region of the disulfide stabilized antibody is a monoclonal antibody selected from any one of the above (1) to (6).
  • the amino acid sequence of the complementarity determining region of the H chain V region and L chain V region of the disulfide stabilized antibody is such that the amino acid sequence of the monoclonal antibody selected from any one of the above (1) to (6) (23)
  • the amino acid sequence of the H chain V region and the V chain region of the disulfide stabilized antibody is the monoclonal antibody KM1668 (FERM BP-6216), the monoclonal antibody K 1992 (FER BP-6217) or the monoclonal antibody KM1995 (FERM BP-6217).
  • the amino acid sequence of the complementarity determining region of the V region of the H and S chains of the disulfide-stabilized antibody is the monoclonal antibody KM 1668 (FERM BP-6216), and the monoclonal antibody KM1992 (FERM BP-6217).
  • the disulfide stability according to (25) which has the same amino acid sequence as the amino acid sequence of the complementarity determining region of the H chain V region and L chain V region of the monoclonal antibody selected from the monoclonal antibody KM1995 (FERM BP-6218). Antibody.
  • (32) A method for inhibiting the binding between human VEGF and human VEGF receptor KDR using the monoclonal antibody according to any one of (1) to (7) and (10) to (27).
  • a diagnostic agent for an angiogenesis disorder comprising the monoclonal antibody according to any one of (1) to (7) and (10) to (27) as an active ingredient.
  • a therapeutic agent for an angiogenesis disorder comprising the monoclonal antibody according to any one of (1) to (7) and (10) to (27) as an active ingredient.
  • the extracellular domain of the human VEGF receptor KDR consists of seven imnoglobulin-like domains.
  • FIG. 7 shows a schematic diagram of the immunoglobulin-like domains from No. 1 to No. 7 from the N-terminal end of the extracellular domain of the human VEGF receptor KDR and the amino acid numbers in the amino acid sequence of the domain.
  • the present invention relates to an antibody that reacts with the human VEGF receptor KDR but does not react with the human VEGF receptor Flt-1, and specifically reacts with the first to fifth immunoglobulin-like domains of the human VEGF receptor KDR.
  • An antibody is provided.
  • Antibodies in the present invention include monoclonal antibodies, recombinant antibodies, and the like.
  • the monoclonal antibody in the present invention may be any antibody as long as it specifically reacts with the human VEGF receptor KDR, and an antibody established by the following production method is preferable. That is, a human VEGF receptor KDR protein is prepared as an antigen, plasma cells having antigen specificity are induced from an animal immunized with the antigen, and a hybridoma is prepared by fusing it with myeloma cells. An anti-human VEGF receptor KDR monoclonal antibody obtained by culturing an hybridoma or administering the hybridoma cells to an animal to cause the animal to undergo ascites carcinoma, and separating and purifying the culture solution or ascites is used. I can give it.
  • the recombinant antibody of the present invention is obtained by modifying the above-described monoclonal antibody of the present invention using a gene recombination technique.
  • the recombinant antibody include antibodies produced by genetic recombination, such as humanized antibodies, single-chain antibodies, and disulfide stabilized antibodies.
  • Recombinant antibodies that have the characteristics of a monoclonal antibody, have low antigenicity, and have an extended half-life in blood are preferred as therapeutic agents.
  • the humanized antibody in the present invention includes a human chimeric antibody and a human CDR (Complementary Determining Region; hereinafter referred to as CDR) transplanted antibody.
  • CDR Complementary Determining Region
  • the human chimeric antibody is composed of an antibody variable region heavy chain (hereinafter, referred to as VH) and a variable region light chain (hereinafter, referred to as VL) of an animal other than a human and a constant region heavy chain (hereinafter, referred to as CH) of a human antibody. ) And the constant region light chain of a human antibody (hereinafter, referred to as CL).
  • VH antibody variable region heavy chain
  • VL variable region light chain
  • CH constant region heavy chain
  • CL constant region light chain of a human antibody
  • the human CDR-grafted antibody means an antibody obtained by replacing the CDRs of VH and VL of an antibody of a non-human animal with the CDR sequence of an antibody of a non-human animal.
  • the human chimeric antibody of the present invention obtains cDNAs encoding VH and VL from a hybridoma producing a monoclonal antibody that binds to the human VEGF receptor KDR and neutralizes the action of KDR, and obtains human antibody CH
  • a human antibody CL can be inserted into an expression vector for animal cells having a gene encoding the same to construct a human-type chimeric antibody expression vector, and the vector can be expressed and produced by introducing the vector into animal cells.
  • the human CDR-grafted antibody of the present invention reacts with human VEGF receptor KDR, binds to human VEGF receptor KDR, and neutralizes the action of KDR.
  • a cDNA encoding a V region in which the CDR sequences of VH and VL of the human antibody are substituted with the CDR sequences of VH and V, respectively, is constructed, and genes encoding CH of human antibody and CL of human antibody are constructed.
  • the antibody can be produced by constructing a human CDR-grafted antibody expression vector by inserting it into an animal cell expression vector having the above, and introducing it into animal cells to allow expression.
  • the human chimeric antibody and the human CDR-grafted antibody of the present invention may belong to any of the immunoglobulin (lg) classes, but are preferably of the IgG type, and more preferably belong to the IgG type. Any of the C regions of immunoglobulins such as IgG4 can be used.
  • the present invention relates to a single chain antibody (single chain Fv; hereinafter, referred to as scFv) or a disulfide stabilized Fv (hereinafter, referred to as dsFv) which exhibits binding to human VEGF receptor KDR.
  • scFv single chain Fv
  • dsFv disulfide stabilized Fv
  • a single-chain antibody is a VH_L—VL or VL—L—VH in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as L). 1 shows a polypeptide.
  • L an appropriate peptide linker
  • the disulfide-stabilized antibody refers to a polypeptide in which one amino acid residue in each of VH and VL has been substituted with a cysteine residue via a disulfide bond.
  • the amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. [Protein 'Engineering (7, 697 (1994)]).
  • Reiter et al. Protein 'Engineering (7, 697 (1994)]
  • any of a mouse anti-human VEGF receptor KDR monoclonal antibody and a human CDR-grafted antibody can be used.
  • Single-chain antibodies that bind to human VEGF receptor KDR are human VEGF receptor
  • cDNAs encoding VH and VL from a hybridoma producing an antibody that binds to the body KDR and neutralize the action of KDR construct a single-chain antibody expression vector, and use it in E. coli, yeast, or animals. It can be expressed and produced by introducing it into cells.
  • a disulfide-stabilized antibody that binds to human VEGF receptor KDR can be obtained by obtaining cDNAs encoding VH and VL from a hybridoma that produces an antibody that reacts with human VEGF receptor KDR, and converting it into an appropriate expression vector.
  • the expression vector can be inserted into Escherichia coli, yeast, or animal cells for expression.
  • the antibody of the present invention binds to human VEGF receptor KDR, inhibits the binding of human VEGF to human VEGF receptor KDR, and neutralizes the action of KDR. It can inhibit promoting activity, migration promoting activity, meta-oral protease secretion promoting activity, perokinase, tPA secretion promoting activity, in vivo angiogenesis promoting activity, vascular permeability enhancing activity, and the like. Therefore, the present invention is useful for the diagnosis and treatment of diseases in which the disease state progresses due to abnormal angiogenesis, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness. Can be used.
  • the anti-human VEGF receptor KDR monoclonal antibody the anti-human VEGF receptor KDR humanized antibody, the anti-human VEGF receptor KDR-main chain antibody and the anti-human VEGF receptor KDR disulfide stabilized antibody of the present invention will be described.
  • the production method and the method for detecting and quantifying human VEGF receptor KDR using the antibody will be described.
  • Antigens required for preparing anti-human VEGF receptor KDR monoclonal antibody include cells expressing human VEGF receptor KDR on the cell surface or cell membrane fractions thereof, or cells having different amino acid lengths.
  • Soluble human VEGF with outer region Examples include a receptor KDR protein or a fusion protein of the protein with the Fc portion of an antibody.
  • Cells expressing the human VEGF receptor KDR on the cell surface include N1H3T3-KDR cells [Cell Growth & Differentiation (Cell Growth &
  • Methods for expressing a soluble human VEGF receptor KDR protein having extracellular regions of different lengths or a fusion protein of the protein and the Fc portion of an antibody include full-length human VEGF receptor KDR or a partial fragment thereof. Creating a recombinant vector in which [Cell Growth & Differentiation (Cell Growth & Differentiation) 7, 213, 1996.] has been inserted downstream of a suitable vector and introducing it into host cells. By culturing the human VEGF receptor KDR-expressing cells obtained in Step 1 in an appropriate medium, the full-length or partial fragment of human VEGF receptor KDR can be produced as is or as a fusion protein in the cells or in the culture supernatant. can do. It can also be prepared by synthesizing a polypeptide having the partial sequence of the protein described above using an amino acid synthesizer.
  • the host may be any host such as bacteria, yeast, animal cells, and insect cells, as long as it can express the gene of interest.
  • bacteria include bacteria of the genus Escherichia such as Escherichia coli and Bacillus subtilis, and bacteria of the genus Bacillus.
  • Yeasts include Saccharomyces cerevisiae and Shizosatsu Tokoguchi. And Nobe (Schizosaccharomyces pombe).
  • animal cells include Namalva cells, which are human cells, COS cells, which are monkey cells, and CHO cells, which are Chinese hamster cells.
  • insect cells include S ⁇ 9, Sfil (Pharmingen), High Five (Invitrogen) and the like.
  • any vector can be used as long as it can incorporate the DNA and can be expressed in a host cell.
  • a bacterium for example, Escherichia coli
  • the expression vector is composed of a promoter, a ribosome binding sequence, the DNA of the present invention, a transcription termination sequence, and, in some cases, a promoter control sequence.
  • pGEX manufactured by Pharmacia
  • pET system manufactured by Novagen
  • Examples of a method for introducing a recombinant vector into a bacterium include a method for introducing DNA into a bacterium, for example, a method using calcium ions [Proceedings of the 'National Academy' of 'Science' * U.s.A. (Proc.
  • examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), and YCp50 (ATCC37419).
  • any method for introducing DNA into yeast can be used, for example, by the electrolysis method [Methods. Enzymol., 194, 182- 187 (1990)], and the Suhloe Plast method [Proc. Natl. Acad. Sci., Proc. Natl. Acad. Sci., "The National Academy of Sciences of the U.S.A.” USA), 84, 1929—1933 (1978)], and the Oki lithium method [J. Bacteriol. (J. Bacteriol.), 153, 163-168 (1983)]. .
  • Any promoter can be used as long as it can be expressed in animal cells.
  • CMV cytomegalovirus
  • lE immedia1 ⁇ 2 early
  • Promote the gene SV40 or metallotionin promoter, etc.
  • the enhancer of the 1E gene of human CMV may be used together with the promoter.
  • a method for introducing DNA into animal cells includes, for example, the elect-portion method [Cytotechnology, 3, 133 (1990)].
  • Calcium Phosphate Method Japanese Patent Laid-Open No. 2-227075
  • Lipofection Method [Procedures of the National Academy of Sciences Ob the U.S.A. (Proc. Natl. Acad. Sci., USA), 84, 7413 (1987)].
  • Noculovirus' Expression ⁇ Proteins can be expressed by the method described in Hexagons, Pfatries, and Manu / J (Baculovirus expression vectors A laboratory manual). That is, the recombinant gene transfer vector and baculovirus described below are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected into insect cells to express the protein. Obtain insect cells.
  • pVL1392 As the gene transfer vector, for example, pVL1392, pVL1393, pBlueBacllI (both manufactured by Invitrogen) and the like are used.
  • Autographa californica nuclear polyhedrosis virus which is a virus that infects insects of the night moth family
  • Autographa californica nuclear polyhedrosis virus is used.
  • proteins are produced by infecting insect cells such as Sf, Sf21 or High Five described above with the recombinant virus. [Bio / Technology, 6, 47 (1988)].
  • secretory production, fusion protein expression, and the like have been developed as gene expression methods, and any method can be used. For example, Molecular Cloning 2nd edition, Cold Spring Harbor Lab. Press New York (1989); hereinafter referred to as "Molecular Cloning 2nd Edition” ] Can be carried out according to the method described in [1].
  • the transformant obtained as described above is cultured in a medium, the protein of the present invention is produced and accumulated in the culture, and collected from the culture to obtain the full-length or partial fragment of the human VEGF receptor DR. It can be produced as it is or as a fusion protein.
  • the method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganism to efficiently culture the transformant.
  • a natural medium or a synthetic medium can be used as long as the medium can be used (Molecular 'Cloning 2nd edition).
  • Cultivation is usually performed at 15-40 ° C for 16-96 hours under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.
  • antibiotics such as ampicillin / tetracycline to the medium as needed May be.
  • a medium for culturing the transformants obtained using animal cells as a host commonly used RPM116 medium, Eag1e MEM medium, or a medium obtained by adding fetal bovine serum or the like to these mediums are used. Cultures, 5% C0 2 presence usually performed 3-7 days at 35 to 37 ° C, the culture if necessary, kanamycin, may be added to the antibiotic penicillamine phosphorus to the medium.
  • TNM-FH medium commonly used TNM-FH medium [Pharmingen], Sf900IISFM [Life Technologies] ExCell400, ExCell405 [all manufactured by JRH Biosciences] and the like.
  • the culture is performed at 25 to 30 ° C. for 1 to 4 days.
  • an antibiotic such as gentamicin may be added to the medium as needed.
  • a serum-free medium in order to directly purify the full-length or partial fragment of human VEGF receptor KDR or to facilitate purification of the fusion protein.
  • the cells are centrifuged, suspended in an aqueous buffer, and subjected to ultrasonic method.
  • the cells are disrupted by a French press method or the like, and the protein is recovered in the supernatant obtained by centrifugation.
  • an insoluble substance when solubilizing the insoluble substance with a protein denaturant, a solution containing no protein denaturant or a concentration of protein denaturant that is so dilute that protein is not denatured is prepared. It can be diluted or dialyzed to form a protein conformation.
  • the expressed protein can be recovered in the culture supernatant.
  • solvent extraction, fractional precipitation with organic solvents, salting out, dialysis, centrifugal separation, ultrafiltration, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography Separation operations such as reverse phase chromatography, crystallization, and electrophoresis can be performed alone or in combination.
  • the protein obtained above is immunized as an antigen.
  • the method of immunization may be to administer the antigen as it is, subcutaneously, intravenously, or intraperitoneally to the animal, or by administering a carrier protein with high antigenicity or administering the antigen together with an appropriate adjuvant. Is preferred.
  • Carrier proteins include keyhole limpet mossin, keyhole limb mossinine, bovine serum albumin, and bovine thyroglobulin.
  • Agile bands include Complete Freund's Adjuvant, aluminum hydroxide gel Pertussis vaccine and the like.
  • Examples of the immunized animal include non-human mammals such as rabbits, goats, mice, rats, and hamsters.
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration.
  • the dosage of the antigen is preferably 50 to 100 g per animal.
  • Blood is collected from the fundus venous plexus or the tail vein of the immune animal on days 3 to 7 after each administration, and the reactivity of the serum with the antigen is determined by enzyme immunoassay [enzyme immunoassay (ELISA): Medical Shoin. Published (1976)].
  • lymphocytes are excised according to the “Antibody's 'Lab' Manual” and the lymphocytes are fused with myeloma cells.
  • myeloma cells include a cell line obtained from a mouse, 8-azazanin-resistant mouse (derived from BALB) myeloma cell line P3-X63Ag8-U UP3-U 1) [G. ohler et al .; journal 'O Bed' Imunorojii, 6, 511 (1976)] , SP2 / 0 - A g 14 (SP2) [M.Shulman al (Europ J. Immunol..); Neichiya one (Nature), 276, 269 (1978)], P3-X63-Ag8653 (653) [JF Kearney et al., Journal of Immunology, J.
  • a cell-aggregating medium such as polyethylene glycol 1000 (PEG-1000) is added, and the cells are fused and suspended in a medium.
  • PEG-1000 polyethylene glycol 1000
  • MEM medium or PBS 1.83 g of sodium sodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of salt, 1 liter of distilled water, pH 7.2
  • a medium for suspending the fused cells a HAT medium (normal medium [Dalmin (1.5 mM), 2-mercaptoethanol in RPMI 1640 medium) so that only the desired fused cells can be selectively obtained.
  • a part of the culture supernatant is removed, and a sample that reacts with the antigen protein but does not react with the non-antigen protein is selected by enzyme immunoassay. Then, by limiting dilution method Cloning is performed, and those with a stable and high antibody titer determined by enzyme immunoassay are selected as monoclonal antibody-producing hybridoma strains.
  • the antigen protein or cells expressing the antigen protein are coated on a plate, and the hybridoma culture supernatant or the purified antibody obtained by the above method is reacted as the first antibody.
  • the plate After the first antibody reaction, the plate is washed and the second antibody is added.
  • the second antibody is an antibody capable of recognizing the first antibody, which is labeled with biotin, an enzyme, a chemiluminescent substance or a radioactive compound. Specifically, if a mouse is used for preparing the hybridoma, an antibody capable of recognizing mouse immunoglobulin is used as the second antibody.
  • the hybridoma is selected as a hybridoma that produces a monoclonal antibody that specifically reacts with the antigen.
  • hybridoma strain of the present invention examples include the hybridoma strains KM1668, KM1992 and KM1995.
  • hybridoma strains KM1668, KM 1992 and KM1995 were registered with FERM BP at the Institute of Biotechnology, Institute of Life Science and Technology (1-1-3 Tsukuba East, Ibaraki, Japan). -6216, deposited as FERM BP-6217, FERM BP-6218.
  • Monoclonal antibodies culture obtained by culturing the High Priestess dormer cells, or is a pristane-treated [2,6,10,14-tetramethyl-pen evening decane (Prist ane) 0.5ml was intraperitoneally administered, two weeks
  • a monoclonal antibody-producing hybridoma cell is intraperitoneally administered to a mouse or nude mouse of 8 to 10 weeks of age, which is then separated and purified from ascites caused by ascites cancer.
  • Methods for separating and purifying monoclonal antibodies include centrifugation, salting out with 40 to 0% ammonium sulfate, force prillic acid precipitation, and DEAE-cephalo-scalar. Chromatography using a column, anion exchange column, protein A or G-column, gel filtration column, etc., alone or in combination. According to this method, an IgG or IgM fraction can be collected to obtain a purified monoclonal antibody.
  • the subclass of the purified monoclonal antibody can be determined using a monoclonal antibody typing kit or the like.
  • the protein content can be calculated by the Lowry method or from the absorbance at 280 nm.
  • the subclass of the antibody by isotype within a class in mice, I g Gl, lgG2a, IgG2b , IgG3, in humans, IgGU lgG2, lgG3, IgG4 and the like, in particular a mouse I g Gl, IgG2a, human
  • the IgGl type has a complement-dependent cytotoxic activity (hereinafter, CDC activity) and an antibody-dependent cytotoxic activity (hereinafter, ADCC activity), and is useful for therapeutic applications.
  • the expression vector for humanized antibody is an expression vector for animal cells into which genes encoding the C regions of human antibodies, CH and CL, are incorporated. It was constructed by inserting genes encoding CL and CL, respectively.
  • a C region of an arbitrary human antibody such as C a1 or C a 4 for a human antibody H chain and C ⁇ for a human antibody L chain can be used.
  • the gene encoding the C region of the human antibody chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used.
  • Any expression vector for animal cells can be used as long as it can incorporate and express the gene encoding the human antibody C region.
  • pAGE107 [Cytotechnology, 3, 133 (1990)]
  • pAGE103 [Journal of Bio Chemist U—Q. Biochem.), 101, 1307 (1987)]
  • pHSG274 [Gene, 223 (1984)]
  • pKCR Procedureing 'ob. The national' academy of science ( Proc. Natl. Acad. Sci.),, 1527 (1981)], pSGl ⁇ d2-4 [Cytotechnology, 173 (1990)], and the like.
  • Promoters and enhancers used in animal cell expression vectors include the SV40 initial promoter and enhancer [Journal of Biochemistry, J.
  • the humanized antibody expression vector can be either a type in which the antibody H-chain or L-chain is present on a separate vector or a type in which the antibody is present on the same vector (tandem type). Tandem humanized antibody expression in terms of the ease of construction of expression vectors, ease of introduction into animal cells, and the ability to balance the expression levels of antibody H chains and stranded chains in animal cells. The use of a vector is more preferred [Journal of Physical Methods], 167, 271 (1994).
  • Antibodies of non-human animals for example, cDNAs encoding VH and VL of mouse anti-human VEGF receptor KDR monoclonal antibody are as follows: Get it like. MRNA is extracted from cells producing an anti-human VEGF receptor KDR monoclonal antibody, for example, a hybridoma producing a mouse human VEGF receptor KDR antibody, and cDNA is synthesized. Insert the synthesized cDNA into a vector such as a phage or a plasmid to prepare a cDNA library. From the library, a non-human animal antibody, for example, the C region or V region of a mouse antibody is used as a probe.
  • a recombinant phage or recombinant plasmid having a cDNA encoding VH and a recombinant phage or recombinant plasmid having a cDNA encoding VL, respectively.
  • the entire VH and VL nucleotide sequences of the target antibody on the recombinant phage or recombinant plasmid are determined, and the entire amino acid sequences of VH and VL are deduced from the nucleotide sequences.
  • a cDNA encoding VH and VL of a non-human animal antibody was inserted upstream of the genes encoding CH and CL of the human antibody in the humanized antibody expression vector constructed in 2 (1) above.
  • a chimeric antibody expression vector can be constructed.
  • a restriction enzyme recognition sequence for cloning cDNA encoding VH and VL of a non-human animal antibody is provided upstream of the gene encoding the CH and CL of the human antibody in the chimeric antibody expression vector.
  • a human chimeric antibody expression vector can be produced by inserting a cDNA encoding the V region of a non-human animal antibody into this cloning site via a synthetic DNA described below.
  • Synthetic DNA consists of a base sequence at the 3 'end of the V region of an antibody of a non-human animal and a base sequence at the 5' end of the C region of a human antibody. It is manufactured using a DNA synthesizer so as to have
  • VH and VL that form the antigen-binding site of the antibody are composed of four relatively conserved framework regions (hereinafter referred to as FR regions) and three sequence-rich complementary sequences that link them. It consists of determining region (CDR) [Shikenshizu * O Breakfast - protein's 'O Breakfast' Imunorojikaru Interest (sequences of proteins of Immunological Interest) , US Dept. Health and Human Services, 1991] 0 and each CDR amino acid sequence ( CDR sequence) is composed of the amino acid sequence of the V region of a known antibody [sequences of proteins] of proteins. (Sequences of Proteins of Immunological Interest), US Dept. Health and Human Services, 1991].
  • CDR determining region
  • CDR sequence is composed of the amino acid sequence of the V region of a known antibody [sequences of proteins] of proteins.
  • CDNAs encoding VH and VL of the human CDR-grafted antibody can be obtained as follows.
  • the FR amino acid sequence of the V region of the human antibody for transplanting the CDR of the V region of the antibody of the animal other than the target human is selected for each of VH and VL.
  • the amino acid sequence of the FR in the V region of the human antibody any amino acid sequence of the FR in the V region of the human antibody can be used.
  • the amino acid sequence of the FR of the V region of the human antibody registered in the Protein Data Bank the common amino acid sequence of each subgroup of the FR of the V region of the human antibody [sequences of proteins, proteins] Immunological Interest (Sequences of Proteins of Immunological Interest), US Dept.
  • the amplified fragment is subcloned in an appropriate vector, its base sequence is determined, and a plasmid containing cDNA encoding the amino acid sequence of the V region of each chain of the target human CDR-grafted antibody is obtained. I do.
  • the V region of each chain of the target human CDR-grafted antibody is synthesized.
  • a cDNA encoding the amino acid sequence of can also be constructed.
  • a human CDR-grafted antibody has the same activity as that of the original non-human animal by simply grafting the CDR of the V region of the target non-human animal antibody between the FRs of the V region of the human antibody. It is known that the activity is lower than that of the antibody [BIO / TECHNOLOGY], 9, 266 (1991)]. Therefore, in the amino acid sequence of the FRs in the V region of the human antibody, amino acid residues that are directly involved in antigen binding, amino acid residues that interact with CDR amino acid residues, or the three-dimensional structure of the antibody Amino acid residues that have a possibility of being involved in maintenance or the like have been modified to amino acid residues found in antibodies of animals other than the original human to increase the activity.
  • the modification of the amino acid sequence of FR in the V region of the selected human antibody can be achieved by performing the PCR described in 2 (5) above using various mutagenesis primers. After subcloning the amplified fragment into a suitable vector after PCR, determine its nucleotide sequence and obtain a vector containing the cDNA into which the desired mutation has been introduced (hereinafter referred to as amino acid sequence modification vector). .
  • modification of the amino acid sequence in a narrow region can be performed by a PCR mutagenesis method using a mutation-introducing primer consisting of 20 to 35 bases. Specifically, a 20- to 35-base nucleotide containing the DNA sequence encoding the amino acid residue after modification Then, a two-step PCR is performed using a plasmid containing cDNA encoding the amino acid sequence of the V region to be modified. After subcloning the final amplified fragment into an appropriate vector, its nucleotide sequence is determined, and an amino acid sequence-modified vector containing cDNA into which the desired mutation has been introduced is obtained.
  • a cDNA encoding VL can be inserted to construct a human CDR-grafted antibody expression vector.
  • a recognition sequence of an appropriate restriction enzyme is added to the ends of the synthetic DNA at the 5 ′ end and 3 ′ end. By introduction, they can be inserted upstream of the gene encoding the C region of the desired human antibody so that they can be expressed in an appropriate form.
  • the human chimeric antibody expression vector of 2 (3) and the human CDR-grafted antibody expression vector of 2 (7) or a modification thereof were used.
  • Transient expression of humanized antibodies by introducing the vector into COS-7 cells (ATCC CRL1651) [Methods in Nucleic Acids Res., CRC Press, p.283, 1991 ] And measure its activity.
  • a transformant capable of stably producing a humanized antibody by introducing the human chimeric antibody expression vector of 2 (3) and the human CDR-grafted antibody expression vector of 2 (7) into appropriate host cells. Obtainable.
  • Examples of a method for introducing an expression vector into a host cell include an electoporation method (Japanese Patent Application Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)) and the like.
  • any cell can be used as long as it can express the humanized antibody.
  • mouse SP2 / 0-Agl4 cells ATCC CRL1581
  • mouse P3X63-Ag8.653 cells ATCC CRL1580
  • CHO cells lacking the dihydrofolate reductase gene hereinafter referred to as the DHFR gene
  • Rat YB2 / 3HL.P2.G11.16Ag.20 cells ATCC CRL1662, Hereinafter, referred to as YB2 / 0 cells).
  • a transformant that stably produces a humanized antibody is selected on an RPMI1640 medium containing G418 and FCS according to the method disclosed in Japanese Patent Application Laid-Open No. 257891/1990.
  • a humanized antibody By culturing the obtained transformant in a medium, a humanized antibody can be produced and accumulated in the culture solution.
  • the activity of the humanized antibody in the culture solution is measured by the method described in 1 (5) or the like.
  • the transformant can increase the production amount of the humanized antibody using a DHFR gene amplification system or the like according to the method disclosed in Japanese Patent Application Laid-Open No. 2-257891.
  • Humanized antibodies can be purified from the culture supernatant of the transformant using a protein A column [Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter s, 1988; hereinafter referred to as "antibodies”].
  • other purification methods used for ordinary proteins can be used.
  • purification can be performed by a combination of gel filtration, ion exchange chromatography, and ultrafiltration.
  • the molecular weight of the purified H-chain, L-chain or whole antibody molecule of the humanized antibody can be determined by polyacrylamide gel electrophoresis (SDS-PAGE) [Nature- (Nature), 227, 680 (1970)] or Western blotting. (Antibody, Chapter 12, 1988).
  • the reactivity of the purified humanized antibody and the inhibitory activity of the humanized antibody on VEGF can be measured by the method described in 1 (4) or the like.
  • the humanized antibody of the present invention specifically binds to human VEGF receptor KDR and neutralizes the action of KDR. Therefore, the humanized antibody provided by the present invention can be used to inhibit the growth or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and angiogenesis due to excessive VEGF production such as dryness. It is considered useful for the treatment of diseases in which the disease state progresses due to abnormalities. Furthermore, compared to antibodies from non-human animals, most of the amino acid sequence is derived from the amino acid sequence of a human antibody, so it does not show immunogenicity in the human body and its effects are expected to last for a long period of time. .
  • the humanized antibodies of the present invention can be used alone or with at least one or more pharmaceutically acceptable auxiliaries.
  • a humanized antibody is dissolved in an aqueous solution of physiological saline, glucose, lactose, mannitol, or the like to prepare a suitable pharmaceutical composition.
  • a humanized antibody is freeze-dried according to a conventional method, and sodium chloride is added thereto to prepare a powder injection.
  • the present pharmaceutical composition can contain, if necessary, additives well known in the pharmaceutical field, for example, pharmaceutically acceptable salts and the like.
  • the dose of the pharmaceutical composition varies depending on the age, symptoms, etc. of the patient, and the human antibody-containing mammal is administered the humanized antibody at 0.1 to 20 mg / kg / day. Dosing once daily (Single or daily administration) or intermittently by intravenous injection 1 to 3 times a week and once a few weeks.
  • an expression vector of a single-chain antibody of a non-human animal antibody or a single-chain antibody of a human CDR-grafted antibody may be any vector as long as it can integrate and express cDNA encoding VH and VL of an animal antibody other than human or a human CDR-grafted antibody. Can be used.
  • an appropriate host can be selected from Escherichia coli, yeast, animal cells, and the like.In such a case, an expression vector may be selected for each host. You need to choose the right one. Insertion of a cDNA encoding an appropriate signal peptide into an expression vector allows the single-chain antibody to be secreted extracellularly, transported to the periplasmic region, or retained inside the cell. it can.
  • cDNA encoding the single-chain antibody consisting of VH—Shi-VL or VSh-VH (L is the peptide ligand) to the selected expression vector at the appropriate promoter and signal peptide.
  • L is the peptide ligand
  • a single-chain antibody expression vector into which cDNA encoding the desired single-chain antibody has been inserted can be constructed.
  • the cDNA encoding the single-chain antibody is ligated to the cDNA encoding VH and the cDNA encoding VL using synthetic DNA encoding a peptide linker having an appropriate restriction enzyme recognition sequence at both ends. Can be obtained.
  • linker peptide be optimized so that its addition does not interfere with the binding of VH, VL to the antigen, such as that shown by Pantoliano et al. [Biochemistry, 30, 10117 ( 1991)] or a modified version thereof can be used.
  • the single-chain antibody expression vector constructed in the above 3 (1) is introduced into an appropriate host cell by a method such as an electoral poration method [Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)]. As a result, a transformant producing the desired single-chain antibody can be obtained. After the introduction of the expression vector, the activity of the single-chain antibody contained in the culture supernatant or the like can be measured by the method described in 1 (4) or the like.
  • Recovery and purification of the single-chain antibody of the present invention can be achieved by combining known techniques. For example, if the single-chain antibody is secreted into the medium, it can be concentrated by ultrafiltration and then achieved by performing antigen affinity chromatography or ion exchange chromatography or gel filtration. Can be. Alternatively, if transported to the periplasmic region of the host cell, the cell can be subjected to osmotic shock and concentrated by ultrafiltration, followed by antigen affinity chromatography or ion-exchange chromatography. This can be achieved by performing gel filtration.
  • Single-chain antibodies which are insoluble and present as granules (inclusion body), lyse cells, repeat centrifugation and washing to isolate granules, solubilize with guanidine-HCl, and It can be achieved by an operation of leading again to a structure having the activity of a single-chain antibody, followed by purification of the active molecule.
  • the activity of the purified single-chain antibody can be measured by the method described in 1 (4) or the like.
  • the single chain antibody of the present invention specifically binds to human VEGF receptor KDR and neutralizes the action of KDR. Therefore, the single-chain antibody provided by the present invention can be used to inhibit the growth or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and angiogenesis due to excessive VEGF production such as dryness. It is considered to be useful for treatment of diseases in which the disease progresses due to abnormalities.
  • the single-chain antibodies of the present invention can be used alone or together with at least one or more pharmaceutically acceptable auxiliaries.
  • a single-chain antibody is dissolved in an aqueous solution of physiological saline, glucose, lactose, mannitol or the like to prepare a suitable pharmaceutical composition.
  • the single-chain antibody is freeze-dried according to a conventional method, and sodium chloride is added thereto to prepare a powder injection.
  • the present pharmaceutical composition can contain additives known in the pharmaceutical field, for example, pharmaceutically acceptable salts and the like, if necessary.
  • the dose of the pharmaceutical composition varies depending on the age, symptoms, etc. of the patient, but the single-chain antibody is administered to mammals including humans at 0.1 to 20 mg / kg / day. Dosing once a day
  • the disulfide-stabilized antibody is composed of one amino acid residue at an appropriate position in the cDNA encoding the VH and V of an antibody from a non-human animal or the cDNA encoding the VH and VL of a human CDR-grafted antibody.
  • the modification of an amino acid residue to a cysteine residue can be performed by the mutagenesis method using PCR in the above 2 (5).
  • a disulfide stabilized antibody H chain expression vector and a disulfide stabilized antibody L chain expression vector can be constructed.
  • any vector can be used as long as it can incorporate and express cDNAs encoding modified VH and modified VL.
  • pAGE107 [Cytotechnology, 3, 133 (1990)]
  • PAGE103 [Journal of Biochemistry G.
  • Suitable hosts for expressing the disulfide-stabilized antibody light chain expression vector and the disulfide-stabilized antibody heavy chain expression vector for forming disulfide-stabilized antibodies are E. coli, yeast, animal cells, etc. However, it is necessary to select a suitable expression vector for each host. Inserting a cDNA encoding an appropriate signal peptide into an expression vector allows the disulfide-stabilized antibody to be secreted extracellularly, transported to the periplasmic region, or retained inside the cell. Can be.
  • the disulfide-stabilized antibody H chain expression vector or the disulfide-stabilized antibody chain expression vector constructed in the above 4 (1) was subjected to elect-portation method (Japanese Patent Laid-Open Publication No. 2525/891, Cytotechnology). , 3, 133 (1990)] to obtain a transformant that produces the desired disulfide stabilized antibody H chain or disulfide stabilized antibody L chain. it can.
  • elect-portation method Japanese Patent Laid-Open Publication No. 2525/891, Cytotechnology. , 3, 133 (1990)] to obtain a transformant that produces the desired disulfide stabilized antibody H chain or disulfide stabilized antibody L chain. it can.
  • the expression of the disulfide-stabilized antibody H chain or the disulfide-stabilized antibody chain contained in the culture supernatant or the like is determined by the method described in 1 (5) above. Can be confirmed by
  • Recovery and purification of the disulfide-stabilized antibody H chain or the disulfide-stabilized antibody L chain can be achieved by a combination of known techniques. For example, if a disulfide-stabilized antibody H chain or disulfide-stabilized antibody L chain is secreted into the medium, it can be concentrated by ultrafiltration, followed by various mouth chromatography or gel filtration. Can be achieved by doing so. In addition, if the cells are transported to the periplasmic region of the host cells, the cells can be subjected to osmotic shock and concentrated by ultrafiltration, and then achieved by performing various types of chromatography or gel filtration. can do.
  • Disulfide stabilized antibody H chains or disulfide stabilized antibody chains that are insoluble and exist as granules (inclusion body) are lysed, centrifuged and washed to isolate granules This can be achieved by, for example, solubilizing with guanidine-hydrochloric acid and then performing various chromatography or gel filtration.
  • the active disulfide-stabilized antibody can be purified by antigen affinity chromatography, ion exchange chromatography or gel filtration.
  • the activity of the disulfide stabilized antibody can be measured by the method described in the above 1 (5) or the like.
  • the disulfide-stabilized antibody of the present invention specifically binds to human VEGF receptor KDR and neutralizes the action of DR. Therefore, the disulfide-stabilized antibody provided by the present invention can be used for the growth or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of premature babies, vascular disease caused by excessive VEGF production such as dry new It is considered to be useful for treatment of diseases in which the disease state progresses due to abnormalities in life.
  • the disulfide stabilized antibodies of the present invention can be used alone or in combination with at least one or more pharmaceutically acceptable auxiliaries.
  • a single-chain antibody or a disulfide-stabilized antibody is dissolved in a physiological saline solution or an aqueous solution of glucose, lactose, mannitol, or the like to prepare a suitable pharmaceutical composition.
  • the disulfide-stabilized antibody is freeze-dried according to a conventional method, and sodium chloride is added thereto to prepare a powder injection.
  • the pharmaceutical composition can contain, if necessary, additives well known in the pharmaceutical field, for example, pharmaceutically acceptable salts and the like.
  • the dose of the present pharmaceutical composition varies depending on the age, symptoms, etc. of the patient, but the disulfide-stabilized antibody is administered to mammals, including humans, at 0.1 to 20 mg / kg / day. Dosing may be by intravenous injection once a day (single dose or daily dose) or intermittently 1-3 times a week, once every few weeks.
  • the present invention provides a method for immunologically detecting and quantifying cells expressing human VEGF receptor KDR or human VEGF receptor KDR on the cell surface using the antibody of the present invention, a soluble human VEGF receptor.
  • the present invention relates to a method for immunologically detecting and quantifying KDR, a method for inhibiting the binding of human VEGF to human VEGF receptor KDR, and a method for neutralizing the action of human VEGF receptor KDR.
  • the method of immunologically detecting and quantifying human VEGF receptor KDR, cells expressing human VEGF receptor KDR on the cell surface or soluble human VEGF receptor KDR using the monoclonal antibody of the present invention includes a fluorescent antibody method.
  • Immunohistochemical staining ELISA
  • radioactive substance-labeled immunoantibody R1A
  • immunohistochemical staining immunocytochemical staining, etc.
  • immunohistochemical staining (ABC, CSA, etc.)
  • western blotting Immunoprecipitation method, enzyme immunoassay described above, sandwich EL1SA method [Monoclonal antibody experiment manual (Kodansha Scientific, 1987), Seisho Chemistry Experiment Course 5 Immunobiochemistry Research Method (Tokyo Kagaku Dojin, 1986)].
  • the fluorescent antibody method is to react the isolated antibody or the monoclonal antibody of the present invention with isolated cells or tissues, and then react with an anti-immunoglobulin antibody or a binding fragment labeled with a fluorescent substance such as fluorescin'isothiocyanate (FITC). After that, the fluorescent dye is measured with a flow cytometer.
  • a fluorescent substance such as fluorescin'isothiocyanate (FITC).
  • the immunoenzyme-linked immunosorbent assay refers to the reaction of the fungus body of the present invention with isolated cells or their lysate, tissue or its lysate, cell culture supernatant, serum, pleural effusion, ascites, eye fluid, etc. This is a method in which a colored dye is measured with an absorptiometer after reacting with an anti-immunoglobulin antibody or a binding fragment labeled with an enzyme such as peroxidase or biotin.
  • the radioactive-labeled immunoantibody method refers to the reaction of the antibody of the present invention with isolated cells or their lysates, tissues or their lysates, cell culture supernatants, serum, pleural effusion, ascites, and eye fluids. This is a method in which a radiolabeled anti-immunoglobulin antibody or a bound fragment is reacted, and the reaction is measured using a scintillation counter or the like.
  • the immunocytostaining and immunohistochemical staining methods are the reaction of the antibody of the present invention with isolated cells or tissues, and further, fluorescent substances such as fluorescin / isothiocynate (FITC), enzymes such as peroxidase and biotin.
  • fluorescent substances such as fluorescin / isothiocynate (FITC), enzymes such as peroxidase and biotin.
  • FITC fluorescin / isothiocynate
  • enzymes such as peroxidase and biotin.
  • the human VEGF receptor KDR present in the cells or tissues of a subject is analyzed by the immunology described above. Methods for quantitatively detecting or quantifying it.
  • the monoclonal antibody of the present invention is used as a diagnostic agent for a disease based on the growth or metastasis of a solid tumor, or a disease in which the disease state progresses due to abnormal vascularization. Can be used.
  • the inhibitory activity of the binding between human VEGF and the human VEGF receptor KDR was determined by measuring the binding between growth factors and receptors. Biochemistry Laboratory Course 7 Growth differentiation factors and their receptors (Tokyo Kagaku Dojin, 1991)] It can be confirmed by performing a VEGF-VEGF receptor KDR binding inhibition test using the antibody of the present invention in accordance with the methods described above.
  • the antibody of the present invention By reacting the antibody of the present invention simultaneously with VEGF labeled with a radioactive substance or the like, the activity of inhibiting binding of VEGF labeled with a radioactive substance or the like to KDR can be measured.
  • the autophosphorylation inhibitory activity of the VEGF receptor KDR can be determined by a method such as the growth factor receptor autophosphorylation assay [Sequence Chemistry Laboratory Course, Signaling and Cell Response (Tokyo Kagaku Dojin, 1986)], etc. It can be confirmed by performing a VEGF-VEGF receptor KDR autophosphorylation inhibition test using a monoclonal antibody.
  • VEGF vascular endothelial growth factor
  • a method in which VEGF is reacted with cells or tissues expressing KDR, and the autophosphorylation of KDR, which is enhanced by the binding of VEGF is detected by immunoprecipitation method and Western blot method.
  • By reacting the antibody of the present invention simultaneously with VEGF it is possible to measure the activity of inhibiting the autophosphorylation of KDR which is enhanced by the binding of VEGF.
  • VEGF vascular endothelial growth factor
  • the VEGF-dependent vascular endothelial cell proliferation test is a method of reacting vascular endothelial cells with VEGF and measuring the number of vascular endothelial cell proliferation-promoting activities that are enhanced by the binding of VEGF. By reacting the antibody of the present invention simultaneously with VEGF, the activity of inhibiting the growth promoting activity of vascular endothelial cells promoted by VEGF can be measured. It is possible.
  • the VEGF-dependent migration test of vascular endothelial cells is a method in which VEGF is reacted with vascular endothelial cells, and the activity of promoting the migration of vascular endothelial cells, which is enhanced by the binding of VEGF, is observed using a microscope.
  • VEGF vascular endothelial growth factor
  • the tube formation test of VEGF-dependent vascular endothelial cells is a method in which vascular endothelial cells are reacted with VEGF and the tube formation promoting activity of vascular endothelial cells, which is enhanced by the binding of VEGF, is observed using a microscope.
  • VEGF vascular endothelial growth factor
  • the present invention provides a method for diagnosing or treating an angiogenesis disorder, comprising using the antibody of the present invention, and a diagnostic or therapeutic agent for an angiogenic disorder comprising the antibody of the present invention as an active ingredient. About.
  • Abnormal angiogenesis disease is caused by abnormal angiogenesis that can be caused by excessive VEGF, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness. Illness.
  • Examples of a method for diagnosing an angiogenesis disorder include a method for immunologically detecting or quantifying the human VEGF receptor KDR present in cells or tissues of a subject. Further, the antibody of the present invention can be used as a diagnostic agent for an angiogenesis disorder.
  • the antibody of the present invention can inhibit the biological activity of human VEGF, it inhibits KDR autophosphorylation by inhibiting the binding of human VEGF to KDR, thereby inhibiting VEGF-dependent proliferation of human vascular endothelial cells. Therefore, it can be used as a therapeutic drug for abnormal angiogenesis disease.
  • FIG. 1 is a view showing a construction process of plasmid pVL-KDR-7N-Fc.
  • FIG. 2 is a schematic diagram of various derivatives of soluble KDR-Fc.
  • FIG. 3 is a schematic diagram of various derivatives of soluble KDR.
  • FIG. 4 is a diagram showing a pattern of purified soluble KDR-Fc various derivatives by SDS polyacrylamide electrophoresis (using a 5 to 20% gradient gel). From left, DR-IN-Fc, KDR-2N-Fc, KDR-3N-Fc, KDR-4N-Fc, KDR-5N_Fc, KDR-7N-Fc, DR-2A 1N-Fc, KDR-4 ⁇ 1 ⁇ — Fc and KDR—5 ⁇ 1 ⁇ —The migration patterns of Fc are shown, respectively. Electrophoresis was performed under reducing conditions.
  • Figure 5 shows the results of analyzing the inhibitory effect of soluble human VEGF receptor KDR-Fc various derivatives on the binding of the plate co-one Bok was to soluble human Bok VEGF receptor KDR-7 N- Fc '25 Bok human VEGF .
  • B shows the results of analyzing the binding of 125 -human VEGF to various derivatives of the soluble human VEGF receptor DR-Fc coated on the plate.
  • FIG. 6 shows the results of analyzing the binding activity of human VEGF receptor KDR monoclonal antibody to soluble human VEGF receptor KDR-Fc various derivatives.
  • FIG. 7 shows the epitope site of the human VEGF receptor KDR monoclonal antibody.
  • FIG. 8 shows the results of analyzing the binding activity of human VEGF receptor KDR monoclonal antibody to soluble human VEGF receptor KDR-Fc various derivatives.
  • FIG. 9 shows the results of analysis of the inhibitory effect of human VEGF receptor KDR monoclonal antibody on binding between VEGF and soluble VEGF receptor KDR-7N-Fc and soluble VEGF receptor Fit-17N.
  • FIG. 10 shows the results of examining KDR autophosphorylation inhibitory activity by a human VEGF receptor KDR monoclonal antibody.
  • FIG. 11 shows the results of examining the VEGF-dependent vascular endothelial cell growth inhibitory activity of a human VEGF receptor KDR monoclonal antibody.
  • Fig. 12 Human VEGF receptor KDR monoclonal antibody expressing human VEGF receptor KDR N1H3T3-Fit-1, control cells NlH3T3-Neo cells, HUVEC and The results of analyzing the reactivity with HMVEC by flow cytometry are shown.
  • Fig. 13 shows the results of analysis of the reactivity of the human VEGF receptor KDR monoclonal antibody with HUVEC and HVEC using a single cytometer.
  • Soluble human VEGF corresponding to the 19 amino acids described in SEQ ID NO: 27 and the mature human VEGF receptor KDR constituting the signal peptide of human VEGF receptor KDR, which corresponds to amino acid sequence 1 to 738 of SEQ ID NO: 26
  • a fusion protein consisting of a receptor KDR fragment, a linker consisting of 6 amino acid residues (linker # 1) and 227 amino acids constituting the human antibody Fc region hereinafter referred to as soluble human VEGF receptor KDR-7N-Fc) ) was prepared by the following procedure.
  • Soluble human VEGF receptor KDR-7N-Fc is a linker (linker #) consisting of 7 immoglobulin-like sites and 6 amino acid residues from the N-terminal side of the extracellular region of soluble human VEGF receptor KDR. 1) and a fusion protein comprising a human antibody Fc region.
  • the cDNA clone BCMGS-neo-DR (A. Sawano et al., Cell Growth & Differentiation 7, 213-221, 1996) encoding the full-length cDNA of the human VEGF receptor KDR was cut with EcoRI, and the extracellular region of KDR and PUC-KDR was generated by incorporating an approximately 2.8 kb fragment encoding the membrane binding region into the EcoRI site of pUC18.
  • pUC-KDR was cut with Xhoi, treated with Klenow, and inserted with Xbal linker (SEQ ID NO: 1) to prepare pUC-KDR-Xb.
  • a fusion protein consisting of a KDR fragment, a linker consisting of 6 amino acid residues (linker # 1) and 227 amino acids constituting the human antibody Fc region hereinafter referred to as soluble human VEGF receptor KDR-6N-Fc
  • a vector for expression was prepared by the following procedure.
  • the soluble human VEGF receptor KDR-6N-Fc is a linker (linker # 6) consisting of six immunoglobulin-like sites and six amino acid residues from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR. 1) and a fusion protein comprising a human antibody Fc region.
  • Primer 10 having the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 10 pmoK pBS-DR-Xb-S (Preparation of antigen (1)) DNA 10 ng and lOmM
  • a fusion gene of soluble human VEGF receptor KDR-6N and human antibody Fc region was inserted into the 5 'Xbal and 3' NoU sites downstream of the transcription start site of the polyhedrin gene of the pVL1393 plasmid to express the fusion gene.
  • pVL-KDR-6N-Fc was constructed.
  • Soluble human VEGF receptor KDR-5N-Fc is a linker (linker # 1) consisting of five immonoglobulin-like sites and six amino acid residues from the N-terminal side of the extracellular region of soluble human VEGF receptor KDR. And a human antibody Fc region.
  • the Xbal / SnaBl (1.9 kbp) fragment of pBS-KDR-5N and SnaBI / Notl (0.7 kbp) encoding the Fc region of the human antibody on pAMoPRFc were ligated to the baculovirus recombinant pVL1393 plasmid. Integrates into the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the hedrin (Polyhedrin) gene, and expresses the fusion gene between soluble human VEGF receptor KDR-5N and human antibody Fc region. -5N-Fc was constructed. (4) Construction of a fusion gene expression vector of soluble human VEGF receptor KDR-4N and human antibody Fc region
  • a fusion protein consisting of a VEGF receptor KDR fragment, a linker consisting of two amino acid residues (linker # 2), and 227 amino acids constituting a human antibody Fc region (hereinafter referred to as soluble human VEGF receptor KDR-4N-Fc A vector for expressing) was prepared by the following procedure.
  • Soluble human VEGF receptor KDR-4N-FC is a linker (linker # 2) consisting of four immoglobulin-like sites and two amino acid residues from the N-terminal side of the extracellular region of soluble human VEGF receptor KDR. ) And a human antibody Fc region.
  • pUC-KDR-Xb (1 kbp) and Xbal / Hindlll (0.7 kbp) of pUC-KDR-Xb are incorporated into the baculovirus-recombinant pVL1393 plasmid at the 5 'Xbai and 3' Notl sites downstream of the transcription start site of the polyhedrin (Polyhedrin) gene.
  • a fusion gene expression vector pVL-KDR-4N-Fc comprising a soluble human VEGF receptor KDR-4N and a human antibody Fc region was constructed.
  • Soluble human VEGF receptor corresponding to the 19 amino acids described in SEQ ID NO: 27 and the amino acid sequence described in SEQ ID NO: 26, which is the mature human VEGF receptor KDR, which constitutes the signal peptide of human VEGF receptor KDR.
  • Fusion protein consisting of a linker KDR fragment (linker # 1) consisting of 6 amino acid residues and 227 amino acids constituting the Fc region of a human antibody (hereinafter referred to as soluble human VEGF receptor KDR-3N-FC) was prepared by the following procedure.
  • the soluble human VEGF receptor KDR-3N-Fc is a linker (linker # 3) consisting of three immunoglobulin-like sites and six amino acid residues from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR. 1) and a fusion protein comprising a human antibody Fc region.
  • the Xbal / SnaBI (1.2 kbp) fragment of pBS-KDR-3N and the SnaBI / Notl (0.7 kbp) fragment encoding the Fc region of the human antibody on pAMoPRFc were ligated to the baculovirus recombinant PVL1393 plasmid.
  • a fusion protein consisting of a KDR fragment, a linker consisting of 6 amino acid residues (linker # 1) and 227 amino acids constituting a human antibody Fc region hereinafter referred to as a soluble human VEGF receptor KDR-2N-Fc
  • Soluble human VEGF Receptor KDR-2N-Fc is a linker (linker # 1) consisting of two immoglobulin-like sites and six amino acid residues from the N-terminal side of the extracellular region of soluble human VEGF receptor KDR, This corresponds to a fusion protein consisting of the antibody Fc region.
  • Soluble human VEGF receptor corresponding to the 19 amino acids described in SEQ ID NO: 27 and the mature human VEGF receptor KDR, which constitutes the signal peptide of human VEGF receptor KDR, and the amino acid sequence described in SEQ ID NO: 26 corresponding to positions 1 to 104 KDR fragment, a fusion protein consisting of a linker consisting of 6 amino acid residues (linker # 1) and 227 amino acids constituting the human antibody Fc region (hereinafter referred to as soluble human VEGF receptor KDR-IN-Fc) was prepared by the following procedure.
  • the soluble human VEGF receptor KDR-IN-Fc is a linker (linker # 1) consisting of one immunoglobulin-like site and 6 amino acid residues from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR. And a human antibody Fc region.
  • a synthetic linker having the nucleotide sequences of SEQ ID NO: 14 and SEQ ID NO: 15 was ligated to a Bglll / otl (2.8 kbp) fragment of PBS-DR-2 (preparation of antigen (6)), and pBS-KDR-1N was constructed.
  • the Xbal / SnaBI (0.4 kbp) fragment of pBS-KDR- and SrmBl / Notl (0.7 kbp) encoding the Fc region of the human antibody on pAMoAPRFc (see Preparation of Antigen (1)) were patched.
  • soluble human VEGF receptor KDR-7N-Fc From the soluble human VEGF receptor KDR-7N-Fc (see antigen preparation (1)), a total of 72 amino acids from the 31st amino acid to the 102nd amino acid forming the 1st immunoglobulin-like site from the N-terminal end A fusion protein consisting of a linker (linker # 1) consisting of 6 amino acid residues and 227 amino acids constituting the human antibody Fc region (hereinafter referred to as soluble human VEGF receptor KDR -7 ⁇ 1 ⁇ -Fc) was prepared by the following procedure.
  • Soluble human VEGF receptor KDR-7D1N-Fc is a linker (linker # 2) consisting of the 2-7th immunoglobulin-like site from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR and a 6-amino acid residue. 1) and a fusion protein comprising a human antibody Fc region.
  • Primer having the nucleotide sequence shown in SEQ ID NO: 16 and SEQ ID NO: 17 10 pmoK PVL-DR-7N (Preparation of antigen (14)) Contains 10 ng of DNA and 10 mM deoxynucleotide triphosphates 2.5 units Taq polymerase was added to 10 mM MgCl 2 , 0.001% (W / V) gelatin solution 100 H 1. The reaction was pretreated at 95 ° C for 5 minutes, followed by 30 cycles of polymerase chain reaction (PCR) at 95 ° C for 90 seconds, 50 ° C for 90 seconds, and finally at 72 ° C for 90 seconds. The DNA fragment was recovered.
  • PCR polymerase chain reaction
  • This DNA fragment was digested with Xbal and Bglll to obtain a 0.8 kbp Xbal / Bglll fragment.
  • This DA fragment and the Bglll / Notl (1.6 kbp) fragment of pVL-KDR-5N (see Preparation of antigen (17)) were inserted into Xbal / Notl of pBluescriptll SK (-), and pBS-KDR-5-5 ⁇ was prepared.
  • the Xbal / Hincn (1.6 kbp) fragment of pBS-KDR-5 ⁇ 1 ⁇ and the Hincll / Notl (1.2 kbp) of pVL-KDR-7N_Fc were recombined with the baculovirus recombinant pVL1393 plasmid. Integrates into the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the polyhedrin gene of Sumid, and expresses the fusion gene expression vector between soluble human VEGF receptor KDR-7D 1N and human antibody Fc region pVL -Constructed KDR-7 ⁇ ⁇ -Fc.
  • soluble human VEGF receptor KDR-5N-Fc From the soluble human VEGF receptor KDR-5N-Fc (see Preparation of Antigen (3)), a total of 72 amino acids from the 31st amino acid to the 102nd amino acid forming the 1st immunoglobulin-like site from the N-terminal end KDR fragment from which a single amino acid has been deleted, a linker consisting of 6 amino acid residues (linker # 1) and a fusion protein consisting of 227 amino acids constituting the human antibody Fc region (hereinafter referred to as soluble human VEGF receptor KDR- 5 ⁇ -Fc) was prepared by the following procedure.
  • Soluble human VEGF receptor KDR-5 ⁇ -Fc is a linker consisting of the second to fifth immunoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR and a 6-amino acid residue (linker # 1) and a fusion protein comprising a human antibody Fc region.
  • soluble human VEGF receptor KDR-4N-Fc From the soluble human VEGF receptor KDR-4N-Fc (see Antigen Preparation (4)), ⁇ : 72 amino acids from the 31st amino acid to the 102nd amino acid forming the first imnoglobulin-like site from the terminal KDR fragment from which the amino acid has been deleted, linker consisting of 6 amino acid residues (linker # 2) and 227 amino acids constituting the human antibody Fc region
  • linker # 2 linker consisting of 6 amino acid residues
  • linker # 227 amino acids constituting the human antibody Fc region A vector for expressing a fusion protein consisting of (hereinafter, referred to as soluble human VEGF receptor KDR-4D 1N-FC) was prepared by the following procedure.
  • Soluble human VEGF receptor DR-4 ⁇ -Fc is a linker consisting of the second to fourth immunoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR and a 2-amino acid residue. This corresponds to a fusion protein consisting of an antibody # 2) and a human antibody Fc region.
  • Baculovirus recombinant pVL1393 plasmid with Xbal / Kpnl (1.0 kbp) fragment of pBS-KDR-4 ⁇ and SnaBI / Notl (0.7 kbp) encoding Fc region of human antibody on pAMoAPRFc (see antigen preparation (1))
  • a fusion gene expression vector pVL that integrates the soluble human VEGF receptor KDR-4 ⁇ and the human antibody Fc region by integrating into the 5 'Xbal and 3 Notl sites downstream of the transcription start site of the Polyhedrin gene -KDR- 4 ⁇ -Fc was constructed.
  • Soluble human VEGF corresponding to the 19 amino acid described in SEQ ID NO: 27 and the mature human VEGF receptor KDR constituting the signal peptide of human VEGF receptor KDR, which corresponds to the 1st to 738th amino acid sequence described in SEQ ID NO: 26
  • a vector for expressing a receptor KDR fragment (hereinafter, referred to as soluble human VEGF receptor KDR-7N) and two amino acid residues derived from a linker was prepared by the following procedure.
  • the soluble human VEGF receptor DR-7N corresponds to ⁇ immunoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR.
  • pBS-DR-Xb-S (see Preparation of antigen (1)) is digested with SnaBl / BamHI, and a synthetic linker (SEQ ID NO: 18 and SEQ ID NO: 19) containing a stop codon and Notl site is incorporated.
  • Xb) -SN was prepared.
  • U (2.3 kb) fragment was inserted into the Xbal and 3 'Notl sites downstream of the transcription start site of the polyhedrin (Polyhedrin) gene of the recombinant Paculovirus PVL1393 plasmid, and A VEGF receptor KDR-7N expression vector pVL-KDR-7N was constructed.
  • Soluble human VEGF corresponding to amino acids 1 to 714 of amino acid sequence described in SEQ ID NO: 26, which is 19 amino acids described in SEQ ID NO: 27 and mature human VEGF receptor KDR that constitutes a signal peptide of human VEGF receptor KDR
  • a vector for expressing a receptor KDR fragment (hereinafter referred to as soluble human VEGF receptor KDR-7N ') was prepared by the following procedure.
  • the soluble human VEGF receptor KDR-7N ' corresponds to up to about 2/3 of the 7th-thick immoglobulin-like site from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR.
  • pUC-DR-Xb was cut with Stul and Sphl and a synthetic linker containing a stop codon and a Notl site (SB column No. 23 and SEQ ID No. 21) was inserted.
  • XbaI Notl (2.2 kbp) fragment was incorporated into the baculovirus recombinant pVL1393 plasmid at the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the polyhedrin (Polyhedrin) gene, expressing the soluble human VEGF receptor KDR-7N '
  • the vector pVL-KDR-7N ' was prepared.
  • a vector for expressing a somatic KDR fragment (hereinafter, referred to as soluble human VEGF receptor KDR-5N) was prepared by the following procedure. Soluble human VEGF receptor KDR-5N corresponds to the five immunoglobulin-like sites from the N-terminal side of the extracellular region of soluble human VEGF receptor KDR.
  • Soluble amino acids corresponding to the 19 amino acids described in SEQ ID NO: 27 and the mature human VEGF receptor KDR, which constitute the signal peptide of the human VEGF receptor KDR, and corresponding to amino acids 1 to 393 of the amino acid sequence described in SEQ ID NO: 26 A vector for expressing a VEGF receptor KDR fragment (hereinafter referred to as soluble human VEGF receptor KDR-4N) and a linker-derived two amino acid residue was prepared by the following procedure.
  • the soluble human VEGF receptor DR-4 corresponds to the four immunoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR.
  • Xbal / Kpnl (1.2 kb) fragment of pAMo-4N-Fc (see Preparation of Antigen (4)) and a synthetic linker having the nucleotide sequence of SEQ ID NO: 24 and SEQ ID NO: 25 were recombined with baculovirus, and polyhedrin of pVL1393 plasmid (Polyhedrin) )
  • a soluble human VEGF receptor KDR-4N expression vector pVL_KDR-4N was constructed by incorporating the gene into the 5 ′ Xbal and 3 ′ Notl sites downstream of the transcription start site of the gene.
  • Soluble human VEGF corresponding to the 19 amino acids described in SEQ ID NO: 27 and the human VEGF receptor KDR described in SEQ ID NO: 26, which constitute the signal peptide of human VEGF receptor KDR.
  • a vector for expressing a receptor KDR fragment (hereinafter referred to as a soluble human VEGF receptor KDR-3N) and a linker-derived two amino acid residue was prepared by the following procedure.
  • the soluble human VEGF receptor DR-3N corresponds to the three immunoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR.
  • soluble human VEGF receptor KDR-7 ⁇ From the soluble human VEGF receptor KDR-7 ⁇ '(see Preparation of Antigen (14)), a total of 72 amino acids from the 31st amino acid to the 102nd amino acid forming the first immunoglobulin-like site from the ⁇ -terminal side KDR fragment from which the amino acid has been deleted, a linker consisting of 6 amino acid residues (linker # 1) and a fusion protein consisting of 227 amino acids constituting the human antibody Fc region (hereinafter referred to as soluble human VEGF receptor KDR -7 ⁇ 1 ⁇ ) was prepared by the following procedure.
  • the soluble human VEGF receptor KDR-7 ⁇ 1 is derived from the 2-7th immoglobulin II site from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR and a linker (linker # 1) consisting of 6 amino acid residues. Corresponding fusion proteins.
  • the production of proteins by insect cells requires the production of a recombinant virus incorporating the target gene.
  • the production of cDXA, which encodes the target protein called transfer vector, into a special plasmid and the production of wild-type virus Cotransfection of insect cells and transfer vectors into insect cells, and undergoes the process of obtaining a recombinant virus by homologous recombination.
  • the above process was performed according to the manual according to the manual using Farmingen's Bakyulogo Star Star Yuichi Kit (product number PM-21001K).
  • 1 Hg of the expression vector prepared in (1) and 20 ng of linear baculovirus DNA were dissolved in 12/1 distilled water, and lipofectin 61 and distilled water 61 were mixed. Was added and left at room temperature for 15 minutes.
  • 1 ⁇ 10 6 Sf9 cells were suspended in 2 ml of S100-11 medium (manufactured by Gibco) and placed in a 35 mm-diameter plastic dish for cell culture. The above plasmid DNA, linear baculovirus virus DNA and lipofectin mixed solution were added thereto, and the mixture was cultured at 27 ° C for 3 days. Then, 1 ml of the culture supernatant containing the recombinant virus was collected.
  • each of the recombinant viruses obtained for use in protein expression was propagated by the following procedure.
  • the virus titer of the obtained recombinant virus solution was calculated according to the method described in the Bakuguchi Gold Star Yuichi Kit 'Manual (Pharmingen). 6 ⁇ 10 s Sf9 cells were suspended in 4 ml of Sf900-Ii medium, placed in a plastic culture dish for cell culture having a diameter of 60 mm, and allowed to stand at room temperature for 1 hour to allow the cells to adhere to the petri dish. Next, remove the supernatant, add the above recombinant virus solution diluted 1000-fold with Sf900-II medium 400 II 1 and Sf900-11 medium, and leave at room temperature for 1 hour.Remove the medium and remove 5 ml of 1% low-melting-point agarose.
  • a medium containing [Agarplaque Agarose, manufactured by Pharmingen] [Sterile lml of 5% agar-plaque plus-agarose aqueous solution and 4 ml of TMN-FH insect medium are mixed. Kept at 42 ° C] was poured into the petri dish. After standing at room temperature for 15 minutes, vinyl tape was spread on a Petri dish to prevent drying, the dish was placed in a sealable plastic container, and cultured at 27 ° C for 6 days. After adding 1 ml of PBS containing 0.01% neutral nut to the petri dish and further culturing for 1 day, the number of plaques that appeared was counted. From the above operations, it was found that each of the recombinant virus solutions contained about 1 ⁇ 10 7 plaque forming units (hereinafter, referred to as PFU) / ml of the virus.
  • PFU plaque forming units
  • Various soluble human VEGF receptor KDR-Fc derivatives and various soluble human VEGF receptor KDR derivatives described in (1) to (16) were obtained as follows. 4 X High Five cells were suspended in 30 ml of EX-CELL TM 400 medium (manufactured by JRH Bioscience) in a 175 cm 2 flask (manufactured by Glyna), allowed to stand at room temperature for 1 hour, and allowed to adhere to the flask.
  • Prosep A Purification was carried out as follows using a system.
  • the column was filled with about 1 ml of Prosep A [manufactured by Bioprocessing], and the column was washed with 10 ml of 20 mM sodium phosphate buffer (pH 7.2) at a flow rate of 1 ml / min. After washing, 500 to 1000 ml of the culture solution containing the soluble human VEGF receptor KDR prepared as described above was passed through a Prosep A column at a flow rate of 100 ml / hour.
  • the collected purified fractions were concentrated using CentriBrep 10 (manufactured by Amicon), and soluble human KDR3N, KDR4N, KDR5N, KDR7 'and KDR7N were used as solutions in 2.8ml, 8ml, 5.5mK, 4ml and 4.8ml, respectively.
  • ml protein concentration / purity is 345.5 ll g / ml / 30%, 264 g / ml / 50-60%, 380.5 fl g / ml / 70, 1.59 mg / ml / 60% and 815 g / ml / 70 -80 %) Obtained.
  • FIGS. 2 and 3 show schematic diagrams of the obtained various derivatives of soluble human VEGF receptor KDR-Fc and various derivatives of soluble human VEGF receptor KDR. (19) Confirmation of purity of soluble human VEGF receptor KDR
  • KDR-7N-Fc, KDR-5N-Fc, KDR-4N-Fc, KDR-3N-Fc, KDR-2N-Fc, KDR-IN-Fc, KDR-5 ⁇ 1c-Fc, KDR-4 ⁇ -Fc was over 95%.
  • the control antigen protein was obtained as follows. 4 ⁇ 10 7 High Five cells were suspended in 30 ml of EX-CELL TM 400 medium (manufactured by JRH Bioscience) in a 175 cm 2 flask (Graina), allowed to stand at room temperature for 1 hour, and allowed to adhere to the flask. The cells were cultured at 27 ° C for 3 to 4 days. After completion of the culture, the culture supernatant was collected and centrifuged at 1,500 X g for 10 minutes to obtain a supernatant.
  • Soluble human VEGF receptor KDR-Fc derivatives obtained in (18) (KDR-7N-Fc, KDR-5N-Fc, DR-4N-Fc, KDR-3N-Fc, KDR-2N-Fc, KDR-IN — Fc, KDR-5 ⁇ 1 ⁇ -Fc, The human VEGF binding activity of KDR-4A1N-Fc and KDR-2 ⁇ 1 ⁇ -Fc) was confirmed by the following (2) 1) VEGF binding inhibition test and (21-2) VEGF binding test.
  • KDR-3N-Fc, KDR-2N-FC, KDR- ⁇ -Fc and KDR-2A 1N-Fc did not show any binding inhibitory activity.
  • the binding inhibitory activities were in the order of KDR-7N-Fc>KDR-5 ⁇ 1 ⁇ -Fc>DR-5i-Fc>DR-4AlN-Fc> KDR-4N-Fc.
  • Methanol was dispensed into a 96-well Immobilon TM -P Filtration Plate (96-well Immobilon TM -P Filtration Plate; manufactured by Millipore) at 100 ⁇ 1 / well to hydrophilize the PVDF membrane at the bottom of the plate.
  • KDR-Fc various derivatives KDR-7N'-Fc, DR-5N-Fc, KDR-4N -Fc, KDR-3N-Fc, KDR-2N-Fc, KDR-IN-Fc, DR-5A1N-Fc, KDR-4AlN_Fc, KDR—2 ⁇ 1 ⁇ -Fc
  • 125 1-labeled human VEGF (final concentration 4 ng / ml: manufactured by Amersham) was added in 50 ⁇ l, and the mixture was reacted at room temperature for 1.5 hours.
  • Tween-PBS dry the wells at 50 ° C, add 10 1 / well of Microscint-0 (manufactured by Packard), and use Topcount (manufactured by Topcount Inc.). , to measure the radioactivity of 125 1-labeled human VEGF bound to each Ueru.
  • KDR- 7N - Fc, DR-5AlN -Fc, KDR- 5N_Fc, KDR-4A IN - Fc, DR-4N-Fc was shown to bind to a concentration dependent manner 125 1-labeled human VEGF.
  • KDR-3N-Fc, KDR-2N-Fc, KDR-IN-Fc, and KDR-2A1N-FC showed no binding activity.
  • Human VEGF was obtained as follows. EX-CE 4 x 10 7 High Five cells on a 175 cm 2 flask (Grainer) and suspend in 30 ml of L TM 400 medium (JRH Bioscience). It became cloudy, was left at room temperature for 1 hour, and was attached to the flask.
  • the human VEGF recombinant baculovirus solution obtained by the method described in the literature [Cell Growth & Differentiation, 7, 213, (1996)] is used for about 1 to 3 ⁇ 10 8 PFU. The solution was added at a concentration of / ml and infected for 2 hours at room temperature.
  • the culture supernatant was removed and a fresh 30 ml of EX-CELL TM 400 medium was added, followed by culturing at 27 ° C for 3 to 4 days. After completion of the culture, the culture supernatant was collected and centrifuged at 1,500 X g for 10 minutes to obtain a supernatant.
  • the column was filled with about 40 ml of heparin-Sepharose CL-6B gel [Pharmacia Biotech AB], and 0.5 ml of a buffer solution containing 400 ml of 20 mM tris-hydrochloric acid (pH 7.5) was used. Washed at a flow rate of / min. After washing, 1500 ml of the culture solution containing human VEGF prepared as described above was passed through a heparin-Sepharose CL-6B column at a flow rate of 0.5 ml / min.
  • Venous plexus, heart Alternatively, blood is collected from the tail vein, and its serum antibody titer is examined by the enzyme immunoassay shown below. From the mouse or rat showing a sufficient antibody titer by the enzyme immunoassay shown in step 3, the spleen is obtained 3 days after the final immunization Was extracted. The 5-week-old female BALB to which NIH3T3-KDR cells were administered was not immunized, and the antibody titer against soluble KDR did not increase.
  • the spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical), loosened with tweezers, centrifuged (l, 200 rpm, 5 minutes), the supernatant is discarded, and tris-ammonium chloride buffer is removed.
  • MEM medium manufactured by Nissui Pharmaceutical
  • the cells were treated with (PH7.65) for 1 to 2 minutes to remove red blood cells, washed three times with MEM medium, and used for cell fusion.
  • Heparin column-adsorbed fraction of High Five cell culture supernatant, or anti-GD3 mouse human chimeric antibody KM871 [Cancer Immunology and Immunotherapy, 36, 373 (1993)] was dispensed at 50 1 / ⁇ , and 4 ° C. It was adsorbed by standing. After washing, add 100% PBS containing 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the 8-azaguanine-resistant mouse myeloma cell line P3-U1 was cultured in a normal medium to secure 2 ⁇ 10 7 or more cells at the time of cell fusion, and used as a parent strain for cell fusion.
  • the mouse spleen cells or rat splenocytes obtained in step 2 and the myeloma cells obtained in step 4 are mixed at a ratio of 10: 1, centrifuged (l, 200 rpm, 5 minutes), and the supernatant is removed. Discard and disintegrate the precipitated cell group well, and stir with agitation at 37 with a mixture of 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM medium and 0.7 rnl of DMSO 0.2-lml / 10 8 mouse spleen Cells were added, 1-2 ml of MEM medium was added several times every 1-2 minutes, and then MEM medium was added to bring the total volume to 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded, the cells were loosened gently, and the cells were suspended in 100 ml of HAT medium gently by aspiration and aspiration with a female pipette.
  • PEG-1000 poly
  • Soluble human VEGF receptor obtained in 1 (18) KDR-Fc derivatives, KDR derivatives and KDR-NIH3T3 cells were immunized with Balb / c mice, B6C3F1 mice, or SD rats.
  • the obtained hybridoma was screened for about 16548 wells, and specifically reacted with the soluble human VEGF receptor KDR-Fc derivatives and KDR derivatives obtained in 1 (18), and 1 (20)
  • a total of 74 clones of anti-human VEGF receptor KDR monoclonal antibody that did not react with the control antigen or KM871 obtained in the above were obtained and named as shown in Table 1, respectively.
  • VEGF receptor KDR monoclonal antibodies 40 monoclonal antibodies (KM1668, 1768, 1825, 1826, 1827, 1828, 1829, 1831, 1835, 1837, 1853, 1856, 1857, 1859, 1860 , 1861, 1862, 1863, 1864, 1865, 1933, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1987, 1988, 1989, 1858, 1832, 1833, 1834, 1836, 1838, 1932 ) was shown to respond to KDR on the cell surface by the immunocytostaining method shown in 15.
  • a monoclonal antibody that inhibits the biological activity of KDR which exhibits the activity of inhibiting the growth promoting activity of vascular endothelial cells by VEGF stimulation, could not be obtained.
  • the specificity of the anti-human VEGF receptor KDR monoclonal antibody described in 6. was confirmed using the hybridoma culture supernatant by the enzyme immunoassay described in 3. above.
  • Fig. 6 shows typical results
  • Fig. 7 summarizes the results.
  • KM 1668 32 other species in response to the first I g-like domain corresponding to 1-104 Amino acid
  • KM1987 other three are the first lg-like domain (1 104 KM 1855 and other 5 species react with the second Ig-like domain (corresponding to 105-194 amino acids) and the second Ig-like domain (corresponding to 105-194 amino acids).
  • KM1858 and 2 other species react to the third Ig-like domain (corresponding to 195-294 amino acids), KM1854 and 3 other species to the fourth Ig-like domain (corresponding to 295-393 amino acids), KM1832 and others 14 species react to the 5th Ig-like domain (corresponding to amino acids 394-518) KM1665 and two other species reacted to the 6th to 7th g-like domains (corresponding to amino acids 519 to 738).
  • the first lg-like domain of KDR is not involved in the binding activity of VEGF, but because of its high immunogenicity, it is difficult to produce a monoclonal antibody showing neutralizing activity in ELISA screening. Was estimated.
  • mice were immunized with the KDR-5A1N-Fc obtained in 1 (18) to eliminate monoclonal antibodies against the first lg-like domain, which were highly immunogenic and unrelated to neutralizing activity.
  • the binding inhibitory activity of mouse antisera between human VEGF and human VEGF receptor KDR was evaluated according to the following procedure.
  • Methanol was dispensed into a 96-well MultiScreen-IP plate (96-well MultiScreen-IP Plate; manufactured by Millipore) at 100 1 / well to hydrophilize the PVDF membrane at the bottom of the plate.
  • soluble human VEGF receptor KDR-7N-Fc diluted to a concentration of 4 / g / ml with PBS was dispensed at 50 1 / well, and allowed to stand at 4 ° C for adsorption. .
  • PBS containing 1% bovine serum albumin (BSA) was added at 200 1 / well, and the remaining active groups were blocked by reacting at room temperature for 30 minutes.
  • BSA bovine serum albumin
  • Table 2 shows the results of examining the activity of the culture supernatant of the hybridoma.
  • KDR-5 5 ⁇ 1 ⁇ - Fc All three sera of mice immunized showed more than 50% binding inhibitory activity at 100-fold dilution, and one out of three antisera had the strongest binding inhibitory activity at 34.3% at 1000-fold dilution showed that.
  • binding inhibition activity most strongly, further do not contain a strong first l g-like domain fin immunogenic KDR- ⁇ ⁇ -Fc was shown to be suitable as an immunogen.
  • VEGF-KDR binding inhibitory assay A hybridoma was prepared from one mouse immunized with KDR-5N-Fc, and the obtained culture supernatant of about 672 ⁇ l was used and indicated in 8. and [125 1] was subscription-learning in VEGF-KDR binding inhibition Atsusi, culture in supernatants 90.1, 66.7, 59.0, 85.7, 86.8, 78.0, 7 clones producing monoclonal antibodies High Priestess showing the binding inhibition activity of 91.2% Domas were obtained and named KM 1991-1997, respectively (Table 1).
  • the specificity of the anti-human VEGF receptor KDR monoclonal antibody described in 9. was confirmed by the enzyme immunoassay described in 3 using 5 g / iPl of the purified antibody.
  • Fig. 8 shows typical results
  • Fig. 7 summarizes the results.
  • KM 1992 were reacted K 1995 the seven monochromator port one monoclonal antibody represented by all the fourth I g-like domain (corresponding to 295 to 393 amino acids). Therefore, the fourth Ig-like domain from the N-terminal of KDR (corresponding to amino acids 295 to 393) was shown to be particularly important for binding to VEGF.
  • KM1994 and KM1995 show the autophosphorylation inhibitory activity of VEGF receptor KDR shown in 13 or the VEGF-dependent vascular endothelial cell growth inhibitory activity shown in 14. It was shown to be a neutralizing monoclonal antibody that inhibits the biological activity of KDR.
  • the monoclonal antibodies established in the present invention were all IgG classes except for KM1659 and KM1942 which are IgM, KM1664 which is IgA, and KM1991, KM1996 and KM1997 which are IgE.
  • Pristane-treated 8-week-old nude female mice (Balb / c) were intraperitoneally injected with each of the 5 to 10 ⁇ 10 6 cells Z of the hybridoma cells obtained in steps 6 and 9. After 10 to 21 days, the hybridoma became ascites cancer. Collect ascites from mice with ascites (1 ⁇ 8 ml / animal), centrifugation (3,000 rpm, 20 minutes) to remove solids, and purification by force prillic acid precipitation method (Antibody's laboratory manual) to obtain a purified monoclonal antibody .
  • the anti-human VEGF receptor KDR monoclonal antibody binding inhibitory activity of the anti-human VEGF receptor KDR monoclonal antibody described in 9 was confirmed in accordance with the procedure shown in 8.
  • the anti-human VEGF receptor KDR monoclonal antibody binding inhibitory activity between human VEGF and human VEGF receptor Fit-1 was confirmed according to the following procedure.
  • Methanol was dispensed into a 96-well MultiScreen-IP plate (96-well MultiScreen-IP Plate; manufactured by Millipore) at 100 1 / well to hydrophilize the PVDF membrane at the bottom of the plate.
  • the soluble human VEGF receptor Fit-17N diluted to a concentration of 1.6 g / ml with PBS was dispensed at 50/21 / well, and allowed to stand at 4 ° C for adsorption. After washing, 50% of PBS containing 1% bovine serum albumin (BSA) was added, and the mixture was reacted at room temperature for 1 hour to block the remaining active groups.
  • BSA bovine serum albumin
  • the culture supernatant of the hybridoma or purified monoclonal antibody (0.01 to 7.29 / g / ml) diluted with 1% BSA-PBS solution containing 0.5 M NaCl is dispensed at 50 1 / well.
  • 125 (Amersham) 1 labeled human VEGF of 3 ng / ml and the 50 mu 1 / Ueru added and reacted at room temperature for 1.5 hours.
  • Tween-PBS dry the wells at 50 ° C, add 30 l / well of Mic-mouth scinti-0 (manufactured by Packard), and use TopCount (manufactured by Packard). Te, and the radioactivity of 125 1-labeled human VEGF bound to each Ueru.
  • KM1992, 1993, 1994, and 1995 did not inhibit the binding of human VEGF receptor Flt-1 to human VEGF used as a control experimental system at all, and KM1992, 1993, 1994, and 1995 were KDR-specific binding inhibitors. It was shown to be.
  • NIH3T3-KDR Human Bok VEGF receptor KDR expressing NIH3T3 cells
  • 10% FCS - were cultured to 5 ⁇ 10 X 10 6 cells / flask in l 7 5 cm 2 flasks using DMEM culture land 20 ml. After culturing, replace with 0.1% FCS_DMEMlOml containing 0.1 mM sodium orthovanadate (V), add anti-VEGF receptor KDR antibody to 10 g / ml, and perform pretreatment on ice for 30 minutes Was. After the pretreatment, human VEGF (manufactured by R & D) was added to a concentration of 50 ng / ml, and stimulation was performed on ice for 45 minutes.
  • cell disruption buffer 20 mM Hepes (pH 7.4), 150 mM NaCl, 0.2% TritonX-100, 10% Glycerol, 2 mM Na 3 V ⁇ 4 , 10 mM Na 4 P 2 ⁇ 7, 5 mM EDTA, 50 mM NaF, 1.5 mM MgCl 2, 1 mM PMSF, 10 ng / ml aprotinin, 5 a (ig / ml leupeptin] was added 2 ml, the cells were disrupted to obtain a cell fracture ⁇ Centrifuge the cell lysate at 15,000 X g for 10 minutes, add 100 1 of goat anti-mouse lgG (H + L) Sepharose 4B (Zymed Laboratories) to the supernatant, and gently incubate at 4 ° C for i hours.
  • cell disruption buffer 20 mM Hepes (pH 7.4), 150 mM NaCl, 0.2% TritonX-100
  • a 48-well microphone mouth plate 1% plate in E-BM medium 5% fetal serum (FBS), human recombinant epidermal growth factor (hEGF) 10ng / ml, hydrocortisone 1 pg / mK Genyumycin 50 ig / MVU Human skin-derived microvascular endothelial cells HMVEC (Kurabo Industries) suspended in a medium (Kurabo) supplemented with 50 ng / ml amphetericin B (Kurabo Co., Ltd.) at 4,000 cells / 800 11 1 / well.
  • FBS fetal serum
  • hEGF human recombinant epidermal growth factor
  • ig / MVU Human skin-derived microvascular endothelial cells HMVEC (Kurabo Industries) suspended in a medium (Kurabo) supplemented with 50 ng / ml amphetericin B (K
  • the results are shown in FIG.
  • the anti-VEGF receptor KDR monoclonal antibodies KM1992 and KM1995 inhibited VEGF-dependent HMVEC proliferation in a concentration-dependent manner.
  • the growth inhibitory activities of KM1992 and KM1995 at the addition of 2 ⁇ g / ml were 74.3% and 70.9%, and the growth inhibition activities of KM1992 and KM1995 at the addition of 20 g / ml were 108.7% and 103.0%, respectively.
  • the KM231 and antibody-free groups used as controls did not show any inhibitory activity.
  • Human VEGF receptor KDR-expressing NIH3T3 cells (N1H3T3-KDR), control NIH3T3 cells (NIH3T3-Neo), human skin-derived microvascular endothelial cells HMVEC (Kurabo), human umbilical vein-derived vascular endothelial cells HUVEC (Kurabo) 2-5 x 10 5 cells are suspended in a round-bottom 96-well plate in 100 II 1 buffer solution (PBS containing 1% BSA, 0.02% EDTA, 0.05% sodium azide) and dispensed. did.
  • PBS containing 1% BSA, 0.02% EDTA, 0.05% sodium azide
  • the supernatant was removed, and the purified antibody (10 ⁇ g / ml) 501 described in 6 and 9 was added and reacted at 4 ° C for 30 minutes. After the reaction, 200 1 of the immunological cell staining buffer was added to each well 4. After centrifugation at 350 X g for 1 minute, the supernatant was removed and the cells were washed.
  • the cells were stained with an FITC-labeled anti-mouse immunoglobulin antibody or F1TC-labeled anti-rat immunoglobulin antibody (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 1 II g / ml.
  • the buffer solution 501 was added and reacted at J ° C for 30 minutes. After the reaction, the same washing operation as described above was performed three times, and the analysis was performed using a flow cytometer (manufactured by Kor Yuichisha).
  • Anti-human VEGF receptor KDR monoclonal antibody KM1668 Did not react with the control cells but specifically reacted with the KDR-expressing cells (A).
  • A human vascular endothelial cells
  • HMVEC and HUVEC it was shown that DR on vascular endothelial cells could be detected.
  • KM1992, KM1993, KM1994, and KM1995 selected by the [ 125 I] VEGF-KDR binding inhibition assay shown in Section 9 react with human vascular endothelial cells HMVEC and HUVEC to reduce KDR on vascular endothelial cells. It was shown to be detectable ( Figure 13). Industrial applicability
  • a monoclonal antibody that specifically binds to a human VEGF receptor KDR that is specifically expressed on vascular endothelial cells in a neovascularization site.
  • the monoclonal antibody of the present invention can be used for immunological detection of human angiogenesis in immune cell staining, growth of solid tumors by neutralizing the effect of KDR, metastasis formation, arthritis in rheumatoid arthritis, diabetic retinopathy, retina of premature infants Diagnosis of diseases in which the disease progresses due to abnormal vascular neoplasia such as intestinal dystrophy and dryness is useful for treatment.

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Abstract

L'invention concerne un anticorps réagissant avec le récepteur KDR humain du VEGF mais pas avec le récepteur Flt-1 humain du VEGF; ainsi qu'un anticorps se liant au récepteur KDR humain du VEGF et empêchant que le VEGF humain ne se lie au récepteur KDR humain du VEGF, ce qui permet de neutraliser l'effet de KDR. L'invention concerne également des procédés d'utilisation de ces anticorps monoclonaux dans le diagnostic et le traitement de maladies se développant avec une néoformation de vaisseaux sanguins anormale, notamment, la prolifération de tumeurs solides, la métastase, la polyarthrite rhumatoïde, la rétinopathie diabétique, le rétinopathie de l'enfant prématuré, le psoriasis, etc.
PCT/JP1999/000478 1998-02-04 1999-02-04 Anticorps diriges contre le recepteur kdr humain du vegf WO1999040118A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
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US6342219B1 (en) 1999-04-28 2002-01-29 Board Of Regents, The University Of Texas System Antibody compositions for selectively inhibiting VEGF
EP1086705A4 (fr) * 1998-05-20 2002-02-06 Kyowa Hakko Kogyo Kk Inhibiteurs de l'activite du facteur de croissance endothelial vasculaire (vegf)
EP1151002A4 (fr) * 1999-01-29 2002-05-02 Imclone Systems Inc Anticorps specifiques au kdr et leurs utilisations
US6703020B1 (en) 1999-04-28 2004-03-09 Board Of Regents, The University Of Texas System Antibody conjugate methods for selectively inhibiting VEGF
WO2004024766A1 (fr) * 2002-09-12 2004-03-25 Oncotherapy Science, Inc. Peptides kdr et vaccins les renfermant
WO2008109433A2 (fr) 2007-03-02 2008-09-12 The Cleveland Clinic Foundation Peptides anti-angiogéniques
WO2010005527A1 (fr) 2008-06-30 2010-01-14 Angioblast Systems, Inc. Traitement de maladies oculaires et d’une néovascularisation excessive utilisant un traitement combiné
JP4689124B2 (ja) * 1999-09-30 2011-05-25 協和発酵キリン株式会社 ガングリオシドｇｄ３に対するヒト型相補性決定領域移植抗体およびガングリオシドｇｄ３に対する抗体の誘導体
US8143025B2 (en) 2004-11-18 2012-03-27 Imclone Llc Antibodies against vascular endothelial growth factor receptor-1
EP2614837A1 (fr) 2007-11-09 2013-07-17 Affitech Research AS Compositions d'anticorps anti-VEGF et procédés
US8703713B2 (en) 2007-08-24 2014-04-22 Onco Therapy Science, Inc. Combination therapy for pancreatic cancer using an antigenic peptide and chemotherapeutic agent
US8975229B2 (en) 2009-06-11 2015-03-10 Oncotherapy Science, Inc. Methods for treating a disease caused by choroidal neovascularization
US10308943B2 (en) 2016-02-08 2019-06-04 Vitrisa Therapeutics, Inc. Compositions with improved intravitreal half-life and uses thereof

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JPH09509414A (ja) * 1994-02-10 1997-09-22 ルードヴィッヒ・インスティテュート・フォア・キャンサー・リサーチ 免疫相互作用性分子−ｉ
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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1086705A4 (fr) * 1998-05-20 2002-02-06 Kyowa Hakko Kogyo Kk Inhibiteurs de l'activite du facteur de croissance endothelial vasculaire (vegf)
EP1151002A4 (fr) * 1999-01-29 2002-05-02 Imclone Systems Inc Anticorps specifiques au kdr et leurs utilisations
US6342221B1 (en) 1999-04-28 2002-01-29 Board Of Regents, The University Of Texas System Antibody conjugate compositions for selectively inhibiting VEGF
US6416758B1 (en) 1999-04-28 2002-07-09 Board Of Regents, The University Of Texax System Antibody conjugate kits for selectively inhibiting VEGF
US6524583B1 (en) 1999-04-28 2003-02-25 Board Of Regents, The University Of Texas System Antibody methods for selectively inhibiting VEGF
US6676941B2 (en) 1999-04-28 2004-01-13 Board Of Regents, The University Of Texas System Antibody conjugate formulations for selectively inhibiting VEGF
US6703020B1 (en) 1999-04-28 2004-03-09 Board Of Regents, The University Of Texas System Antibody conjugate methods for selectively inhibiting VEGF
US6342219B1 (en) 1999-04-28 2002-01-29 Board Of Regents, The University Of Texas System Antibody compositions for selectively inhibiting VEGF
US6887468B1 (en) 1999-04-28 2005-05-03 Board Of Regents, The University Of Texas System Antibody kits for selectively inhibiting VEGF
US7056509B2 (en) 1999-04-28 2006-06-06 Board Of Regents The University Of Texas System Antibody methods for selectively inhibiting VEGF
JP4689124B2 (ja) * 1999-09-30 2011-05-25 協和発酵キリン株式会社 ガングリオシドｇｄ３に対するヒト型相補性決定領域移植抗体およびガングリオシドｇｄ３に対する抗体の誘導体
US8206719B2 (en) 2002-09-12 2012-06-26 Oncotherapy Science, Inc. KDR peptides and vaccines comprising the same
WO2004024766A1 (fr) * 2002-09-12 2004-03-25 Oncotherapy Science, Inc. Peptides kdr et vaccins les renfermant
US7514084B2 (en) 2002-09-12 2009-04-07 Oncotherapy Science, Inc. KDR peptides and vaccines comprising the same
US8574585B2 (en) 2002-09-12 2013-11-05 Oncotherapy Science, Inc. KDR peptides and vaccines comprising the same
US7695720B2 (en) 2002-09-12 2010-04-13 Oncotherapy Science, Inc. KDR peptides and vaccines comprising the same
CN100352843C (zh) * 2002-09-12 2007-12-05 肿瘤疗法科学股份有限公司 Kdr肽和包括该肽的疫苗
US8574586B2 (en) 2002-09-12 2013-11-05 Oncotherapy Science, Inc. KDR peptides and vaccines comprising the same
CN103073620A (zh) * 2002-09-12 2013-05-01 肿瘤疗法科学股份有限公司 Kdr肽和包括该肽的疫苗
US8143025B2 (en) 2004-11-18 2012-03-27 Imclone Llc Antibodies against vascular endothelial growth factor receptor-1
WO2008109433A2 (fr) 2007-03-02 2008-09-12 The Cleveland Clinic Foundation Peptides anti-angiogéniques
US8703713B2 (en) 2007-08-24 2014-04-22 Onco Therapy Science, Inc. Combination therapy for pancreatic cancer using an antigenic peptide and chemotherapeutic agent
EP2614837A1 (fr) 2007-11-09 2013-07-17 Affitech Research AS Compositions d'anticorps anti-VEGF et procédés
WO2010005527A1 (fr) 2008-06-30 2010-01-14 Angioblast Systems, Inc. Traitement de maladies oculaires et d’une néovascularisation excessive utilisant un traitement combiné
US8975229B2 (en) 2009-06-11 2015-03-10 Oncotherapy Science, Inc. Methods for treating a disease caused by choroidal neovascularization
US10308943B2 (en) 2016-02-08 2019-06-04 Vitrisa Therapeutics, Inc. Compositions with improved intravitreal half-life and uses thereof

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