WO1999040118A1 - Antibodies against human vegf receptor kdr - Google Patents

Antibodies against human vegf receptor kdr 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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French (fr)
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/en

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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.

Abstract

An antibody reacting with human VEGF receptor KDR but not with human VEGF receptor Flt-1; an antibody binding to human VEGF receptor KDR and preventing human VEGF from binding to the human VEGF receptor KDR to thereby neutralize the effect of KDR; and methods with the use of these monoclonal antibodies in diagnosing and treating diseases which advance with abnormal neovascularization, for example, proliferation of solid tumor, metastasis, arthritis in chronic rheumatoid arthritis, diabetic retinopathy, premature infant retinopathy, psoriasis, etc.

Description

明 細  Details
ヒ卜 VEGF受容体 KDRに対する抗体  Antibody to human VEGF receptor KDR
技術分野 Technical field
本発明は、 固形腫瘍の増殖、 転移形成、 慢性関節リュウマチにおける関節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など異常な血管新生により病態が進行す る疾患の診断あるいは治療に有用な、 ヒト VEGF受容体 KDRに特異的に結合す る抗体、 該抗体を生産するハイプリ ドーマあるいは形質転換体、 および該抗体 を用いてヒト VEGF受容体 KDRを免疫学的に検出する方法、 並びに該抗体を用 いる固形腫瘍、 慢性関節リュウマチ、 糖尿病性網膜症、 未熟児網膜症、 乾鮮な どの診断法、 治療法に関する。 血管新生は、 脊椎動物の胎生期における循環器系の形成や多くの組織の構築 に重要な役割を果たすとともに、 成熟個体 （雌） においても性周期における黄 体形成、 子宮内膜の一過性の増殖、 胎盤形成などに密接に関与する。 さらに、 病的状態としては、 固形腫瘍の増殖、 転移形成、 糖尿病性網膜症、 慢性関節リ ユウマチの病態形成、促進に血管新生が深く関与している O.Folkmanら； J. Biol. Chem., 267, 10931 , 1992) 。 血管新生は、 血管新生因子が分泌されることが引き 金となり、 近傍にある既存の血管の内皮細胞からプロテアーゼが分泌され、 基 底膜、 間質が破壊され、 続いて血管内皮細胞の遊走、 増殖がはじまり、 管腔が 形成されることで血管が新生される過程よりなる （J.Folkmanら ； J. Biol. Chem., 267, 10931 , 1992)。血管新生を誘導する因子としては、 Vascular permeability factor (VPF)/Vascular endothelial growth factor (VEGF)が上記発生段階における血管新生 および病的な状態における血管新生において最も重要な因子として知られてい る ( .Shibuya; Advances in Cancer Research 67, 281 , 1995.) 。 VPF/VEGFはホモ ダイマ一よりなる分子量約 4万のタンパクであり、 1983年に血管透過性促進因 子 (Vascular permeability factor: VPF)として （D.R.Senger ら ； Science 219, 983, 1983.)、 1989年に血管内皮細胞増殖因子 (Vascular endothelial growth factor: VEGF) として独立した分子として報告されたが （N.Ferraraら ； Biochem. Biophys. Res. Comm., 161 , 851 , 1989.) 、 cDNAクロ一ニングの結果、 両者は同一の物質である ことが明らかとなった (D.W丄 eung ら ； Science, 246, 1306, 1989; P.J.Keck ら ； Science, 246, 1309, 1989.) (以下 VEGFと記載） 。 VEGFの活性としてはこれまで に、 血管内皮細胞に対し、 増殖促進活性 (ED50 = 2- 3 PM) ( .Ferraraら； Biochem. Biophys. Res. Comm.,巡, 851 , 1989.)、遊走促進活性 (A.E.Kochら； J. Immunology, 152, 4149, 1994.)、 メタ口プロテア一ゼ分泌促進活性 （E.N.Unemori ら ； J. Cell Physiol., 153, 557， 1992.)、 ゥロキナーゼ、 tPA 分泌促進活性 (M.S.Pepper ら ； Biochem. Biophys. Res. Comm., 181, 902， 1991.)を示し、 in vivoにおいては血管新 生促進活性 (T.Asaharaら ； Circulation, 92 suppl Π, 365， 1995.)、 血管透過性促進 活性 (D.R.Sengerら ； Science, 219, 983， 1983.) が報告されている。 VEGFは血 管内皮細胞に極めて特異性の高い増殖因子であることが報告されている (N.Ferraraら ； Biochem. Biophys. Res. Comm., 161 , 851 , 1989.) 。 VEGFには Alternative splicing により 4 種類のタンパクが存在することが報告さわている (K.A.Houckら ； J. Biol. Chem., 267, 26031， 1991.)。 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 (. Shibuya; Advances in Cancer Research 67, 281, 1995.). 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. ), CDNA cloning revealed that both were the same substance (DW eung et al .; Science, 246, 1306, 1989; PJKeck et al .; Science, 246, 1309, 1989.) VEGF). So far as the activity of VEGF, compared vascular endothelial cell growth promoting activity (ED50 = 2- 3 P M) (.Ferrara al;.... Biochem Biophys Res Comm, cruiser, 851, 1989.), migration Stimulating activity (AEKoch et al .; J. Immunology, 152, 4149, 1994.), Meta-oral protease secretion promoting activity (ENUnemori et al., J. Cell Physiol., 153, 557, 1992.), Perokinase, tPA secretion promoting activity (MSPepper et al .; Biochem. Biophys. Res. Comm., 181, 902, 1991.), and angiogenesis promoting activity in vivo (T. Asahara et al .; Circulation, 92 suppl III, 365, 1995.) A vascular permeability promoting activity (DRSenger et al .; Science, 219, 983, 1983.) has been reported. 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.).
血管新生を伴う疾患の中で、 固形腫瘍の増殖、 転移形成、 糖尿病性網膜症、 慢性関節リュウマチの病態形成に VEGFが深く関与していることが報告されて いる。固形腫瘍については、 これまでに腎癌 (A.Takahashiら； Cancer Research, ， 4233， 1994)、 乳癌（し. F.Brown ら ； Human Pathology, 26, 86, 1995)、 脳腫瘍 (R.A.Berkmanら； J. Clinical Investigation, 91 , 153, 1993)、消化器癌 (L.F.Brownら ； Cancer Research, 53, 4727, 1993)、 卵巣癌 (T.A.Olsonら ； Cancer Research, 54, 276, 1994)などの多くのヒト腫瘍組織において VEG Fが産生されていることが報告さ れている。乳癌については VEGFと患者の予後との関係が検討された結果、 VEGF 高発現腫瘍は、 低発現腫瘍に比べ、 腫瘍血管新生が盛んであり生存率が低いこ とが明らかとなっている（M.Toiら ； Japanese J. Cancer Research, 85, 1045, 1994.)。 また、 ヌードマウスにヒト腫瘍を皮下移植したゼノグラフトモデル実験系にお いて、 抗 VEGFモノクローナル抗体は腫瘍増殖抑制効果を示すことが報告され ている（J.K.Kimら ； Nature, 362, 841 , 1993.)。 さらに、 ヌードマウスにおけるヒ ト腫瘍の転移癌モデルにおいて、 抗 VEGF モノクローナル抗体は癌転移を抑制 できることが報告されている (O.Meh ykら ； Cancer Research, 56, 921， 1996)。 従 つて、 VEGF活性を抑制することができれば癌患者における腫瘍の増殖、 転移形 成を抑制できるものと期待される。 また、 ヒ卜の癌性胸水、 腹水中に高濃度の VEGFが検出されることから、 胸水、 腹水貯留の主要な因子である可能性も示さ れ (S.Kondoら ； Biochimica et Biophysica Acta, 1221 , 211 , 1994.)、 VEGFをブロッ クすることで癌性胸水、 腹水の貯留を防ぐことも期待される。 Among diseases associated with angiogenesis, VEGF has been reported to be deeply involved in the growth of solid tumors, metastasis, diabetic retinopathy, and pathogenesis of rheumatoid arthritis. For solid tumors, kidney cancer (A. Takahashi et al .; Cancer Research,, 4233, 1994), breast cancer (S. F. Brown et al .; Human Pathology, 26, 86, 1995), brain tumor (RABerkman et al .; J Many human tumor tissues such as Clinical Investigation, 91, 153, 1993), gastrointestinal cancer (LFBrown et al .; Cancer Research, 53, 4727, 1993), ovarian cancer (TAOlson et al .; Cancer Research, 54, 276, 1994). It has been reported that VEGF is produced in E. coli. Investigation of the relationship between VEGF and prognosis of patients with breast cancer revealed that tumors with high VEGF expression had more active tumor angiogenesis and lower survival rates than tumors with low expression (M .Toi et al .; Japanese J. Cancer Research, 85, 1045, 1994.). In addition, it has been reported that an anti-VEGF monoclonal antibody has a tumor growth inhibitory effect in a xenograft model experimental system in which human tumors are subcutaneously implanted into nude mice (JKKim et al .; Nature, 362, 841, 1993.) . Furthermore, it has been reported that 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. In addition, 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.
糖尿病網膜症においては、 異常な血管新生により網膜剥離や硝子体出血をお こして失明にいたるが、糖尿病性網膜症における血管新生と患者眼球内の VEGF レベルが正相関することが報告されている (し P.Aielloら； New England J. Medicine, 331 , 1480, 1994)。 また、 サルの網膜症モデルにおいて抗 VEGF中和モノクロ一 ナル抗体の眼内投与により VEGF活性を抑制すると血管新生が抑制されること が報告されている (A.P.Adamisら ； Arch Opthalmol., 114, 66, 1996.) 。 従って、 過剰に産生される VEGF活性を抑制することで糖尿病性網膜症における血管新 生を抑制できることが期待される。  In diabetic retinopathy, abnormal angiogenesis leads to blindness due to retinal detachment and vitreous hemorrhage, but it has been reported that angiogenesis in diabetic retinopathy is positively correlated with VEGF levels in the patient's eye. (And P. Aiello et al .; New England J. Medicine, 331, 1480, 1994). In addition, it has been reported that in a monkey retinopathy model, 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.
慢性関節リュウマチの関節炎の病態の進展 （骨、 軟骨の破壊） には血管新生 を伴うが、 慢性関節リュウマチ患者の関節液中には VEGFが高濃度で含まれて いること、 関節中のマクロファージが VEGF を産生することが報告されている (A.E.Koch b； Journal of Immunology, 152, 4149, 1994; R.A.Favaら； J. Experimental Medicine, 180, 341 , 1994.)。 過剰に産生される VEGF活性を抑制することで関節 炎における血管新生を抑制できることが期待される。  The development of arthritis in rheumatoid arthritis (destruction of bone and cartilage) is accompanied by angiogenesis, but the synovial fluid of patients with rheumatoid arthritis contains high levels of VEGF and macrophages in joints. It has been reported to produce VEGF (AEKoch b; Journal of Immunology, 152, 4149, 1994; RAFava et al .; J. Experimental Medicine, 180, 341, 1994.). It is expected that suppressing an excessively produced VEGF activity can suppress angiogenesis in arthritis.
ヒ卜の VEGF受容体としては、 これまでに受容体型チロシンキナーゼフアミ リ一に属 る受容体乙、'め "O Fit- 1 (fins— like tyrosine kinase)( .Shibuyaら；Oncogene, 5, 519, 1990; C.Vries ら ； Science, 255, 989, 1992.)および KDR(kinase insert domain-containing receptor) (B.I.Termanら ； W092/ 14748, Priolity Feb.22, 1991; B.I.Terman ら； Biochem. Biophys. Res. Comm., 187, 1579, 1992)が報告されている。 ヒ卜型 VEGF受容体 KDRのマウス型ホモログは Flk- K W.Matthewsら； Proc. Natl. Acad. Science, USA, 88, 9026, 1991; A.Ullichら； W094/11499 Priolity Nov. 13, 1992; B.Millauerら ； Cell, 72, 835, 1993.)と命名されている。 Fit- 1および KDR/Flk- 1の 細胞外ドメインは 7個のィムノグロブリン様ドメインよりなり、 細胞内ドメイ ンはチ口シンキナーゼドメインを有する分子量 180〜200キロダルトンの膜タン パクよりなる。 VEGFは、 Fit- 1および KDR/Flk - 1に対して、 それぞれ KD値が 20 pMおよび 75 pMで特異的に結合する。 Fk- 1および KDR/Flk-1は血管内皮細 胞に特異的に発現していると報告されている (T.P.Quinnら； Proc. Natl. Acad. Sci. USA, 90, 7533, 1993; R丄. Kendailら ； Proc. Natl. Acad. Sci. USA, 90, 8915, 1993)。 ヒト型受容体 KDRとマウス型受容体 Flk-1のアミノ酸配列のホモロジ一は、 細 胞内の N末端側のチロシンキナーゼドメインおよび C末端側のチロシンキナー ゼドメインは、 それぞれ 95 %および 97 %と非常に高いが、 細胞外ドメイン間の ホモロジ一は 80 %と少し低い (B, Barleonら ； J. Cellular Biochemistry, 54, 56, 1994.) 。 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. Matthews et al .; Proc. Natl. Acad. Science, USA, 88, 9026, 1991; A. Ullich et al .; W094 / 11499 Priolity Nov. 13, 1992 B. Millauer et al .; Cell, 72, 835, 1993.). 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 丄. 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. However, the homology between extracellular domains is slightly lower at 80% (B, Barleon et al .; J. Cellular Biochemistry, 54, 56, 1994.).
様々なヒ卜の疾患における KDRの発現については、 ヒ卜脳腫瘍組織の腫瘍血 管内皮細胞 (E,Hatvaら ； American J. Pathology, 146, 368, 1995.)、 ヒト消化器癌組 織の腫瘍血管内皮細胞 (し F.Brownら ； Cancer Research, 53, 4727, 1993.)において は正常組織の血管内皮細胞に比べ KDRの mRNAレベルの発現が上昇しているこ とが報告されている。 これらの結果は、 腫瘍血管新生において VEGF- VEGF レ セプター KDR 系が重要な役割を果たしていることを強く示唆するものである。 さらに、 慢性関節リュウマチ患者の関節の血管内皮細胞においても in situ hybridization により KDR mRNA の発現が認められることが報告されており (R.A.Favaら ； J. Experimental Medicine, 180, 341， 1994·)、 VEGF— VEGFレセプ夕 一 KDR系の重要性を示唆している。 Regarding the expression of KDR in various human diseases, tumor vascular endothelial cells of human brain tumor tissues (E, Hatva et al .; American J. Pathology, 146, 368, 1995.), tumors of human gastrointestinal cancer tissues It has been reported that vascular endothelial cells (F. Brown et al .; Cancer Research, 53, 4727, 1993.) have increased expression of KDR mRNA levels as compared to vascular endothelial cells in normal tissues. These results strongly suggest that the VEGF-VEGF receptor KDR system plays an important role in tumor angiogenesis. In addition, it has been reported that 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 レセプ夕一 KDR/Rk-1 の機能については、 ブ夕動脈の血管内皮細胞に KDRを発現させると VEGFに反応し増殖、 遊走することから、 VEGFの多様な 活性の中で KDR は血管内皮細胞の増殖、 遊走に関与すると報告されている （J. Waltenbergerら ； J. Biol. Chem., 269, 26988, 1994.) 。 また、 マウス型 flk-1遺伝 子を破壊した flk- 1ノックァゥトマウスは成熟した血管内皮細胞が全く認められ ず、 卵黄嚢の血島も形成されず、 子宮内で死亡したことから、 動物個体におい ても KDR/flk - 1は血管内皮細胞の増殖、分化に関与することが報告されている（F, Shalabyら ； Nature, 376， 62, 1995.) 。  Regarding the function of KGF / Rk-1, 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.).
Flk-lの細胞内チロシンキナーゼドメインを除去した不活性型の Flk-lミュー タン を用い、 ウィルスベクターにより内皮細胞に導入する方法を試みたとこ ろ、不活性型 Flk- 1ミュータントウィルスと腫瘍細胞を混合して動物細胞に移植 すると腫瘍の増殖が抑制されることが報告されており （B， Millauer ら ； Nature, 367, 576, 1994.) 、 Flk- 1のシグナル伝達を阻害することで腫瘍増殖が抑制され ることが示されている。  Using an 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.
マウス型 Flk-l細胞外ドメインに対するモノクローナル抗体については、ラッ 卜モノクローナル抗体が報告されている。 マウス型 Flk- 1 の細胞外ドメインと FLAG™融合タンパクを免疫原に用いて、 ラッ卜よりハイプリ ドーマを作製し、 取得した 3種のラットモノク口一ナル抗体 4H3, 3B6, 3C8は細胞上に発現したマ ウス Flk-lを免疫沈降できると報告されているが、 VEGFの機能阻害活性につい ては解析されていない (A, Wilksら ； WO 95/21865, Priolity, 10 Feb. 1994.) 。 ま た、 マウス型 Flk-lの細胞外ドメインと SEAPs(Secretory alkaline phosphatase)融 合夕ンパクを免疫原に用いてラッ 卜において作製したモノクロ一ナル抗体 DC101 は、 細胞表面のマウス Flk-1/ftns キメラレセプ夕一 （細胞外ドメインが Flk-lであり、 細胞内ドメインが finsであるキメラ分子） に反応することが報告 されている。 DC 101は、 VEGF刺激によるマウス Flk - 1/fVnsキメラレセプ夕一の ftnsチロシンキナーゼの自己リン酸化を阻害する活性を有する。 しかしながら、 Flk-1 チロシンキナ一ゼの自己リン酸化を阻害する活性については示されてい ない。 ヌードマウスにヒト腫瘍を皮下移植したゼノグラフトモデル実験系にお いて、 DC101 は抗腫瘍効果を示すことが報告されている （P, Rockwell ら ； WO 95/21868, Priolity, 10 Feb. 1994.) 。 DC101はマウス型 Flk - 1に反応するが、 ヒ卜 型 KDRには反応しない。 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 ™ 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.). In addition, a monoclonal antibody, 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. However, no activity has been shown to inhibit the autophosphorylation of Flk-1 tyrosine kinase. It has been reported that 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.
ハイブリド一マ (Hybridoma, ， 465 - 471, 1997.) には、 ヒト KDR のィムノグロ ブリン様ドメインの 6 、 7 番目に特異的に反応する 2 種のマウスモノクローナ ル抗体 2-7- 9 および 2-10- 1が報告されているが、 ヒト型 VEGF受容体 KDR の ィムノグロプリン様ドメインの 1〜 5番目に特異的に反応する抗体、 あるいは ヒト型 VEGF受容体 KDR に結合し、 かつ KDRの作用を中和する抗体は知られ ていない。  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.
以上のことから、 ヒ卜型 VEGF受容体 KDR に結合し、 かつ KDRの作用を中 和する抗体はヒトにおける固形腫瘍の増殖、 転移形成、 慢性関節リュウマチに おける関節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など異常な血管新生によ り病態が進行する疾患の診断、 治療に有用であることが期待される。 しかしな がら、 ヒト VEGF受容体 KDR を発現している細胞を検出でき、 かつ、 VEGFの 作用を中和する抗ヒト VEGF受容体 KDR モノクローナル抗体はこれまで確立さ れていない。 先に述べたとおり、 抗マウス型 Flk- 1 に対するモノクローナル抗 体を作製できることは報告されているが、 ヒ卜の場合に有用である、 リガンド である VEGFの結合を阻害する抗ヒト型 VEGF受容体 KDR抗体を作製すること はこれまでに知られていない。  Based on the above, 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. However, 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. As mentioned earlier, it has been reported that a monoclonal antibody against anti-mouse Flk-1 can be produced, but an anti-human VEGF receptor that inhibits the binding of ligand VEGF, which is useful in humans, has been reported. The production of KDR antibodies has not been known so far.
発明の開示  Disclosure of the invention
本発明者らは、 固形腫瘍の増殖、 転移形成、 慢性関節リュウマチにおける関 節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など異常な血管新生により病態が 進行する疾患を診断、治療するために有用なヒト型 VEGF受容体 KDR に結合し、 かつ KDRの作用を中和するモノクローナル抗体の開発に関して鋭意検討し、 本 発明を完成するに至った。 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.
すなわち、 本発明は、 以下の （ 1 ) 〜 （37) に関するものである。  That is, the present invention relates to the following (1) to (37).
(1) ヒ卜 VEGF受容体 KDR に反応するが、ヒ卜 VEGF受容体 Fit- 1 には反応 しないモノクローナル抗体。  (1) A monoclonal antibody that reacts with human VEGF receptor KDR but does not react with human VEGF receptor Fit-1.
(2) モノクローナル抗体が、 ヒト VEGF受容体 KDRの細胞外領域に特異的に 結合するモノクローナル抗体である、 前記 （1 ) 記載のモノクローナル抗体。  (2) The monoclonal antibody according to (1), wherein the monoclonal antibody specifically binds to an extracellular region of human VEGF receptor KDR.
(3) モノクローナル抗体が、 ヒト VEGFのヒ卜 VEGF受容体 KDRへの結合を 阻害し、 かつ KDR の作用を中和するモノクローナル抗体である、 前記（1 )記載 のモノクローナル抗体。  (3) The monoclonal antibody according to (1), wherein the monoclonal antibody is a monoclonal antibody that inhibits binding of human VEGF to human VEGF receptor KDR and neutralizes the action of KDR.
(4) モノクローナル抗体が、 ヒト VEGF受容体 KDRの、 配列番号 26に記載 されたアミノ酸配列 1〜518 番目の領域にあるェピ I ^一プを認識するモノクロ ーナル抗体である、 前記 （1 ) 〜 （3 ) のいずれか 1項に記載のモノクローナ ル抗体。  (4) The monoclonal antibody according to the above (1), wherein the monoclonal antibody recognizes an epitope I ^ -ip in the amino acid sequence of positions 1 to 518 of the human VEGF receptor KDR, which is set forth in SEQ ID NO: 26. The monoclonal antibody according to any one of (1) to (3).
(5) モノクローナル抗体が、 ヒト VEGF受容体 KDRの、 配列番号 26に記載 されたアミノ酸配列 1〜104、 1〜194、 105〜393、 295〜393および 394〜518 番 目から選ばれるァミノ酸配列の領域にあるェピト一プを認識するモノクローナ ル抗体である、 前記 （ 1 ) 〜 （3 ) のいずれか 1項に記載のモノクロ一ナル抗 体。  (5) an amino acid sequence selected from the amino acid sequences 1-104, 1-194, 105-393, 295-393 and 394-518 of the amino acid sequence of SEQ ID NO: 26 of the human VEGF receptor KDR, wherein 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).
(6) モノクローナル抗体が、 マウス lgG lサブクラスまたはマウス IgG2bサブ クラスに属する、 前記 （1 ) 〜 （5 ) のいずれか 1項に記載のモノクローナル 抗体。 (6) 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.
(7) モノクローナル抗体が、 モノクローナル抗体 KM1668、 KM1992 および KM1995から選ばれる、 前記 （1 ) 〜 （6 ) のいずれか 1項に記載のモノクロ一 ナル抗体。  (7) The monoclonal antibody according to any one of (1) to (6), wherein the monoclonal antibody is selected from monoclonal antibodies KM1668, KM1992, and KM1995.
(8) 前記 （1) 〜 （7) のいずれか 1項に記載のモノクローナル抗体を生産 ドーマ。 (8) Producing the monoclonal antibody according to any one of (1) to (7) above Dorma.
(9) ハイブリド一マが KM1668(FERM BP- 6216)、 KM1992(FERM BP - 6217)ま たは KM1995(FERM BP- 6218)である、 前記 （8) 記載のハイブリドーマ。  (9) The hybridoma according to (8), wherein the hybridoma is KM1668 (FERM BP-6216), KM1992 (FERM BP-6217) or KM1995 (FERM BP-6218).
(10) モノクローナル抗体が、 遺伝子組換え抗体である、 前記 （ 1 ) 〜 （7) のいずれか 1項に記載のモノクローナル抗体。  (10) The monoclonal antibody according to any one of (1) to (7) above, wherein the monoclonal antibody is a recombinant antibody.
(11) 遺伝子組換え抗体が、 ヒト化抗体、 一本鎖抗体およびジスルフイ ド安 定化抗体から選ばれるモノクローナル抗体である、 前記 （10) 記載のモノクロ ーナル抗体。  (11) The monoclonal antibody according to (10), wherein the recombinant antibody is a monoclonal antibody selected from a humanized antibody, a single-chain antibody, and a disulfide-stabilized antibody.
(12) ヒト化抗体がヒト型キメラ抗体である前記 （11) 記載の抗体。  (12) The antibody according to (11), wherein the humanized antibody is a human chimeric antibody.
(13) ヒト型キメラ抗体が、 前記 （1) 〜 （6) のいずれか 1項に記載のモノ クローナル抗体の抗体重鎖 （H鎖） 可変領域 (V領域)および抗体軽鎖 (L鎖) V領域 と、 ヒト抗体の H鎖定常領域 (C領域)および L鎖 C領域とからなるキメラ抗体 である前記 （12) 記載のヒト型キメラ抗体。  (13) 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 human chimeric antibody according to the above (12), which is a chimeric antibody comprising a V region, an H chain constant region (C region) and an L chain C region of a human antibody.
(14) H鎖 V領域およびし鎖 V領域のアミノ酸配列が、 モノクローナル抗体 KM1668(FERM BP- 6216)、 モノクローナル抗体 KM1992(FERM BP- 6217)またはモ ノクローナル抗体 KM1995(FERM BP-6218)から選ばれるモノクローナル抗体の H鎖 V領域および L鎖 V領域のァミノ酸配列と同じアミノ酸配列を有する、 前 記 (13)記載のヒ卜型キメラ抗体。  (14) 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.
(15) ヒ卜化抗体が CDR (相補性決定領域） 移植抗体である前記 （11) 記載 の抗体。  (15) The antibody according to the above (11), wherein the humanized antibody is a CDR (complementarity determining region) -grafted antibody.
(16) CDR移植抗体が前記 (1)〜 （6) のいずれか 1項に記載のモノクローナル 抗体の H鎖および L鎖の V領域相補性決定領域と、 ヒト抗体の H鎖および L鎖 の C領域および V領域フレームワーク領域とからなる抗体である前記 ( )記載の CDR移植抗体。  (16) 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.
(17) H鎖 V領域およびし鎖 V領域の相補性決定領域のアミノ酸配列が、モノ クロ一ナル抗体 KM1668(FERM BP- 6216)、 モノクローナル抗体 KM1992(FERM BP - 6217)またはモノクローナル抗体 KM1995(FERM BP- 6218)から選ばれるモノ クローナル抗体の H鎖 V領域および L鎖 V領域の相補性決定領域のアミノ酸配 列と同じアミノ酸配列を有する、 前記 (16)記載の CDR移植抗体。 (17) 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.
(18) —本鎖抗体が、 抗体の H鎖 V領域およびし鎖 V領域を含む、 前記 (11) 記載の一本鎖抗体。  (18) The single-chain antibody according to the above (11), wherein the single-chain antibody comprises the H chain V region and the V chain region of the antibody.
(19) 一本鎖抗体の H鎖 V領域およびし鎖 V領域のアミノ酸配列が、前記（ 1 ) 〜（ 6 )から選ばれるモノクローナル抗体の H鎖 V領域および L鎖 V領域のアミ ノ酸配列と同じアミノ酸配列を有する、 前記 (18)記載の一本鎖抗体。  (19) The amino acid sequence of the H chain V region and L chain V region of the monoclonal antibody selected from the above (1) to (6), wherein the amino acid sequence of the H chain V region and the side chain V region of the single chain antibody is The single-chain antibody according to (18), having the same amino acid sequence as that of (18).
(20) 一本鎖抗体の H鎖 V領域および L鎖 V領域の相補性決定領域のアミノ 酸配列が、 （1 )〜（ 6 )の 1項から選ばれるモノクローナル抗体の H鎖 V領域およ び L鎖 V領域の相補性決定領域のァミノ酸配列と同じアミノ酸配列を有する、 前記 (18)記載の一本鎖抗体。  (20) 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); (18) The single-chain antibody according to (18), which has the same amino acid sequence as the amino acid sequence of the complementarity-determining region of L-chain V region.
(21) 一本鎖抗体の H鎖 V領域およびし鎖 V領域のアミノ酸配列が、 モノク ローナル抗体 KM1668(FERM BP- 6216)、 モノクローナル抗体 M1992(FERM BP - 6217)またはモノクローナル抗体 KM1995(FERM BP-6218)から選ばれるモノ クローナル抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列と同じアミノ酸 配列を有する、 前記 (19)記載の一本鎖抗体。  (21) 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).
(22) 一本鎖抗体の H鎖および L鎖の V領域の相補性決定領域のアミノ酸配 列が、 モノクローナル抗体 KM1668(FERM BP - 6216)、 モノクローナル抗体 KM1992(FERM BP - 6217)またはモノクローナル抗体 K 1995(FERM BP-6218)から 選ばれるモノク口一ナル抗体の H鎖 V領域および L鎖 V領域の相補性決定領域 のアミノ酸配列と同じアミノ酸配列を有する、 前記 （20) 記載の一本鎖抗体。  (22) Monoclonal antibody KM1668 (FERM BP-6216), monoclonal antibody KM1992 (FERM BP-6217) or monoclonal antibody K The single-chain antibody according to (20), having the same amino acid sequence as that of the H chain V region and the L chain V region complementarity determining region of the monoclonal antibody selected from 1995 (FERM BP-6218). .
(23) ジスルフィ ド安定化抗体が、 抗体の H鎖 V領域および L鎖 V領域を含 む、 前記 (1 1)記載のジスルフイ ド安定化抗体。  (23) The disulfide-stabilized antibody according to (11), wherein the disulfide-stabilized antibody comprises an H chain V region and an L chain V region of the antibody.
(24) ジスルフィ ド安定化抗体の H鎖 V領域および L鎖 V領域のァミノ酸配 列が、 前記 （1 ) 〜 （6 ) のいずれか 1項から選ばれるモノクローナル抗体の H 鎖 V領域および L鎖 V領域のァミノ酸配列と同じアミノ酸配列を有する、 前記 (23)記載のジスルフィ ド安定化抗体。 (24) 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 disulfide-stabilized antibody according to (23), having the same amino acid sequence as the amino acid sequence of the chain V region and the L chain V region.
(25) ジスルフィ ド安定化抗体の H鎖 V領域および L鎖 V領域の相補性決定 領域のアミノ酸配列が、 前記 （ 1 ) 〜 （6 ) のいずれか 1項から選ばれるモノ クローナル抗体の H鎖 V領域およびし鎖 V領域の相補性決定領域のアミノ酸配 列と同じアミノ酸配列を有する、 前記 (23)記載のジスルフィ ド安定化抗体。  (25) 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 disulfide-stabilized antibody according to (23), which has the same amino acid sequence as the amino acid sequence of the complementarity determining region of the V region and the chain V region.
(26) ジスルフィ ド安定化抗体の H鎖 V領域およびし鎖 V領域のアミノ酸配 列が、 モノクローナル抗体 KM1668(FERM BP- 6216)、 モノクローナル抗体 K 1992(FER BP- 6217)またはモノクローナル抗体 KM1995(FERM BP-6218)から 選ばれるモノクローナル抗体の H鎖 V領域およびし鎖 V領域のアミノ酸配列と 同じアミノ酸配列を有する、 前記 (24)記載のジスルフィ ド安定化抗体。  (26) 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). (24) The disulfide-stabilized antibody according to (24), which has the same amino acid sequence as the H chain V region and the V chain region of a monoclonal antibody selected from BP-6218).
(27) ジスルフィ ド安定化抗体の H鎖およびし鎖の V領域の相補性決定領域 のアミノ酸配列が、 モノクローナル抗体 KM 1668(FERM BP- 6216)、 モノクロ一ナ ル抗体 KM1992(FERM BP- 6217)またはモノクローナル抗体 KM1995(FERM BP - 6218)から選ばれるモノクローナル抗体の H鎖 V領域および L鎖 V領域の相補性 決定領域のアミノ酸配列と同じアミノ酸配列を有する、 前記 (25)記載のジスルフ ィド安定化抗体。  (27) 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). Or 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.
(28) 前記 (1)〜 （7) 、 (10)〜(27)のいずれか 1項に記載のモノクローナル抗体 を用いてヒト VEGF受容体 KDRを免疫学的に検出する方法。  (28) A method for immunologically detecting human VEGF receptor KDR using the monoclonal antibody according to any one of (1) to (7) and (10) to (27).
(29) 前記 (1)〜 （7) 、 （10)〜(27)のいずれか 1項に記載のモノクローナル抗体 を用いてヒ卜 VEGF受容体 KDRを免疫学的に定量する方法。  (29) A method for immunologically quantifying human VEGF receptor KDR using the monoclonal antibody according to any one of (1) to (7) and (10) to (27).
(30) 前記 (1)〜 （7) 、 （10)〜(27)のいずれか 1項に記載のモノクローナル抗体 を用いてヒト VEGF受容体 KDRを細胞表面に発現した細胞を免疫学的に検出す る方法。  (30) Immunologically detecting cells expressing human VEGF receptor KDR on the cell surface using the monoclonal antibody according to any one of (1) to (7) and (10) to (27). how to.
(31) 前記ひ)〜 （7) 、 （i0)〜(27)のいずれか 1項に記載のモノクローナル抗体を 用いてヒト VEGF受容体 KDRを細胞表面に発現した細胞を免疫学的に定量する 方法。 (31) Immunologically quantifying cells expressing human VEGF receptor KDR on the cell surface using the monoclonal antibody according to any one of (i) to (7) and (i0) to (27). Method.
(32) 前記 (1)〜 （7) 、 （10)〜(27)のいずれか 1項に記載のモノクローナル抗体を 用いてヒト VEGFとヒト VEGF受容体 KDRとの結合を阻害する方法。  (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).
(33) 前記 (1)〜 （7) 、 （10)〜(27)のいずれか 1項に記載のモノクローナル抗体を 用いてヒト VEGF受容体 KDRを中和する方法。  (33) A method for neutralizing human VEGF receptor KDR using the monoclonal antibody according to any one of (1) to (7) and (10) to (27).
(34) 前記 (1)~ (7) 、 （10)〜(2ァ)のいずれか 1項に記載のモノクローナル抗体 を用いる、 血管新生異常疾患の診断方法。  (34) A method for diagnosing an angiogenesis disorder, using the monoclonal antibody according to any one of (1) to (7) and (10) to (2a).
(35) 前記 (1)〜 (7) 、 （10)〜(27)のいずれか 1項に記載のモノク口一ナル抗体 を用いる、 血管新生異常疾患の治療方法。  (35) A method for treating an angiogenesis disorder using the monoclonal antibody according to any one of (1) to (7) and (10) to (27).
(36) 前記 (1)〜 （7) 、 （10)〜(27)のいずれか 1項に記載のモノクローナル抗体 を有効成分とする、 血管新生異常疾患の診断薬。  (36) 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.
(37) 前記 (1)〜 （7) 、 （10)〜(27)のいずれか 1項に記載のモノクローナル抗 体を有効成分とする、 血管新生異常疾患の治療薬。  (37) 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.
前述したように、 ヒト VEGF受容体 KDRの細胞外ドメインは 7個のィムノグ ロブリン様ドメインよりなる。 ヒ卜 VEGF受容体 KDRの細胞外ドメインの N末 端側から 1番から 7番までのィムノグロプリン様ドメインの模式図および該ド メインのアミノ酸配列におけるアミノ酸番号を第 7図に示した。  As mentioned above, 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.
本発明は、 ヒト VEGF受容体 KDR に反応するが、 ヒ卜 VEGF受容体 Flt-1 に は反応しない抗体、 ヒ卜 VEGF受容体 KDRの 1〜 5番目のィムノグロブリン様 ドメインに特異的に反応する抗体を提供する。 本発明における抗体は、 モノク 口一ナル抗体、 遺伝子組換え抗体などを包含する。  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.
本発明におけるモノクローナル抗体は、 ヒト VEGF受容体 KDRに特異的に反 応すればいかなるものでもよいが、 以下に述べる製造法によって確立したもの が好適なものとしてあげられる。 すなわち、 ヒト VEGF受容体 KDRタンパクを 抗原として調製し、 抗原を免疫した動物より抗原特異性をもつ形質細胞を誘導 し、 さらに、 それと骨髄腫細胞とを融合させてハイプリ ドーマを調製し、 該ハ イブリドーマを培養するか、 あるいは該ハイプリ ドーマ細胞を動物に投与して 該動物を腹水癌化させ、 該培養液または腹水を分離、 精製することにより取得 された、 抗ヒト VEGF受容体 KDRモノクローナル抗体をあげることができる。 本発明の遺伝子組換え抗体は、 上記本発明のモノクローナル抗体を遺伝子組 換え技術を用いて改変したものである。 遺伝子組換え抗体としては、 ヒト化抗 体、 一本鎖抗体およびジスルフイ ド安定化抗体など、 遺伝子組換えにより製造 される抗体をあげることができる。 遺伝子組換え抗体において、 モノクローナ ル抗体の特徴を有し、 抗原性が低く、 血中半減期が延長されたものは、 治療薬 として好ましい。 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. Examples of 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.
本発明におけるヒト化抗体とは、 ヒト型キメラ抗体およびヒト型 CDR (Complementary Determining Region;相補性決定領域 以下、 CDRと記す） 移植 抗体を包含する。  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.
ヒ卜型キメラ抗体は、 ヒ卜以外の動物の抗体可変領域重鎖（以下、 VHと称す） および可変領域軽鎖 （以下、 VLと称す） とヒト抗体の定常領域重鎖 （以下、 CH と称す） およびヒト抗体の定常領域軽鎖 （以下、 CLと称す） とからなる抗体を 意味する。 ヒ卜型 CDR 移植抗体は、 ヒ卜抗体に、 ヒ卜以外の動物の抗体の VH および VLの CDR をヒト以外の動物の抗体の CDR 配列でそれぞれ置換した抗 体を意味する。  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). 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.
本発明のヒト型キメラ抗体は、 ヒト VEGF受容体 KDRに結合し、 KDRの作用 を中和するモノクローナル抗体を生産するハイブリ ド一マより、 VHおよび VL をコードする cDNAを取得し、 ヒト抗体 CHおよびヒト抗体 CLをコ一ドする遺 伝子を有する動物細胞用発現ベクターにそれぞれ挿入してヒト型キメラ抗体発 現ベクターを構築し、 動物細胞へ導入することにより発現させ製造することが できる。  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 And 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.
本発明の ヒト型 CDR 移植抗体は、 ヒト VEGF受容体 KDR に反応し、 ヒ卜 VEGF受容体 KDRに結合し、 KDRの作用を中和する、 ヒト以外の動物の抗体の VHおよび Vしの CDR 配列で任意のヒト抗体の VHおよび VLの CDR 配列をそ れぞれ置換した V 領域をコードする cDNAを構築し、 ヒト抗体の CHおよびヒ ト抗体の CL をコードする遺伝子を有する動物細胞用発現ベクターにそれぞれ 挿入してヒト型 CDR 移植抗体発現べクタ一を構築し、 動物細胞へ導入し、 発現 させることにより製造することができる。 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.
本発明のヒト型キメラ抗体およびヒ卜型 CDR 移植抗体はいずれのィムノグ ロブリン (lg)クラスに属するものでもよいが IgG 型のものが好適であり、 さらに IgG 型に属する IgGl、 IgG2、 IgG 3, IgG4等のィムノグロブリンの C 領域のいず れも用いることができる。  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.
さらに、 本発明はヒト VEGF受容体 KDR に対して結合性を示す一本鎖抗体 (single chain Fv; 以下、 scFvと称す） あるいはジスルフィ ド安定化抗体 (disulfide stabilized Fv; 以下、 dsFvと称す） を提供する。  Further, 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. provide.
一本鎖抗体 （scFv) とは、 一本の VHと一本の VLとを適当なペプチドリンカ ― (以下、 Lと称す） を用いて連結した、 VH _ L— VL ないしは VL— L— VH ポリペプチドを示す。本発明の scFvに含まれる VHおよび VLは、 抗ヒト VEGF 受容体 KDRモノクローナル抗体あるいはヒト型 CDR 移植抗体のいずれをも用 いることができる。  A single-chain antibody (scFv) 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. As the VH and VL contained in the scFv of the present invention, either an anti-human VEGF receptor KDR monoclonal antibody or a human CDR-grafted antibody can be used.
ジスルフィ ド安定化抗体 （dsFv) とは VHおよび VL中のそれぞれ 1アミノ酸 残基をシスティン残基に置換したポリペプチドをジスルフィ ド結合を介して結 合させたものをいう。 システィン残基に置換するアミノ酸残基は Reiter らによ り示された方法 [プロテイン 'エンジニアリング （Protein Engineering ) , 7, 697 (1994) ] に従って、 抗体の立体構造予測に基づいて選択することができる。 本 発明のジスルフィ ド安定化抗体に含まれる VH あるいは VL はマウス型抗ヒ卜 VEGF受容体 KDR モノクローナル抗体あるいはヒト型 CDR 移植抗体のいずれ をも用いることができる。  The disulfide-stabilized antibody (dsFv) 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)]). . As the VH or VL contained in the disulfide-stabilized antibody of the present invention, any of a mouse anti-human VEGF receptor KDR monoclonal antibody and a human CDR-grafted antibody can be used.
ヒ卜 VEGF受容体 KDR に対して結合性を示す一本鎖抗体は、ヒ卜 VEGF受容 体 KDR に結合し、 KDR の作用を中和する抗体を生産するハイブリド一マより VHおよび VLをコードする cDNAを取得し、一本鎖抗体発現べクタ一を構築し、 大腸菌、 酵母、 あるいは動物細胞へ導入することにより発現させ製造すること ができる。 Single-chain antibodies that bind to human VEGF receptor KDR are human VEGF receptor Obtain 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.
ヒト VEGF受容体 KDRに対して結合性を示すジスルフィ ド安定化抗体は、 ヒ ト VEGF受容体 KDR に反応する抗体を生産するハイブリドーマより VHおよび VLをコードする cDNAを取得し、 適当な発現ベクターに挿入し、 該発現べクタ 一を大腸菌、 酵母、 あるいは動物細胞へ導入し発現させることにより製造する ことができる。  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.
本発明の抗体は、 ヒト VEGF受容体 KDRに結合し、 ヒト VEGFのヒト VEGF 受容体 KDRへの結合を阻害し、 KDRの作用を中和するため、 ヒト VEGFが有す る血管内皮細胞に対する増殖促進活性、 遊走促進活性、 メタ口プロテアーゼ分 泌促進活性、 ゥロキナーゼ、 tPA分泌促進活性、 in vivo血管新生促進活性、 血管 透過性亢進活性等を阻害することができる。 したがって、 本発明は、 固形腫瘍 の増殖もしくは転移形成、 慢性関節リウマチにおける関節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など血管新生の異常により病態が進行する疾患の診断およ び治療に利用することができる。  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.
以下に、本発明の抗ヒ卜 VEGF受容体 KDRモノクローナル抗体、抗ヒ卜 VEGF 受容体 KDRヒト化抗体、抗ヒト VEGF受容体 KDR—本鎖抗体および抗ヒト VEGF 受容体 KDRジスルフィ ド安定化抗体の製造法、ならびに該抗体によるヒト VEGF 受容体 KDRの検出および定量法について説明する。  Hereinafter, 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.
1 . 抗ヒト VEGF受容体 KDRモノクローナル抗体の作製方法  1. Preparation of anti-human VEGF receptor KDR monoclonal antibody
( 1 ) 抗原の調製  (1) Preparation of antigen
抗ヒ卜 VEGF受容体 KDRモノクローナル抗体を作製するために必要な抗原と しては、 ヒ卜 VEGF受容体 KDRを細胞表面に発現した細胞あるいはその細胞膜 画分、 または、 アミノ酸の長さの異なる細胞外領域を有する可溶性ヒト VEGF 受容体 KDR蛋白質あるいは該蛋白質と抗体の Fc部分との融合蛋白質などがあ げられる。 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.
ヒ卜 VEGF受容体 KDRを細胞表面に発現する細胞としては、 N1H3T3- KDR細 胞 [セルグロ一ス アンド ディファレンシェ一シヨン （Cell Growth & Cells expressing the human VEGF receptor KDR on the cell surface include N1H3T3-KDR cells [Cell Growth & Differentiation (Cell Growth &
Differentiation) 7, 213, 1996.] があげられる。 長さの異なる細胞外領域を有する 可溶性ヒト VEGF受容体 KDR蛋白質あるいは該蛋白質と抗体の Fc部分との融 合蛋白質として発現させる方法としては、 ヒト VEGF受容体 KDRをコードする 全長あるいはその部分断片 cDNA [セルグロース アンド ディファレンシエー シヨン （Cell Growth & Differentiation) 7, 213, 1996.] を適当なベクタ一のプロ モー夕—下流に挿入した組み換え体ベクターを造成し、 それを宿主細胞に導入 することにより得られたヒト VEGF受容体 KDR発現細胞を、 適当な培地中で培 養することにより細胞内あるいは培養上清中にヒト VEGF受容体 KDRの全長あ るいは部分断片をそのままあるいは融合蛋白質として生産することができる。 また、 上述の蛋白質の部分配列を有するポリペプチドをアミノ酸合成機を用い て合成することによつても調製することができる。 Differentiation) 7, 213, 1996.]. 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.
宿主としては、 細菌、 酵母、 動物細胞、 昆虫細胞など、 目的とする遺伝子を 発現できるものであれば、 いずれでもよい。 細菌としては、 ェシエリヒア *コ リ (Escherichia coli) 、 バチルス ·ズブチリス （Bacillus subtilis ) 等のェシエリ ヒア属、 バチルス属等の細菌が例示される。 酵母としては、 サッカロミセス - セレヒンェ ( Saccharomyces cerevisiae ) 、 シゾサッ刀口ミセス . 。、ノべ (Schizosaccharomyces pombe) 等が例示される。 動物細胞としては、 ヒ卜の細胞 であるナマルバ細胞、 サルの細胞である COS細胞、 チャイニーズ 'ハムスター の細胞である CHO細胞等が例示される。 昆虫細胞としては、 S{9、 Sfi l (ファー ミンジェン社製） 、 High Five (インビトロジェン社製） 等が例示される。  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. Examples of the 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). Examples of animal cells include Namalva cells, which are human cells, COS cells, which are monkey cells, and CHO cells, which are Chinese hamster cells. Examples of insect cells include S {9, Sfil (Pharmingen), High Five (Invitrogen) and the like.
本発明の DNAを導入するベクターとしては、 該 DNAを組み込むことができ、 宿主細胞で発現できるものであればいかなるベクターでも用いることができる。 細菌、 例えばェシエリヒア 'コリ (Escherichia coli) を宿主として用いる場合 の発現べクタ一としては、 プロモー夕一、 リボゾーム結合配列、 本発明の DNA、 転写終結配列、 場合によってはプロモーターの制御配列より構成されているの が好ましいが、 例えば、 市販の pGEX (フアルマシア社製） 、 pET システム （ノ バジェン社製） などが例示される。 As a vector into which the DNA of the present invention is introduced, any vector can be used as long as it can incorporate the DNA and can be expressed in a host cell. When a bacterium, for example, Escherichia coli is used as a host, 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. For example, commercially available pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen) and the like are exemplified.
細菌への組換えベクターの導入方法としては、 細菌に DNAを導入する方法で あれば、 例えば、 カルシウムイオンを用いる方法 [プロシーデイングス ·ォブ' ザ'ナショナル ·アカデミー'ォブ 'サイエンス ·ォブ ·ザ * U. s . A. (Proc. 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.
Natl. Acad. Sci" USA), 69, 2110-2114 (1972) ] 、 プロトプラスト法 （特開昭 63 - 248394) 等、 いずれの方法も用いられる。 Natl. Acad. Sci "USA), 69, 2110-2114 (1972)], and the protoplast method (JP-A-63-248394).
酵母を宿主として用いる場合には、 発現ベクターとして、 例えば、 Y E p l 3 (ATCC37115) 、 Y E p 2 4 (ATCC37051) 、 Y C p 5 0 (ATCC37419) 等 が用いられる。  When yeast is used as a host, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), and YCp50 (ATCC37419).
酵母への組換えベクターの導入方法としては、 酵母に DNAを導入する方法で あれば、 例えば、 エレクトロボレ一シヨン法 [メソッズ .ォブ.ェンザィモロ ジ一 (Methods. Enzymol.), 194, 182- 187 (1990)] 、 スフエロプラスト法 [プロシ一 ディングス 'ォブ 'ザ 'ナショナル ·アカデミー ·ォブ ·サイエンス ·ォブ · ザ · U. S . A . (Proc. Natl. Acad. Sci., USA), 84, 1929—1933 (1978) ] 、 隱リ チウム法 [ジャーナル ·ォブ ·バクテリオロジ一（J. Bacteriol.), 153, 163-168 (1983) ] 等、 いずれの方法も用いられる。  As a method for introducing a recombinant vector into yeast, 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)]. .
動物細胞を宿主として用いる場合には、 発現ベクターとして、 例えば、 p A G E 1 0 7 [特開平 3 - 22979 ；サイ トテクノロジー (Cytotechnology), 3, 133 (1990) ] ， P A G E 1 0 3 [ジャーナル'ォブ ·バイオケミストリー G.Biochem.) 101 , 1307(1987) ] 等が用いられる。  When an animal cell is used as a host, as an expression vector, for example, pAGE107 [Japanese Unexamined Patent Publication No. 3-22979; Cytotechnology, 3, 133 (1990)], PAGE 103 [Journal '] G. Biochem.) 101, 1307 (1987)].
プロモーターとしては、 動物細胞中で発現できるものであればいかなるもの を用いてもよい力 ^例えば、サイトメガロウィルス（ C M V )の lE(immedia½ early) 遺伝子のプロモー夕一、 SV40あるいはメタロチォネィンのプロモーター等があ げられる。 また、 ヒト C M Vの 1E遺伝子のェンハンサ一をプロモ一夕一ととも に用いてもよい。 Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (CMV) lE (immedia½ early) Promote the gene, SV40 or metallotionin promoter, etc. Alternatively, the enhancer of the 1E gene of human CMV may be used together with the promoter.
動物細胞への組換えべクタ一の導入方法としては、 動物細胞に DNAを導入す る方法であれば、 例えば、 エレクト口ポレーシヨン法 [サイ トテクノロジ一 (Cytotechnology), 3, 133(1990)] 、 リン酸カルシウム法 (特開平 2- 227075) 、 リ ポフエクシヨン法 [プロシーディングス .ォブ.ザ.ナショナル .アカデミー . ォブ 'サイエンス ·ォブ ·ザ · U . S . A . (Proc. Natl. Acad. Sci., USA), 84, 7413 (1987)] 等、 いずれの方法も用いられる。  As a method for introducing a recombinant vector into animal cells, 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)].
昆虫細胞を宿主として用いる場合には、 例えばカレント ·プロ卜コールズ · イン 'モレキュラー 'バイオロジー、 サプルメント 1 ~ 3 4 (Current Protocols in Molecular Biology Supplement 1—34)、ノ キュロウィルス 'イクスプレツシヨン · へク夕——ズ、 · フ ヽフトリ—— ·マニュ / Jレ (Baculovirus expression vectors A laboratory manual) 等に記載された方法によって、 タンパク質を発現することが できる。 すなわち、 以下に述べる組換え遺伝子導入ベクターおよびバキュロウ ィルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウィルスを得たの ち、 さらに組換えウィルスを昆虫細胞に感染させ、 タンパク質発現昆虫細胞を 取得する。  When insect cells are used as hosts, for example, Current Protocols in 'Molecular' Biology, Supplements 1-34 (Current Protocols in Molecular Biology Supplement 1-34), 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.
遺伝子導入べクタ一としては、 例えば、 pVL1392、 pVL1393 、 pBlueBacllI (と もにインビトロジェン社製） 等が用いられる。  As the gene transfer vector, for example, pVL1392, pVL1393, pBlueBacllI (both manufactured by Invitrogen) and the like are used.
バキュロウィルスとしては、 例えば、 夜盗蛾科昆虫に感染するウィルスであ るアウトグラファ ·カリフォルニ力 ·ヌクレア一 'ボリへドロシス . ウィルス (Autographa californica nuclear polyhedrosis virus) なとが用いられる。  As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects insects of the night moth family, Autographa californica nuclear polyhedrosis virus, is used.
組換えウィルスを調製するための、 昆虫細胞への上記組換え遺伝子導入べク 夕一と上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシゥ ム法 （特開平 2- 227075) 、 リボフェクシヨン法 [プロシーディングス .ォブ · ザ'ナショナル'アカデミー 'ォブ'サイエンス 'ォブ 'ザ · U . S . A . (Proc. Natl. Acad. Sci" USA), 84, 7413 (1987)] 等が用いられる。 For the preparation of recombinant viruses, as Cotransfection of the above recombinant gene transfer base click evening one and the baculovirus into insect cells, for example, phosphoric acid Karushiu beam method (JP-A-2 2270 75), Ribofekushiyon Law [Proceedings. The 'National' Academy 'Ob' Science 'Ob' The U.S.A. (Proc. Natl. Acad. Sci "USA), 84, 7413 (1987)].
また、 ファーミンジェン社製バキュ口ゴールドスターターキッ卜などを用い て組み換えバキュロウィルスを作製したのち、 前述した Sf 、 Sf21あるいは High Five 等の昆虫細胞に該組み換えウィルスを感染させることにより蛋白質を生産 させることもできる [バイオテクノロジー （Bio/Technology) , 6, 47(1988)] 。 遺伝子の発現方法としては、 直接発現以外に、 分泌生産、 融合蛋白質発現等 が開発されており、 いずれの方法も用いることができる。 例えば、 モレキユラ —'クロ一ニング 第 2版、コールドスプリングハーバーラボ.プレス [Molecular Cloning 2nd edition, Cold Spring Harbor Lab. Press New York (1989)；以下、 「モレ キユラ一 ' クローニング 第 2版」 と記す] に記載されている方法に準じて行 うことができる。  In addition, after producing a recombinant baculovirus using Farmingen's Bacchus Gold Starter Kit or the like, 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)]. In addition to direct expression, 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].
以上のようにして得られる形質転換体を培地に培養し、 培養物中に本発明の 蛋白質を生成蓄積させ、 該培養物から採取することにより、 ヒ卜 VEGF受容体 DR の全長あるいは部分断片をそのままあるいは融合蛋白質として製造するこ とができる。  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.
本発明の形質転換体を培地に培養する方法は、 宿主の培養に用いられる通常 の方法に従って行われる。  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.
大腸菌あるいは酵母等の微生物を宿主として得られた形質転換体を培養する 培地としては、 微生物が資化し得る炭素源、 窒素源、 無機塩類等を含有し、 形 質転換体の培養を効率的に行える培地であれば天然培地、 合成培地のいずれを 用いてもよい （モレキュラー ' クローニング 第 2版） 。 培養は、 通常振盪培 養または深部通気攪拌培養などの好気的条件下、 15〜40°Cで 16〜96時間行う。 培養期間中、 pHは 3.0〜9.0に保持する。 pHの調整は、 無機または有機の酸、 アルカリ溶液、 尿素、 炭酸カルシウム、 アンモニアなどを用いて行う。 培養中 は必要に応じて、 アンピシリンゃテトラサイクリン等の抗生物質を培地に添加 してもよい。 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. Either 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. During the culture period, 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. During culture, add antibiotics such as ampicillin / tetracycline to the medium as needed May be.
動物細胞を宿主として得られた形質転換体を培養する培地としては、 一般に 使用されている R P M 1 1 6 4 0培地、 E a g 1 eの M E M培地またはこれら 培地に牛胎児血清等を添加した培地等が用いられる。 培養は、 通常 5%C02存在 下、 35〜37°Cで 3〜7日間行い、 培養中は必要に応じて、 カナマイシン、 ぺニシ リン等の抗生物質を培地に添加してもよい。 As 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.
昆虫細胞を宿主として得られた形質転換体を培養する培地としては、 一般に 使用されてレ ^る TNM-FH培地 [ファーミンジェン（Pharmingen)社製]、 Sf900IISFM [ライフテクノロジ一ズ (Life Technologies)社製] 、 ExCell400 、 ExCell405 [い ずれも JRH バイオサイエンシーズ （JRH Biosciences) 社製] 等が用いられる。 培養は、 25〜30°Cで 1〜4日間行い、 培養中は必要に応じて、 ゲンタマイシン等 の抗生物質を培地に添加してもよい。  As a medium for culturing a transformant obtained by using an insect cell as a host, 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. During the culture, an antibiotic such as gentamicin may be added to the medium as needed.
上記において、 動物細胞および昆虫細胞の培養において可能であれば、 ヒト VEGF受容体 KDRの全長あるいは部分断片をそのままあるいは融合蛋白質の精 製を容易にするため、 血清無添加の培地を用いることが好ましい。  In the above, if possible in culturing animal cells and insect cells, it is preferable to use 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. .
ヒト VEGF受容体 KDR の全長あるいは部分断片をそのままあるいは融合蛋白 質として宿主細胞内に蓄積された場合には、 培養終了後、 細胞を遠心分離し、 水系緩衝液にけん濁後、 超音波法、 フレンチプレス法などにより細胞を破砕し、 その遠心分離上清に該蛋白質を回収する。  If the full-length or partial fragment of the human VEGF receptor KDR is accumulated in the host cell as it is or as a fusion protein, after the culture is completed, 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.
さらに、 細胞内に不溶体を形成した場合には、 不溶体をタンパク質変性剤で 可溶化後、 タンパク質変性剤を含まないあるいはタンパク質変性剤の濃度が夕 ンパク質が変性しない程度に希薄な溶液に希釈、 或いは透析し、 タンパク質の 立体構造を形成させることができる。  Furthermore, when an insoluble substance is formed in cells, after 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.
ヒト VEGF受容体 KDR の全長あるいは部分断片をそのままあるいは融合蛋白 質として細胞外に分泌された場合には、 培養上清中に発現蛋白質を回収するこ とができる。 単離精製については、 溶媒抽出、 有機溶媒による分別沈殿、 塩析、 透析、 遠 心分離、 限外ろ過、 イオン交換クロマトグラフィー、 ゲルろ過クロマトグラフ ィー、 疎水性クロマトグラフィー、 ァフィ二ティークロマトグラフィー、 逆相 クロマトグラフィー、 結晶化、 電気泳動などの分離操作を単独あるいは組み合 わせて行うことができる。 When the full-length or partial fragment of the human VEGF receptor KDR is secreted extracellularly as it is or as a fusion protein, the expressed protein can be recovered in the culture supernatant. For isolation and purification, 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.
( 2 ) 動物の免疫と抗体産生細胞の調製  (2) Animal immunization and preparation of antibody-producing cells
上記で得られた該蛋白質を抗原として免疫する。 免疫する方法としては、 動 物の皮下、 静脈内または腹腔内に抗原をそのまま投与してもよいが、 抗原性の 高いキャリアタンパク質を結合させて投与したり、 あるいは適当なアジュバン トとともに抗原を投与することが好ましい。  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.
キャリアタンパク質としては、 スカシガイへモシァニン、 キーホールリンべ ッ卜へモシァニン、 牛血清アルブミン、 牛チログロブリン等があげられ、 アジ ュバンドとしては、 フロインドの完全アジュパント（Complete Freund's Adjuvant), 水酸化アルミニウムゲルと百日咳菌ワクチン等があげられる。  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.
免疫動物としては、 ゥサギ、 ャギ、 マウス、 ラット、 ハムスターなどの非ヒ ト哺乳動物があげられる。  Examples of the immunized animal include non-human mammals such as rabbits, goats, mice, rats, and hamsters.
抗原の投与は、 1回目の投与の後、 1〜2週間毎に 3〜10 回行う。 抗原の投 与量は動物 1匹当たり 50〜100 gが好ましい。各投与後、 3 〜7 日目に免疫動 物の眼底静脈叢あるいは尾静脈より採血し、 該血清の抗原との反応性について、 酵素免疫測定法 [酵素免疫測定法 （ELISA 法） ：医学書院刊 （1976年） ] など で確認する。  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)].
そして、 該血清が十分な抗体価を示した非ヒト哺乳動物を、 抗体産生細胞の 供給源とする。  Then, a non-human mammal whose serum shows a sufficient antibody titer is used as a source of the antibody-producing cells.
抗原の最終投与後 3 〜7 日目に、 免疫動物より公知の方法 [アンティボディ ーズ ·ァ · ラボラトリー 'マニュアル、 コールド ·スプリングハ一バー · ラボ フ トリ一 Antibodies - A Laboratory Manual Cold Spring Harbor Laboratory, 1988) 、 以下、 アンチボディ一ズ 'ァ ' ラボラトリー 'マニュアルと記す] に準じてリ ンパ球を摘出し、 リンパ球と骨髄腫細胞とを融合させる。 Three to seven days after the last administration of the antigen, a method known from the immunized animal [Antibodies-Laboratory 'Manual, Cold Spring Haver-Laboratory Antibodies-A Laboratory Manual Cold Spring Harbor Laboratory , 1988), Hereinafter, the lymphocytes are excised according to the “Antibody's 'Lab' Manual” and the lymphocytes are fused with myeloma cells.
( 3 ) 骨髄腫細胞の調製  (3) Preparation of myeloma cells
骨髄腫細胞としては、 マウスから得られた株化細胞である、 8 -ァザグァニン 耐性マウス（BALBん由来）骨髄腫細胞株 P3- X63Ag8- U UP3-U 1) [G . ohlerら ； ョ一口ビアン · ジャーナル ' ォブ ' ィムノロジィ（Europ. J. Immunol.), 6, 511 (1976) ] 、 SP2/0 - Ag14(SP - 2) [ M.Shulman ら ； ネィチヤ一（Nature), 276, 269(1978)] 、 P3-X63-Ag8653(653) [J.F.Kearney ら ； ジャーナル 'ォブ.ィムノ ロジィ（J. Immunol.) , 123, 1548(1979)] 、 Ρ3- Χ63- Ag8(X63) [G.Kohlerら ；ネイチ ャ一(Nature), 256, 495(1975)] など、 イン 'ビトロ (in vitro) で増殖可能な骨髄腫 細胞であればいかなるものでもよい。 これらの細胞株の培養および継代につい ては公知の方法 （アンチボディ一ズ ·ァ · ラボラトリー .マニュアル） に従い、 細胞融合時までに 2 X 107個以上の細胞数を確保する。 Examples of 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. Immunol., 123, 1548 (1979)], Ρ3-Χ63-Ag8 (X63) [G [Kohler et al .; Nature, 256, 495 (1975)], and any other myeloma cells that can grow in vitro. Culture and passage of these cell lines are performed according to a known method (Antibody-z-Laboratory. Manual), and a cell number of 2 × 10 7 or more is secured by the time of cell fusion.
( 4 ) 細胞融合とモノクローナル抗体の選択  (4) Cell fusion and selection of monoclonal antibody
上記で得られた抗体産生細胞と骨髄腫細胞とを洗浄したのち、 ポリエチレン グライコ一ルー 1000(PEG- 1000)などの細胞凝集性媒体を加え、 細胞を融合させ、 培地中に懸濁させる。 細胞の洗浄には M E M培地または P B S (リン酸ニナト リウム 1.83g 、リン酸一カリウム 0.21g 、食塩 7.65g 、蒸留水 1 リットル、 pH7.2 ) などを用いる。 また、 融合細胞を懸濁させる培地としては、 目的の融合細胞の みを選択的に得られるように、 HAT 培地 {正常培地 [RPM卜 1640 培地にダル夕 ミン (1.5mM) 、 2-メルカプトエタノール（5 X 10—5M )、 ジェン夕マイシン（10 g/ml) および牛胎児血清 (FCS) (CSL 社製、 10% ) を加えた培地] にヒボキサン チン （10— 4M ) 、 チミジン （1 .5 X 10— 5M ) およびアミノプテリン （4 X 10"7M ) を加えた培地） を用いる。 After washing the antibody-producing cells and myeloma cells obtained above, a cell-aggregating medium such as polyethylene glycol 1000 (PEG-1000) is added, and the cells are fused and suspended in a medium. For washing the cells, use 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) or the like. As 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. (5 X 10- 5 M), Jen evening mycin (10 g / ml) and fetal calf serum (FCS) (CSL Ltd., 10%) was added medium] in Hibokisan Chin (10- 4 M), thymidine ( 1 .5 X 10- 5 M) and aminopterin (4 X 10 "7 M) was added medium) is used.
培養後、 培養上清の一部をとり、 酵素免疫測定法により、 抗原蛋白質に反応 し、 非抗原蛋白質に反応しないサンプルを選択する。 ついで、 限界希釈法によ りクローニングを行い、 酵素免疫測定法により安定して高い抗体価の認められ たものをモノク口一ナル抗体産生ハイプリドーマ株として選択する。 After the culture, 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.
酵素免疫測定法 Enzyme immunoassay
抗原蛋白質あるいは抗原蛋白質を発現した細胞などをプレートにコートし、 ハイプリ ドーマ培養上清もしくは上述の方法で得られる精製抗体を第一抗体と して反応させる。  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.
第一抗体反応後、 プレートを洗浄して第二抗体を添加する。  After the first antibody reaction, the plate is washed and the second antibody is added.
第二抗体とは、 第一抗体を認識できる抗体に、 ピオチン、 酵素、 化学発光物 質あるいは放射線化合物等で標識したものである。 具体的には、 ハイプリドー マ作製の際にマウスを用いたのであれば、 第二抗体としては、 マウスィムノグ ロブリンを認識できる抗体を用いる。  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.
反応後、 第二抗体を標識した物質に応じた反応を行い、 抗原に特異的に反応す るモノクローナル抗体を生産するハイプリドーマとして選択する。 After the reaction, a reaction is performed according to the substance labeled with the second antibody, and the hybridoma is selected as a hybridoma that produces a monoclonal antibody that specifically reacts with the antigen.
本発明ハイプリ ドーマ株の具体例としては、 ハイプリ ドーマ株 KM1668、 KM1992および KM1995が挙げられる。 ハイブリドーマ株 KM1668、 KM 1992お よび KM1995は、平成 10年 1月 21日付で、それぞれ工業技術院生命工学工業技 術研究所（日本国茨城県つくば巿東 1丁目 1番 3号） に、 FERM BP- 6216、 FERM BP-6217, FERM BP- 6218として寄託されている。  Specific examples of the hybridoma strain of the present invention include the hybridoma strains KM1668, KM1992 and KM1995. On January 21, 1998, 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.
( 5 ) モノクローナル抗体の調製  (5) Preparation of monoclonal antibody
モノクローナル抗体は、 ハイプリドーマ細胞を培養して得られる培養液、 ま たはプリスタン処理 〔2,6,10,14-テトラメチルペン夕デカン (Pristane)0.5ml を腹 腔内投与し、 2 週間飼育する〕 した 8 〜10週令のマウスまたはヌードマウスに、 モノクローナル抗体産生ハイプリドーマ細胞を腹腔内投与して腹水癌化させた 腹水から、 分離、 精製することにより調製できる。 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.
モノクローナル抗体を分離、 精製する方法としては、 遠心分離、 40〜 0% 飽 和硫酸アンモニゥムによる塩析、 力プリル酸沈殿法、 DEAE-セファロ一スカラ ム、 、 陰イオン交換カラム、 プロテイン Aまたは G- カラムあるいはゲル濾過 カラム等を用いるクロマトグラフィー等を、 単独または組み合わせて行う方法 があげられる。 この方法により、 IgG あるいは IgM 画分を回収し、 精製モノク ローナル抗体を取得することができる。 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.
精製モノクローナル抗体のサブクラスの決定は、 モノクロ一ナル抗体タイピ ングキットなどを用いて行うことができる。 蛋白質量は、 ローリー法あるいは 280nm での吸光度より算出することができる。  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.
抗体のサブクラスとは、 クラス内のアイソタイプのことで、 マウスでは、 IgGl、 lgG2a 、 IgG2b 、 IgG3, ヒトでは、 IgGU lgG2、 lgG3、 IgG4 があげられるが、 特にマウス IgGl、 IgG2a 、 ヒト IgGlタイプは、 補体依存性細胞傷害活性 （以下、 CDC 活性） および抗体依存性細胞傷害活性 （以下、 ADCC活性） を有し、 治療 への応用上、 有用である。 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.
2 . 抗ヒト VEGF受容体 KDR ヒト化抗体の作製  2. Production of anti-human VEGF receptor KDR humanized antibody
( 1 ) ヒト化抗体発現用ベクターの構築  (1) Construction of humanized antibody expression vector
ヒ卜以外の動物の抗体からヒト化抗体を作製するために必要なヒ卜化抗体発 現用ベクターを構築する。 ヒ卜化抗体発現用ベクターとは、 ヒト抗体の C 領域 である CHおよび CLをコ一ドする遺伝子が組み込まれた動物細胞用発現べクタ —であり、動物細胞用発現ベクターにヒト抗体の CHおよび CLをコードする遺 伝子をそれぞれ挿入することにより構築されたものである。 ヒ卜抗体の C 領域 としては、 例えば、 ヒト抗体 H 鎖では C ァ 1 や C ァ 4 、 ヒト抗体 L鎖では C κ等の任意のヒト抗体の C 領域を用いることができる。 ヒト抗体の C 領域 をコードする遺伝子としてはェキソンとィントロンより成る染色体 DNA を用 いることができ、 また、 cDNAを用いることもできる。 動物細胞用発現べクタ一 としては、 ヒ卜抗体 C 領域をコードする遺伝子を組込み発現できるものであれ ばいかなるものでも用いることができる。 例えば、 pAGE107 [サイトテクノロ ジー (Cytotechnology), 3, 133 (1990) ] 、 pAGE103 [ジャーナル ·ォブ ·バイオ ケミスト U— Q.Biochem.), 101 , 1307 (1987)] 、 pHSG274 [ジーン (Gene), ， 223 (1984)] 、 pKCR [プロシーデイング 'ォブ.ザ .ナショナル ' アカデミー ·ォ ブ ·サイエンス (Proc.Natl.Acad.Sci.), ， 1527 (1981)] 、 pSG l β d2- 4 [サイトテ クノロジー (Cytotechnology), ， 173 (1990) ] 等があげられる。 動物細胞用発現べ クタ一に用いるプロモ一夕一とェンハンサ一としては、 SV40の初期プロモ一夕 一とェンハンサ一 [ジャーナル · ォブ · バイオケミストリ一 (J.Biochem.), 101,1307(1987)]、モロニ一マウス白血病ウィルスの LTR プロモーターとェンハ ンサー [バイオケミカル ·アンド ·バイオフィジカル . リサーチ . コミュニケ ーシヨンズ (Biochem.Biophys.Res.Comun.), 149, 960(1987) ] 、 および免疫グロブ リン H鎖のプロモ一夕一 [セル (Cell), ， 479 (1985)]とェンハンサー [セル (Cell), 33, 717 (1983)] 等があげられる。 Construct a humanized antibody expression vector necessary for producing a humanized antibody from antibodies of animals other than human. 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. As the C region of the human antibody, for example, a C region of an arbitrary human antibody such as C a1 or C a 4 for a human antibody H chain and Cκ for a human antibody L chain can be used. As 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. For example, 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)], pSGlβd2-4 [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. Biochem., 101, 1307 (1987). )], Moroni murine leukemia virus LTR promoter and enhancer [Biochemical and Biophysical. Research. Communications (Biochem. Biophys. Res. Comun.), 149, 960 (1987)], and immunoglobulin. Promoters of the H-chain [Cell,, 479 (1985)] and Enhancer [Cell, 33, 717 (1983)].
ヒ卜化抗体発現用ベクターは、 抗体 H鎖、 L鎖が別々のベクター上に存在す るタイプあるいは同一のベクター上に存在するタイプ （タンデム型） のどちら でも用いることができるが、 ヒト化抗体発現べクタ一の構築のしゃすさ、 動物 細胞への導入のし易さ、動物細胞内での抗体 H 鎖およびし鎖の発現量のバラン スがとれる等の点でタンデム型のヒト化抗体発現用べクタ一の方が好ましい [ジャーナル' ォブ' ィムノロジカル · メソッズ hnmunol.Methods) , 167， 271(1994)] 。  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).
( 2 ) ヒト以外の動物の抗体の VHおよび VLをコードする cDNAの取得 ヒ卜以外の動物の抗体、 例えば、 マウス抗ヒト VEGF受容体 KDR モノクロ一 ナル抗体の VHおよび VLをコードする cDNAは以下のようにして取得する。 抗ヒト VEGF受容体 KDR モノクローナル抗体を産生する細胞、 例えば、 マウ スヒト VEGF受容体 KDR 抗体産生ハイブリ ドーマ等より mRNAを抽出し、 cDNA を合成する。 合成した cDNA を、 ファ一ジあるいはプラスミドなどのベクター に挿入し、 cDNAライブラリーを作製する。 該ライブラリ一より、 ヒト以外の動 物の抗体、 例えば、 マウス抗体の C領域部分あるいは V領域部分をプローブと して用い、 VHをコードする cDNAを有する組換えファージあるいは組換えプラ スミド、および VLをコードする cDNAを有する組換えファージあるいは組換え プラスミドをそれぞれ単離する。 組換えファージあるいは組換えプラスミド上 の目的とする抗体の VHおよび VLの全塩基配列を決定し、 塩基配列より VHお よび VLの全アミノ酸配列を推定する。 (2) Obtaining cDNAs encoding VH and VL of non-human animal antibodies 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. To isolate 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.
( 3 ) ヒト型キメラ抗体発現ベクターの構築  (3) Construction of human-type chimeric antibody expression vector
前記 2 ( 1 ) で構築したヒト化抗体発現用ベクターのヒト抗体の CH および CLをコードする遺伝子の上流に、 ヒト以外の動物の抗体の VHおよび VLをコ ードする cDNA を挿入し、 ヒト型キメラ抗体発現ベクターを構築することがで きる。 例えば、 キメラ抗体発現用ベクターのヒト抗体の CHおよび CLをコード する遺伝子の上流に らかじめヒト以外の動物の抗体の VHおよび VLをコード する cDNA をクローニングするための制限酵素の認識配列を設けておき、 この クローニングサイトにヒト以外の動物の抗体の V領域をコードする cDNAを下 記に述べる合成 DNAを介して挿入することにより、 ヒト型キメラ抗体発現べク ターを製造することができる。 合成 DNAは、 ヒト以外の動物の抗体の V領域の 3'末端側の塩基配列とヒ卜抗体の C領域の 5'末端側の塩基配列とからなるもの であり、 両端に適当な制限酵素部位を有するように DNA合成機を用いて製造す る。  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. For example, 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
( 4 ) ヒト以外の動物の抗体の CDR 配列の同定  (4) Identification of CDR sequences of non-human animal antibodies
抗体の抗原結合部位を形成する VH及び VLは、 配列の比較的保存された 4個 のフレームワーク領域 （以下、 FR領域と称す） とそれらを連結する配列の変化 に富んだ 3個の相補性決定領域 （CDR) から成っている [シーケンシズ* ォブ- プロテインズ' ォブ' ィムノロジカル · インタレスト （Sequences of Proteins of Immunological Interest) , US Dept. Health and Human Services, 1991 ] 0 そして各 CDR アミノ酸配列 （CDR 配列） は、 既知の抗体の V 領域のアミノ酸配列 [シ 一ケンシズ' ォブ' プロテインズ' ォブ · ィムノロジカル. インタレスト (Sequences of Proteins of Immunological Interest) , US Dept. Health and Human Services, 1991] と比較することにより同定することができる。 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].
( 5 ) ヒト型 CDR 移植抗体の V領域をコ一ドする cDNAの構築  (5) Construction of cDNA encoding V region of human CDR-grafted antibody
ヒト型 CDR 移植抗体の VHおよび VLをコードする cDNAは以下のようにし て取得することができる。  CDNAs encoding VH and VL of the human CDR-grafted antibody can be obtained as follows.
まず、目的のヒ卜以外の動物の抗体の V領域の CDR を移植するためのヒト抗 体の V領域の FRのアミノ酸配列を VH、 VLそれぞれについて選択する。 ヒト抗 体の V領域の FRのアミノ酸配列としては、 ヒト抗体由来の V領域の FRのアミ ノ酸配列であればいかなるものでも用いることができる。 例えば、 Protein Data Bank に登録されているヒト抗体の V領域の FRのアミノ酸配列、 ヒト抗体の V 領域の FRの各サブグループの共通アミノ酸配列 [シ一ケンシズ' ォブ · プロテ インズ' ォブ · ィムノロジカル · インタレス卜 （ Sequences of Proteins of Immunological Interest ) , US Dept. Health and Human Services, 1991] があ られ るが、 充分な活性を有するヒト型 CDR 移植抗体を創製するためには、 目的のヒ ト以外の動物の抗体の V領域のアミノ酸配列と高い相同性、 好ましくは 65%以 上の相同性を有することが望ましい。次に、選択したヒト抗体の V領域の FRの アミノ酸配列をコードする DNA配列と目的のヒ卜以外の動物の抗体の V領域 の CDR のアミノ酸配列をコードする DNA配列を連結させて、 VH、 VLそれぞ れのアミノ酸配列をコードする DNA 配列を設計する。 CDR移植抗体可変領域 遺伝子を構築するために設計した DNA 配列を得るためには、 全 DNA配列を力 バ一するように各鎖について数本の合成 DNAを設計し、 それらを用いてポリメ ラーゼ ·チエイン · リアクンヨ ン (Polymerase し hain Reaction； ^卜、 Pし Rと 5じ す） を行う。 PCR での反応効率および合成可能な DNA の長さから各鎖につい て、 好ましくは、 6本の合成 DNA を設計する。 反応後、 増幅断片を適当なべク 夕一にサブクローニングし、 その塩基配列を決定し、 目的のヒト型 CDR 移植抗 体の各鎖の V 領域のアミノ酸配列をコードする cDNAを含むプラスミドを取得 する。 また、 約 100塩基よりなる合成 DNAを用いてセンス、 アンチセンスとも に全配列を合成し、それらをァ二一リング、連結することで、 目的のヒ卜型 CDR 移植抗体の各鎖の V 領域のアミノ酸配列をコードする cDNAを構築することも できる。 First, 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. As 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. For example, 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. Health and Human Services, 1991], but in order to create human CDR-grafted antibodies with sufficient activity, It is desirable to have high homology with the amino acid sequence of the V region of an antibody of an animal other than the above, preferably at least 65%. Next, the DNA sequence encoding the amino acid sequence of the FR of the V region of the selected human antibody and the DNA sequence encoding the amino acid sequence of the CDR of the V region of the antibody of the animal other than the target human are ligated to obtain VH, Design a DNA sequence that encodes the amino acid sequence of each VL. In order to obtain a DNA sequence designed for constructing the CDR-grafted antibody variable region gene, several synthetic DNAs are designed for each chain so that the entire DNA sequence is supported, and the resulting Perform chainin reaction (Polymerase and hain Reaction; 5 times with P and R). From the reaction efficiency in PCR and the length of DNA that can be synthesized, preferably, six synthetic DNAs are designed for each strand. After the reaction, 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. In addition, using a synthetic DNA consisting of about 100 bases, synthesizing the entire sequence for both sense and antisense, and combining and ligating them, 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.
( 6 ) ヒ卜型 CDR 移植抗体の V 領域のアミノ酸配列の改変  (6) Amino acid sequence modification of V region of human CDR-grafted antibody
ヒ卜型 CDR 移植抗体は目的のヒト以外の動物の抗体の V 領域の CDR のみ をヒト抗体の V 領域の FR間に、 単純に移植しただけでは、 その活性はもとの ヒト以外の動物の抗体の活性に比べて低下してしまうことが知られている [バ ィォテクノロジ— （BIO/TECHNOLOGY) , 9, 266 (1991) ] 。 そこでヒト抗体の V 領域の FRのアミノ酸配列のうち、直接抗原との結合に関与しているアミノ酸 残基、 CDR のアミノ酸残基と相互作用をしているアミノ酸残基、 あるいは抗体 の立体構造の維持に関与している等の可能性を有するアミノ酸残基をもとのヒ 卜以外の動物の抗体に見出されるアミノ酸残基に改変し、 活性を上昇させるこ とが行われている。 そして、 それらのアミノ酸残基を効率よく同定するため、 X 線結晶解析あるいはコンピュータ一モデリング等を用いた抗体の立体構造の構 築および解析を行っている。 しかし、 いかなる抗体にも適応可能なヒト型 CDR 移植抗体の製造法は未だ確立されておらず、 現状では個々の抗体によって種々 の試行錯誤が必要である。  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. In order to identify these amino acid residues efficiently, we are constructing and analyzing the three-dimensional structure of antibodies using X-ray crystallography or computer modeling. However, a method for producing a human CDR-grafted antibody that can be applied to any antibody has not yet been established, and at present, various trials and errors are required for each antibody.
選択したヒト抗体の V領域の FRのアミノ酸配列の改変は各種の変異導入ブラ イマ一を用いて前記 2 ( 5 ) に記載の PCR を行うことにより達成できる。 PCR 後の増幅断片を適当なベクタ一にサブクロ一ニング後、 その塩基配列を決定し、 目的の変異が導入された cDNA を含むベクター （以下、 アミノ酸配列改変べク 夕一と称す） を取得する。  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). .
また、 狭い領域のアミノ酸配列の改変であれば、 20〜35塩基からなる変異導 入プライマ一を用いた PCR変異導入法により行うことができる。 具体的には、 改変後のアミノ酸残基をコードする DNA 配列を含む 20〜35塩基からなるセン ス変異プライマー及びアンチセンス変異プライマーを合成し、 改変すべき V領 域のアミノ酸配列をコードする cDNAを含むプラスミドを铸型として 2段階の PCR を行う。 最終増幅断片を適当なベクタ一にサブクローニング後、 その塩基 配列を決定し、 目的の変異が導入された cDNA を含むアミノ酸配列改変べクタ 一を取得する。 In addition, 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.
( 7 ) ヒ卜型 CDR移植抗体発現ベクターの構築  (7) Construction of human CDR-grafted antibody expression vector
前記 2 ( 1 )のヒト化抗体発現用べクタ一のヒト抗体の CH及び CLをコード する遺伝子の上流に、 前記 2 ( 5 )および 2 ( 6 )で取得したヒト型 CDR移植抗 体の VH及び VLをコードする cDNAを挿入し、 ヒ卜型 CDR移植抗体発現べク 夕一を構築することができる。 例えば、 ヒト型 CDR移植抗体の VH及び VLの アミノ酸配列をコ一ドする cDNAを構築するための PCRの際に 5'末端および 3' 末端の合成 DNAの末端に適当な制限酵素の認識配列を導入することで、 所望の ヒ卜抗体の C領域をコードする遺伝子の上流にそれらが適切な形で発現するよ うに挿入することができる。  The VH of the human CDR-grafted antibody obtained in 2 (5) and 2 (6) above the gene encoding CH and CL of the human antibody in the vector for humanized antibody expression of 2 (1) above And a cDNA encoding VL can be inserted to construct a human CDR-grafted antibody expression vector. For example, at the time of PCR for constructing a cDNA encoding the amino acid sequence of VH and VL of a human CDR-grafted antibody, 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.
( 8 ) ヒト化抗体の一過性 （トランジェン卜） 発現および活性評価  (8) Evaluation of transient expression and activity of humanized antibody
多種類のヒト化抗体の活性を効率的に評価するために、 前記 2 ( 3 ) のヒト 型キメラ抗体発現ベクター、 および前記 2 ( 7 ) のヒト型 CDR移植抗体発現べ クタ一あるいはそれらの改変ベクターを COS-7 細胞 （ATCC CRL1651) に導入 してヒト化抗体の一過性発現 [メソッズ ·イン ·ヌクレイック ·ァシッド · リ サーチ (Methods in Nucleic Acids Res. ) , CRC Press, p.283, 1991] を行い、 その 活性を測定することができる。  In order to efficiently evaluate the activity of various types of humanized antibodies, 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.
COS- 7 細胞への発現ベクターの導入法としては、 DEAE- デキストラン法 [メ ソッズ' ン' ヌクレつ ック' アシッド' リサーチ (Methods in Nucleic Acids Res. ) , CRC Press, ρ·283， 1991 ] 、 リポフエクシヨン法 [プロシーデイング'ォ ブ ·ザ ·ナショナル 'アカデミー 'ォブ ·サイエンス [Pro Natl. Acad. Sci., 84, 7413 ( 1987) ] 等があげられる。 ベクターの導入後、 培養上清中のヒト化抗体の活性は前記 1 ( 5 ) に記載の 酵素免疫測定法 （ELISA法） 等により測定することができる。 As a method for introducing an expression vector into COS-7 cells, the DEAE-dextran method [Methods in Nucleic Acids Res.], CRC Press, ρ · 283, 1991] And the Lipoff execution method [Proceeding of the National 'Academy' of Science] [Pro Natl. Acad. Sci., 84, 7413 (1987)]. After the introduction of the vector, the activity of the humanized antibody in the culture supernatant can be measured by the enzyme immunoassay (ELISA) described in 1 (5) or the like.
( 9 ) ヒト化抗体の安定 （ステーブル） 発現および活性評価  (9) Evaluation of stable (stable) expression and activity of humanized antibody
前記 2 ( 3 ) のヒト型キメラ抗体発現ベクターおよび前記 2 ( 7 ) のヒト型 CDR移植抗体発現べクターを適当な宿主細胞に導入することによりヒ卜化抗体 を安定に生産する形質転換株を得ることができる。  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.
宿主細胞への発現べクタ一の導入法としては、エレクト口ポレーション法〔特 開平 2-257891、 サイトテクノロジー （Cytotechnology) , 3, 133 (1990) 〕 等が あげられる。  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.
ヒト化抗体発現ベクターを導入する宿主細胞としては、 ヒト化抗体を発現さ せることができる宿主細胞であれば、 いかなる細胞でも用いることができる。 例えば、 マウス SP2/0- Agl4細胞 (ATCC CRL1581) 、 マウス P3X63- Ag8.653 細 胞 （ATCC CRL1580) 、 ジヒドロ葉酸還元酵素遺伝子 （以下、 DHFR遺伝子と称 す） が欠損した CHO 細胞 [プロシーディング ·ォブ ·ザ .ナショナル ·ァカデ ミー .ォブ'サイエンス （Proc. Natl. Acad. Sci.) , 77, 4216 (1980) ] 、 ラット YB2/3HL.P2.G11.16Ag.20細胞 （ATCC CRL1662、 以下、 YB2/0 細胞と称す） 等 があげられる。  As the host cell into which the humanized antibody expression vector is introduced, any cell can be used as long as it can express the humanized antibody. For example, 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) [Proceeding , The National Academy of Sciences (Proc. Natl. Acad. Sci.), 77, 4216 (1980)], Rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, Hereinafter, referred to as YB2 / 0 cells).
ベクターの導入後、 ヒト化抗体を安定に生産する形質転換株は、 特開平 2- 257891 に開示されている方法に従い、 G418および FCS を含む RPMI1640培地 により選択する。 得られた形質転換株を培地中で培養することで培養液中にヒ ト化抗体を生産蓄積させることができる。 培養液中のヒト化抗体の活性は前記 1 ( 5 )に記載の方法などにより測定する。また、形質転換株は、特開平 2-257891 に開示されている方法に従い、 DHFR遺伝子増幅系等を利用してヒト化抗体の生 産量を上昇させることができる。  After the introduction of the vector, 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. 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. In addition, 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.
ヒト化抗体は、 形質転換株の培養上清よりプロテイン A カラムを用いて精製 することができる [アンチボディ (Antibodies) , A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter s, 1988 ；以下、 「アンチボディズ」 と記す] 。 また、 その他に、 通常の蛋白質で用いられる精製方法を使用することができる.。 例え ば、 ゲル濾過、 イオン交換クロマトグラフィーおよび限外濾過等を組合せて行 い、 精製することができる。 精製したヒ卜化抗体の H鎖、 L鎖あるいは抗体分 子全体の分子量は、 ボリアクリルアミドゲル電気泳動 （SDS-PAGE) [ネィチヤ ― (Nature) , 227, 680 (1970) ] やウエスタンブロッテイング法 （アンチボデ ィズ， Chapter 12, 1988) 等で測定する。 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"]. In addition, other purification methods used for ordinary proteins can be used. For example, 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).
精製したヒト化抗体の反応性、 また、 ヒト化抗体の VEGF に対する阻害活性 の測定は前記 1 ( 4 ) に記載の方法などにより測定することができる。  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.
( 1 0 ) ヒト化抗体の使用方法  (10) How to use humanized antibodies
本発明のヒト化抗体はヒト VEGF受容体 KDR と特異的に結合し、 KDRの作 用を中和する。 このため、 本発明により提供されるヒト化抗体は固形腫瘍の増 殖もしくは転移形成、 慢性関節リウマチにおける関節炎、 糖尿病性網膜症、 未 熟児網膜症、 乾鮮など過剰な VEGF産生による血管新生の異常により病態が進 行する疾患においてその治療等に有用であると考えられる。 また、 ヒト以外の 動物の抗体に比べ、 ヒ卜抗体のアミノ酸配列に由来する部分がほとんどである ため、 ヒト体内で免疫原性を示さず、 その効果が長期間にわたり持続すること が期待される。 本発明のヒト化抗体は単独でまたは少なくとも 1 種以上の製剤 上許容される補助剤と共に用いることができる。 例えば、 ヒト化抗体を、 生理 食塩水やグルコース、 ラクト一ス、 マンニ卜一ル等の水溶液に溶解して適当な 医薬組成物とする。 または、 ヒト化抗体を常法に従って凍結乾燥し、 これに塩 化ナトリゥムを加えることによって粉末注射剤を作製する。 本医薬組成物は必 要に応じ、 製剤分野で周知の添加剤、 例えば、 製剤上許容される塩等を含有す ることができる。  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. For example, a humanized antibody is dissolved in an aqueous solution of physiological saline, glucose, lactose, mannitol, or the like to prepare a suitable pharmaceutical composition. Alternatively, 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.
本医薬組成物の投与量は、 患者の年齢、 症状等によって異なるが、 ヒ卜を含 む哺乳動物に対し、 ヒ卜化抗体を 0.1 〜20mg/kg/日投与する。投与は、 1 日 1 回 (単回投与または連日投与） または間歇的に 1 週間に 1 〜3 回、 2 、 3 週間に 1 回静脈注射により行う。 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.
3 . 抗ヒト VEGF受容体 KDR —本鎖抗体の作製  3. Production of anti-human VEGF receptor KDR-main chain antibody
( 1 ) 一本鎖抗体発現ベクターの構築  (1) Construction of single-chain antibody expression vector
前記 2 ( 2 ) 、 2 ( 5 ) および 2 ( 6 ) に記載のヒト以外の動物の抗体ある いはヒト型 CDR 移植抗体の VHおよび VLをコードする cDNAを一本鎖抗体発 現用ベクターに挿入することによりヒト以外の動物の抗体の一本鎖抗体あるい はヒト型 CDR 移植抗体の一本鎖抗体の発現べクタ一を構築することができる。 ここで用いる一本鎖抗体発現用べクタ一としてはヒ卜以外の動物の抗体あるい はヒト型 CDR 移植抗体の VHおよび VLをコードする cDNAを組込み発現でき るものであれば、 いかなるものでも用いることができる。例えば、 pAGE107 [サ ィトテクノロジ一 (Cytotechnology), 133 (1990) ] 、 pAGE103 [ジャーナル ' ォブ'バイオケミストリー（J.Biochem.)，上 01，1307 (1987)]、pHSG274 [ジーン (Gene), 27, 223(1984) ] 、 p CR [プロシ一ディング .ォブ .ザ .ナショナル ·ァカデミ 一 ·ォブ ·サイエンス (Proc.Natl.Acad.Sci.U.S.A.), 78, 1527 (1981)] 、 pSG l d2 - 4 Insert the cDNA encoding the VH and VL of the antibody of a non-human animal or the human CDR-grafted antibody according to 2 (2), 2 (5) and 2 (6) into a single-chain antibody expression vector. By doing so, it is possible to construct 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. The vector for single-chain antibody expression used herein 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. For example, pAGE107 [Cytotechnology, 133 (1990)], pAGE103 [Journal 'ob' Biochemistry (J. Biochem.), 01, 1307 (1987)], pHSG274 [Gene , 27, 223 (1984)], pCR [Proceding of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSG l d2-4
[サイトテクノロジ一 (Cytotechnology), ， 173 (1990) ] 等があげられる。 一本鎖 抗体を発現させるための宿主としては、 大腸菌、 酵母、 動物細胞等の中から適 切なものを選択することができるが、 その場合の発現用ベクターとしては、 そ れぞれの宿主に適切なものを選択する必要がある。 また、 適切なシグナルぺプ チドをコードする cDNA を発現用べクタ一に挿入することで一本鎖抗体を細胞 外に分泌させ、 ペリプラズマ領域に輸送させ、 あるいは細胞内に留まらせるこ とができる。 [Sytotechnology, 173 (1990)]. As a host for expressing a single-chain antibody, 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.
選択された発現用ベクターに、 VH—し 一 VLあるいは Vし一し — VH ( Lはぺプ チドリン力一） からなる一本鎖抗体をコードする cDNAを適切なプロモ一夕一、 シグナルぺプチドの下流に挿入することにより、 目的の一本鎖抗体をコードす る cDNAが挿入された一本鎖抗体発現べク夕一を構築することができる。 一本鎖抗体をコ一ドする cDNAは、 VHをコードする cDNAと VLをコードす る cDNA とを、 両端に適当な制限酵素の認識配列を有するペプチドリンカ一を コードする合成 DNAを用いて連結することにより得ることができる。 リンカー ペプチドは、 その付加が VH、 VL の抗原への結合に対して妨害しないように最 適化することが重要で、 例えば Pantolianoらにより示されたもの [パイオケミス トリー (Biochemistry) , 30, 10117(1991) ] あるいはそれを改変したものを用い ることができる。 Add the 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. Thus, 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. It is important that the 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.
( 2 ) 一本鎖抗体の発現および活性評価  (2) Single chain antibody expression and activity evaluation
前記 3 ( 1 ) で構築した一本鎖抗体発現ベクターをエレクト口ポレーシヨン法 [特開平 2-257891、 サイトテクノロジー (Cytotechnology), 3, 133 (1990) ] 等の 方法により適切な宿主細胞へ導入することにより、 目的の一本鎖抗体を生産す る形質転換株を取得することができる。 発現ベクターの導入後、 培養上清等に 含まれる一本鎖抗体の活性は前記 1 ( 4 ) に記載の方法等により測定すること ができる。  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.
本発明の一本鎖抗体の回収および精製は公知の技術を組み合わせることによ り達成することができる。 例えば、 一本鎖抗体が培地中に分泌されるならば、 限外濾過により濃縮することができ、 次いで抗原ァフィ二ティークロマトグラ フィーもしくはイオン交換クロマトグラフィーまたはゲル濾過を実行すること により達成することができる。 また、 宿主細胞のペリプラズマ領域へと輸送さ れるならば、 その細胞に浸透圧ショックを与え、 限外濾過により濃縮すること ができ、 次いで抗原ァフィ二ティークロマトグラフィーもしくはイオン交換ク 口マトグラフィ一またはゲル濾過を実行することにより達成することができる。 不溶性であり、 かつ顆粒 （インクルージョン .ボディ一） として存在している 一本鎖抗体は、 細胞の溶解、 顆粒を単離するための遠心分離と洗浄を繰り返し、 例えばグァニジン -塩酸による可溶化、 および再度一本鎖抗体の活性を有する 構造へと導く操作、 それに続く活性分子の精製によって達成することができる。 そして、 精製された一本鎖抗体の活性は前記 1 ( 4 ) に記載の方法等により 測定することができる。 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.
( 3 ) 一本鎖抗体の使用方法  (3) How to use single chain antibody
本発明の一本鎖抗体はヒ卜 VEGF受容体 KDR と特異的に結合し、 KDRの作 用を中和する。 このため、 本発明により提供される一本鎖抗体は、 固形腫瘍の 増殖もしくは転移形成、 慢性関節リウマチにおける関節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など過剰な VEGF産生による血管新生の異常により病態が 進行する疾患においてその治療等に有用であると考えられる。 本発明の一本鎖 抗体は単独でまたは少なくとも 1 種以上の製剤上許容される補助剤と共に用い ることができる。 例えば、 一本鎖抗体を、 生理食塩水やグルコース、 ラクト一 ス、 マンニトール等の水溶液に溶解して適当な医薬組成物とする。 または、 一 本鎖抗体を常法に従って凍結乾燥し、 これに塩化ナ卜リゥムを加えることによ つて粉末注射剤を作製する。 本医薬組成物は必要に応じ、 製剤分野で周知の添 加剤、 例えば、 製剤上許容される塩等を含有することができる。  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. For example, 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. Alternatively, 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.
本医薬組成物の投与量は、 患者の年齢、 症状等によって異なるが、 ヒトを含 む哺乳動物に対し、 一本鎖抗体を 0.1〜20mg/kg/日投与する。 投与は、 1日 1回 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
(単回投与または連日投与） または間歇的に 1週間に 1〜3回、 2、 3週間に 1回 静脈注射により行う。 (Single dose or daily dose) or intermittently by intravenous injection 1 to 3 times a week and once every 2 or 3 weeks.
4 . 抗ヒト VEGF受容体 KDR ジスルフイ ド安定化抗体の作製  4. Preparation of anti-human VEGF receptor KDR disulfide stabilized antibody
( 1 ) ジスルフィ ド安定化抗体の作製  (1) Preparation of disulfide stabilized antibody
ジスルフィ ド安定化抗体は、 ヒ卜以外の動物の抗体の VHおよび Vしをコード する cDNAあるいはヒト型 CDR移植抗体の VHおよび VLをコ一ドする cDNA のそれぞれの適切な位置の 1アミノ酸残基に相当する DNA 配列をシスティン 残基に相当する DNA 配列に改変し、 発現および精製したのち、 ジスルフィ ド結 合を形成させることで作製することができる。 ァミノ酸残基のシスティン残基 への改変は前記 2 ( 5 ) の PCR を用いた変異導入法により行うことができる。 得られた改変 VHおよび改変 Vしをコードする cDNAを適切な発現用ベクター に挿入することによりジスルフィ ド安定化抗体 H鎖発現ベクターおよびジスル フィ ド安定化抗体 L 鎖発現ベクターを構築することができる。 ここで用いるジ スルフイ ド安定化抗体発現用ベクタ一としては改変 VHおよび改変 VLをコード する cDNA を組込み発現できるものであれば、 いかなるものでも用いることが できる。例えば、 pAGE107 [サイトテクノロジ一 (Cytotechnology), 3， 133 (1990) ]、 PAGE103 [ジャーナル ·ォブ'バイオケミストリー G.Biochem.), 101, 1307 (1987)]、 PHSG274 [ジーン (Gene), ， 223(1984) ] 、 pKCR [プロシーデイング 'ォブ · ザ ' ナショナル · アカデミー · ォブ ' サイエンス（Proc.Natl.Acad.Sci.), 78, 1527(1981) ]、pSGl β d2- 4 [サイトテクノロジ一 (Cytotechnology), 4, 173 (1990) ] 等があげられる。 ジスルフィ ド安定化抗体を形成させるためにジスルフィ ド安 定化抗体 L 鎖発現ベクターおよびジスルフィ ド安定化抗体 H 鎖発現ベクター を発現させるための宿主としては、 大腸菌、 酵母、 動物細胞等の中から適切な ものを選択することができるが、 その場合の発現用べクタ一としては、 それぞ れの宿主に適切なものを選択する必要がある。 また、 適切なシグナルペプチド をコードする cDNA を発現用べクタ一に挿入することでジスルフィ ド安定化抗 体を細胞外に分泌させ、 ペリプラズマ領域に輸送させ、 あるいは細胞内に留ま らせることができる。 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. Can be prepared by modifying a DNA sequence corresponding to cysteine residue to a DNA sequence corresponding to cysteine residue, expressing and purifying, and then forming a disulfide bond. 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). By inserting the resulting cDNA encoding the modified VH and the modified VH into an appropriate expression vector, a disulfide stabilized antibody H chain expression vector and a disulfide stabilized antibody L chain expression vector can be constructed. . As the disulfide-stabilized antibody expression vector used herein, any vector can be used as long as it can incorporate and express cDNAs encoding modified VH and modified VL. For example, pAGE107 [Cytotechnology, 3, 133 (1990)], PAGE103 [Journal of Biochemistry G. Biochem., 101, 1307 (1987)], PHSG274 [Gene,, 223 (1984)], pKCR [Proceeding 'ob the' National Academy of Sciences' (Proc.Natl.Acad.Sci.), 78, 1527 (1981)], pSGl β d2-4 [site Technology (Cytotechnology), 4, 173 (1990)]. 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.
( 2 ) ジスルフイ ド安定化抗体の発現、 活性評価  (2) Expression and activity evaluation of disulfide stabilized antibodies
前記 4 ( 1 ) で構築されたジスルフィ ド安定化抗体 H 鎖発現ベクターあるい はジスルフィ ド安定化抗体し 鎖発現ベクターをエレクト口ポレーシヨン法 [特 開平 2- 25了891、 サイトテクノロジ一 (Cytotechnology), 3, 133 (1990) ] 等の方法 により宿主細胞へ導入することにより、 目的のジスルフィ ド安定化抗体 H 鎖あ るいはジスルフィ ド安定化抗体 L 鎖を生産する形質転換株を取得することがで きる。発現べクタ一の導入後、培養上清等に含まれるジスルフィ ド安定化抗体 H 鎖あるいはジスルフイ ド安定化抗体し 鎖の発現は前記 1 ( 5 ) に記載の方法等 により確認することができる。 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. After the introduction of the expression vector, 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
ジスルフィ ド安定化抗体 H 鎖あるいはジスルフィ ド安定化抗体 L 鎖の回収 および精製は公知の技術を組み合わせることにより達成することができる。 例 えば、ジスルフィ ド安定化抗体 H 鎖あるいはジスルフィ ド安定化抗体 L 鎖が培 地中に分泌されるならば、 限外濾過により濃縮することができ、 次いで各種ク 口マトグラフィ一あるいはゲル濾過を実行することにより達成することができ る。 また、 宿主細胞のペリプラズマ領域へと輸送されるならば、 その細胞に浸 透圧ショックを与え、 限外濾過により濃縮することができ、 次いで各種クロマ トグラフィ一あるいはゲル濾過を実行することにより達成することができる。 不溶性であり、 かつ顆粒 （インクル一ジョン ·ボディー） として存在している ジスルフィ ド安定化抗体 H 鎖あるいはジスルフィ ド安定化抗体し鎖は、細胞の 溶解、顆粒を単離するための遠心分離と洗浄の繰り返し、例えばグァニジン- 塩 酸による可溶化後、 各種クロマトグラフィーあるいはゲル濾過を実行すること により達成することができる。  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.
そして、精製されたジスルフィ ド安定化抗体 H鎖とジスルフィ ド安定化抗体 L 鎖を混合し、 活性を有する構造へと導く操作 [refolding 操作， モレキュラー - ィムノロジー （Molecular Immunology) , 32, 249 (1995)] によりジスルフイ ド結合 を形成させた後、 抗原ァフィ二ティークロマ卜グラフィ一もしくはイオン交換 クロマトグラフィーまたはゲルろ過により活性を有するジスルフィ ド安定化抗 体を精製することができる。 ジスルフイ ド安定化抗体の活性は前記 1 ( 5 ) に 記載の方法等により測定することができる。  Then, an operation of mixing the purified disulfide-stabilized antibody H chain and disulfide-stabilized antibody L chain to produce an active structure [refolding operation, Molecular-Immunology (Molecular Immunology), 32, 249 (1995) To form a disulfide bond, 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.
( 3 ) ジスルフイ ド安定化抗体の使用方法  (3) How to use disulfide stabilized antibodies
本発明のジスルフィ ド安定化抗体はヒト VEGF受容体 KDR と特異的に結合し、 DR の作用を中和する。 このため、 本発明により提供されるジスルフイ ド安定 化抗体は、 固形腫瘍の増殖もしくは転移形成、 慢性関節リウマチにおける関節 炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など過剰な VEGF産生による血管新 生の異常により病態が進行する疾患においてその治療等に有用であると考えら れる。 本発明のジスルフィ ド安定化抗体は単独でまたは少なくとも 1 種以上の 製剤上許容される補助剤と共に用いることができる。 例えば、 一本鎖抗体また はジスルフイ ド安定化抗体を、 生理食塩水やグルコース、 ラクトース、 マンニ トール等の水溶液に溶解して適当な医薬組成物とする。 または、 ジスルフィ ド 安定化抗体を常法に従って凍結乾燥し、 これに塩化ナトリウムを加えることに よって粉末注射剤を作製する。 本医薬組成物は必要に応じ、 製剤分野で周知の 添加剤、 例えば、 製剤上許容される塩等を含有することができる。 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. For example, 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. Alternatively, 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.
本医薬組成物の投与量は、 患者の年齢、 症状等によって異なるが、 ヒトを含 む哺乳動物に対し、 ジスルフィド安定化抗体を 0.1〜20mg/kg/日投与する。 投与 は、 1 日 1回 （単回投与または連日投与） または間歇的に 1週間に 1〜3回、 2、 3週間に 1回静脈注射により行う。 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.
5 . 抗ヒト VEGF受容体 KDR 抗体を用いたヒ卜 VEGF受容体 KDR の検出およ び定量方法  5. Detection and quantification of human VEGF receptor KDR using anti-human VEGF receptor KDR antibody
また、 本発明は、 本発明の抗体を用いて、 ヒト VEGF受容体 KDRまたはヒ卜- VEGF受容体 KDRを細胞表面に発現した細胞を免疫学的に検出および定量する 方法、 可溶性ヒト VEGF受容体 KDRを免疫学的に検出および定量する方法、 ヒ 卜 VEGFとヒト VEGF受容体 KDRとの結合を阻害する方法およびヒト VEGF受 容体 KDR作用を中和する方法に関する。  Further, 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.
本発明のモノクローナル抗体を用いて、 ヒト VEGF受容体 KDR、 ヒト VEGF 受容体 KDRを細胞表面に発現した細胞または可溶性ヒト VEGF受容体 KDRを免 疫学的に検出および定量する方法としては、 蛍光抗体法、 免疫酵素抗体法 (ELISA) 、 放射性物質標識免疫抗体法 （R1A) 、 免疫組織染色法、 免疫細胞染 色法などの免疫組織化学染色法 （ABC法、 CSA法等） 、 ウエスタンプロッティ ング法、 免疫沈降法、 上記に記した酵素免疫測定法、 サンドィツチ EL1SA 法 [単 クローン抗体実験マニュアル （講談社サイエンティフィック、 1987年） 、 続生 化学実験講座 5 免疫生化学研究法 （東京化学同人、 1986 年） ] などがあげら れる。 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)].
蛍光抗体法とは、 分離した細胞あるいは組織などに、 本発明のモノクロ一ナ ル抗体を反応させ、 さらにフルォレシン 'イソチオシァネ一卜 （FITC) などの 蛍光物質でラベルした抗ィムノグロプリン抗体あるいは結合断片を反応させた 後、 蛍光色素をフローサイトメーターで測定する方法である。  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.
免疫酵素抗体法 （ELISA) とは、 分離した、 細胞あるいはその破砕液、 組織あ るいはその破砕液、 細胞培養上清、 血清、 胸水、 腹水、 眼液などに、 本発明の 枋体を反応させ、 さらにペルォキシダーゼ、 ピオチンなどの酵素標識などを施 した抗ィムノグロブリン抗体あるいは結合断片を反応させた後、 発色色素を吸 光光度計で測定する方法である。  The immunoenzyme-linked immunosorbent assay (ELISA) 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.
放射性物質標識免疫抗体法 （R1A) とは、 分離した、 細胞あるいはその破碎液、 組織あるいはその破砕液、 細胞培養上清、 血清、 胸水、 腹水、 眼液などに、 本 発明の抗体を反応させ、 さらに放射線標識を施した抗ィムノグロプリン抗体あ るいは結合断片を反応させた後、 シンチレーシヨンカウンタ一などで測定する 方法である。  The radioactive-labeled immunoantibody method (R1A) 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.
免疫細胞染色法、 免疫組織染色法とは、 分離した、 細胞あるいは組織などに、 本発明の抗体を反応させ、 さらにフルォレシン ·イソチオシァネート （FITC) などの蛍光物質、 ペルォキシダーゼ、 ピオチンなどの酵素標識を施した抗ィム ノグロプリン抗体あるいは結合断片を反応させた後、 顕微鏡を用いて観察する 方法である。  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. This is a method in which a labeled anti-immunoglobulin antibody or a binding fragment is reacted and then observed using a microscope.
また、 固形腫瘍の増殖もしくは転移形成に基づく疾患、 異常な血管新生によ り病態が進行する疾患の診断方法としては、 被験者の細胞あるいは組織に存在 するヒト VEGF受容体 KDRを、 上述した免疫学的に検出または定量する方法が あげられる。 また、 本発明のモノクローナル抗体は、 固形腫瘍の増殖もしくは 転移形成に基づく疾患、 異常な血管新生により病態が進行する疾患の診断薬と して用いることができる。 In addition, as a method for diagnosing a disease based on the growth or metastasis of a solid tumor or a disease progressing due to abnormal angiogenesis, 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. Further, 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.
また、 ヒト VEGFとヒ卜 VEGF受容体 KDRとの結合の阻害活性は、 増殖因子 と受容体の結合測定法 生化学実験講座 7 増殖分化因子とその受容体 （東 京化学同人、 1991年） ]等の方法に準じて、 本発明の抗体を用いた VEGF- VEGF 受容体 KDR結合阻害試験を行うことにより確認することができる。  In addition, 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.
すなわち、 KDRを発現している細胞あるいは組織に放射性物質等を標識した VEGFを反応させ、 KDR発現細胞あるいは組織に結合した VEGFをシンチレ一 シヨンカウンターなどで測定する方法である。 放射性物質等を標識した VEGF と同時に本発明の抗体を反応させることで、 放射性物質等を標識した VEGFが KDRに結合するのを阻害する活性を測定することが可能である。  That is, a method in which a cell or tissue expressing KDR is reacted with VEGF labeled with a radioactive substance or the like, and VEGF bound to the cell or tissue expressing KDR is measured using a scintillation counter or the like. 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.
VEGF受容体 KDRの自己リン酸化阻害活性は、 増殖因子受容体の自己リン酸 化測定法 [続生化学実験講座 情報伝達と細胞応答 （東京化学同人、 1986年） ] 等の方法に準じて、 モノクローナル抗体を用いた、 VEGF-VEGF受容体 KDRの 自己リン酸化阻害試験を行うことにより確認することができる。  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.
すなわち、 KDRを発現している細胞あるいは組織に VEGFを反応させ、 VEGF が結合することで亢進する KDRの自己リン酸化を免疫沈降法およびウエスタン ブロッ卜法などで検出する方法である。 VEGFと同時に本発明の抗体を反応させ ることで、 VEGFが結合することにより亢進される KDRの自己リン酸化を阻害 する活性を測定することが可能である。  That is, 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.
ヒ卜 VEGFの生物活性の阻害活性は、 VEGF依存的な血管内皮細胞の増殖、 遊走、 およびチューブ形成試験 生化学実験講座 1 0 血管 （内皮と平滑筋） (東京化学同人、 1991年） ] を行うことにより確認することができる。  The inhibitory activity of the biological activity of human VEGF has been demonstrated in VEGF-dependent proliferation, migration and tube formation test of vascular endothelial cells. Biochemistry Laboratory Course 10 Blood vessels (endothelium and smooth muscle) (Tokyo Kagaku Dojin, 1991)] It can be confirmed by doing.
VEGF依存的な血管内皮細胞の増殖試験とは、 血管内皮細胞に VEGFを反応 させ、 VEGFが結合することで亢進する血管内皮細胞の増殖促進活性を細胞数を 測定する方法である。 VEGFと同時に本発明の抗体を反応させることで、 VEGF により亢進する血管内皮細胞の増殖促進活性を阻害する活性を測定することが 可能である。 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.
VEGF依存的な血管内皮細胞の遊走試験とは、 血管内皮細胞に VEGFを反応 させ、 VEGFが結合することで亢進する血管内皮細胞の遊走促進活性を顕微鏡を 用いて観察する方法である。 VEGFと同時に本発明の抗体を反応させることで、 VEGFにより亢進する血管内皮細胞の遊走促進活性を阻害する活性を測定する ことが可能である。  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. By reacting the antibody of the present invention simultaneously with VEGF, it is possible to measure the activity of inhibiting the migration promoting activity of vascular endothelial cells promoted by VEGF.
VEGF依存的な血管内皮細胞のチューブ形成試験とは、血管内皮細胞に VEGF を反応させ、 VEGFが結合することで亢進する血管内皮細胞のチューブ形成促進 活性を顕微鏡を用いて観察する方法である。 VEGFと同時に本発明の抗体を反応 させることで、 VEGFにより宂進する血管内皮細胞のチューブ形成促進活性を阻 害する活性を測定することが可能である。  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. By reacting the antibody of the present invention simultaneously with VEGF, it is possible to measure the activity of inhibiting the tube formation promoting activity of vascular endothelial cells promoted by VEGF.
さらに、 本発明は本発明の抗体を用いることを特徴とする、 血管新生異常疾 患の診断方法あるいは治療方法、 本発明の抗体を有効成分とする、 血管新生異 常疾患の診断薬あるいは治療薬に関する。  Furthermore, 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.
血管新生異常疾患とは、 固形腫瘍の増殖もしくは転移形成、 慢性関節リウマ チにおける関節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など、 過剰な VEGF で起こりうる血管新生の異常により病態が進行する疾患を示す。  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.
血管新生異常疾患の診断方法としては、 被験者の細胞あるいは組織に存在す るヒト VEGF受容体 KDRを免疫学的に検出または定量する方法があげられる。 また、 本発明の抗体は、 血管新生異常疾患の診断薬として用いることができ る。  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.
さらに、 本発明の抗体はヒト VEGFの生物活性を阻害できるため、 ヒト VEGF の KDR への結合を阻害することで、 KDR 自己リン酸化を阻害した結果、 VEGF 依存的ヒト血管内皮細胞の増殖を阻害できるため、 血管新生異常疾患の治療薬 として用いることができる。  Further, since 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.
図面の簡単な説明 第 1図 プラスミド pVL- KDR- 7N-Fcの造成工程を示した図である。 BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a view showing a construction process of plasmid pVL-KDR-7N-Fc.
第 2図 可溶性 KDR- Fc各種誘導体の模式図である。  FIG. 2 is a schematic diagram of various derivatives of soluble KDR-Fc.
第 3図 可溶性 KDR各種誘導体の模式図である。  FIG. 3 is a schematic diagram of various derivatives of soluble KDR.
第 4図 精製した可溶性 KDR-Fc各種誘導体の SDSポリアクリルアミド電気 泳動 （5 〜20 %グラジェントゲルを使用） のパターンを示した図である。 左よ り、 DR-IN-Fc, KDR - 2N- Fc、 KDR - 3N-Fc、 KDR - 4N- Fc、 KDR- 5N_Fc、 KDR- 7N - Fc、 DR-2A 1N-Fc, KDR- 4Δ 1Ν— Fc、 KDR— 5Δ 1Ν— Fc の泳動パターンをそ れぞれ示す。 還元条件下で電気泳動を行った。  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-4Δ1Δ— Fc and KDR—5Δ1Ν—The migration patterns of Fc are shown, respectively. Electrophoresis was performed under reducing conditions.
第 5図 はプレートコ一卜した可溶性ヒ卜 VEGF受容体 KDR- 7N- Fcへの'25卜 ヒト VEGFの結合に及ぼす可溶性ヒト VEGF受容体 KDR- Fc各種誘導体の阻害 効果を解析した結果を示す。 B.はプレートコートした可溶性ヒト VEGF受容体 DR-Fc各種誘導体への 125トヒト VEGFの結合を解析した結果を示す。 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.
第 6図 ヒト VEGF受容体 KDRモノクローナル抗体の可溶性ヒト VEGF受容 体 KDR-Fc各種誘導体への結合活性を解析した結果を示す。  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.
第 7図 ヒ卜 VEGF受容体 KDRモノクローナル抗体のェピト一プ部位を示す。 第 8図 ヒト VEGF受容体 KDRモノクローナル抗体の可溶性ヒ卜 VEGF受容 体 KDR-Fc各種誘導体への結合活性を解析した結果を示す。  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.
第 9図 ヒト VEGF受容体 KDRモノクローナル抗体による VEGF と可溶性 VEGF受容体 KDR- 7N-Fcおよび可溶性 VEGF受容体 Fit- 1 7Nの結合阻害効果を 解析した結果を示す。  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.
第 10図 ヒト VEGF受容体 KDRモノクロ一ナル抗体による KDR自己リン酸 化阻害活性を検討した結果を示す。  FIG. 10 shows the results of examining KDR autophosphorylation inhibitory activity by a human VEGF receptor KDR monoclonal antibody.
第 11図 ヒト VEGF受容体 KDRモノクローナル抗体による VEGF依存的な 血管内皮細胞の増殖阻害活性を検討した結果を示す。  FIG. 11 shows the results of examining the VEGF-dependent vascular endothelial cell growth inhibitory activity of a human VEGF receptor KDR monoclonal antibody.
第 12図 ヒ卜 VEGF受容体 KDRモノクローナル抗体のヒト VEGF受容体 KDR 発現細胞 N1H3T3- Fit- 1、 コントロール細胞 NlH3T3-Neo細胞、 HUVECおよび HMVECとの反応性をフローサイトメ一夕一にて解析した結果を示す。 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.
第 13 図 ヒト VEGF 受容体 KDR モノクローナル抗体の HUVEC および H VECとの反応性をフ口一サイトメ一夕一にて解析した結果を示す。  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.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
実施例 1 Example 1
1 . 抗原の調製  1. Preparation of antigen
抗原として可溶性ヒト VEGF 受容体 KDR-Fc 各種誘導体および可溶性ヒト VEGF受容体 KDR各種誘導体を以下のようにして調製した。  Various derivatives of soluble human VEGF receptor KDR-Fc and various derivatives of soluble human VEGF receptor KDR were prepared as antigens as follows.
(1)可溶性ヒ卜 VEGF受容体 KDR-7Nとヒ卜抗体 Fc領域との融合遺伝子発現べ クタ一の構築  (1) Construction of a fusion gene expression vector between soluble human VEGF receptor KDR-7N and human antibody Fc region
ヒ卜 VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜738番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片、 6アミノ酸 残基からなるリンカ一 （リンカ一 # 1) 及びヒト抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR-7N- Fcと 称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒ卜 VEGF 受容体 KDR- 7N-Fcは、 可溶性ヒ卜 VEGF受容体 KDRの細胞外領域の N末端側 から 7個のィムノグロブリン様部位及び 6アミノ酸残基からなるリンカ一 （リ ンカー # 1) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。  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.
ヒ卜 VEGF 受容体 KDR の完全長 cDNA をコードする cDNA クローン BCMGS-neo- DR(A. Sawanoら ； Cell Growth & Differentiation 7, 213 - 221 , 1996)を EcoRIで切断し、 KDRの細胞外領域及び膜結合領域をコードする約 2.8 kbの断 片を pUC18 の EcoRI 部位に組み込むことによって、 pUC- KDR を作製した。 pUC-KDRを Xhoiで切断し、 Klenow処理後、 Xbal リンカ一 （配列番号 1) を挿 入することによって pUC- KDR- Xbを作製した。 pUC- KDR- Xbの Xbaト BamHl (2.3 kbp)断片を pBluescriptll KS(+)の Xbal/BamHl部位に挿入した後、 Sphl-BamHI (5.2 kbp)断片を調製し、 SnaBI部位を含む合成リンカー（配列番号 2及び配列番号 3) を挿入し、 pBS- KDR- Xb- Sを作製した。 pBS- KDR- Xb- Sの Xbal/SnaBl (2.3 kbp) 断片、 プラスミド pAMoPRFc [T. Yagoら ； The Journal of Immunology 158，707 - 714(1997)] 上のヒ卜抗体の Fc領域をコードする SnaBl/Notl (0.7 kbp) 断片をバ キュロウィルス組み換え PVL1393プラスミドのポリヘドリン （Polyhedrin) 遺伝 子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込み、可溶性ヒト VEGF 受容体 KDR-7N とヒト抗体 Fc領域との融合遺伝子発現べクタ一PVL- KDR- 7N - Fcを構築した （第 1図） 。 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. After inserting the Xba to BamHl (2.3 kbp) fragment of pUC-KDR-Xb into the Xbal / BamHl site of pBluescriptll KS (+), Sphl-BamHI (5.2 kbp) fragment was prepared, and a synthetic linker (SEQ ID NO: 2 and SEQ ID NO: 3) containing a SnaBI site was inserted to prepare pBS-KDR-Xb-S. Xbal / SnaBl (2.3 kbp) fragment of pBS-KDR-Xb-S, SnaBl encoding the Fc region of the human antibody on plasmid pAMoPRFc [T. Yago et al., The Journal of Immunology 158, 707-714 (1997)] / Notl (0.7 kbp) fragment was incorporated into the Xbal and 3 'Notl sites downstream of the transcription start site of the polyhedrin gene of the baculovirus-recombinant PVL1393 plasmid, and the soluble human VEGF receptor KDR-7N and human Kuta base fusion gene expression of the antibody Fc region wherein any one P VL- KDR-7N - was constructed Fc (Figure 1).
(2)可溶性ヒト VEGF受容体 KDR-6Nとヒト抗体 Fc領域との融合遺伝子発現べ クタ一の構築  (2) Construction of fusion gene expression vector between soluble human VEGF receptor KDR-6N and human antibody Fc region
ヒト VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜638番目に相当する可溶性ヒト VEGF受容体 KDR断片、 6アミノ酸 残基からなるリンカ一 （リンカ一 # 1) 及びヒト抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR-6N - Fcと 称す） を発現するためのベクタ一を以下の手順で作製した。 可溶性ヒ卜 VEGF 受容体 KDR-6N- Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 6個のィムノグロブリン様部位及び 6アミノ酸残基からなるリンカ一 （リ ンカ一 # 1) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。  Soluble human VEGF receptor 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 human VEGF receptor KDR, and the amino acid sequence described in SEQ ID NO: 26 corresponding to positions 1 to 638 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.
配列番号 4 及び配列番号 5 に示した塩基配列を有するプライマー 10 pmoK pBS- DR-Xb-S (抗原の調製 (1)参照） DNA 10ng、 及び、 lOmM  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
ォチド三リン酸（deoxynucleotide triphosphates)を含む 10mM MgCl2、 0. 10 mM MgCl 2 containing deoxynucleotide triphosphates, 0.
ゼラチン溶液 100 II 1に 2.5 units Taqポリメラ一ゼを加えた。 反応は 95°Cで 5 分間の前処理した後に、 95°Cで 90秒間、 50°Cで 90秒間、 最後に 72°Cで 90秒間 のポリメラーゼ ·チェイン · リアクション （PCR) を 30回繰り返し、 DNA断片 を回収した。 本 DNA断片を EcoT22iと SnaBlで切断し、 80 bpの EcoT22I/SnaBl 断片を得た。 本 DNA 断片及び pBS-KDR- Xb-S (抗原の調製（1)参照） の EcoT22I/SnaBI (5.2 kbp) 断片を連結させ pBS- KDR(6N)L を作製した。 pBS - KDR(6N)Lの Xbal/SnaBl (2.0 kbp) 及び pAMoPRFc (抗原の調製 (1)参照） 上のヒ ト抗体の Fc領域をコードする SnaBI/Notl (0.7 kbp) をバキュロウィルス組み換 え pVL1393プラスミドのポリヘドリン （Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3'側 NoU部位に組み込み、 可溶性ヒト VEGF受容体 KDR-6Nとヒ 卜抗体 Fc領域との融合遺伝子発現べク夕一 pVL- KDR-6N- Fcを構築した。 2.5 units Taq polymerase was added to the gelatin solution 100 II1. 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. This DNA fragment is digested with EcoT22i and SnaBl, and 80 bp EcoT22I / SnaBl A fragment was obtained. This DNA fragment and the EcoT22I / SnaBI (5.2 kbp) fragment of pBS-KDR-Xb-S (see Preparation of Antigen (1)) were ligated to prepare pBS-KDR (6N) L. Recombinant Xbal / SnaBl (2.0 kbp) of pBS-KDR (6N) L and SnaBI / Notl (0.7 kbp) encoding the Fc region of the human antibody on pAMoPRFc (see Preparation of Antigen (1)) 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.
(3)可溶性ヒト VEGF受容体 KDR-5Nとヒト抗体 Fc領域との融合遺伝子発現べ クタ一の構築  (3) Construction of a fusion gene expression vector between soluble human VEGF receptor KDR-5N and human antibody Fc region
ヒ卜 VEGF受容体 KDRのシグナルぺプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒ卜 VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜518番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片、 6アミノ酸 残基からなるリンカ一 （リンカ一 # 1) 及びヒ卜抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR-5N - Fcと 称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 KDR- 5N-Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 5個のィムノグロブリン様部位及び 6アミノ酸残基からなるリンカ一 （リ ンカー # 1) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。  19 amino acids described in SEQ ID NO: 27 constituting the human VEGF receptor KDR signal peptide and soluble amino acids corresponding to amino acids 1 to 518 of the amino acid sequence described in SEQ ID NO: 26, which is the mature human VEGF receptor KDR A fusion protein consisting of a VEGF receptor 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 soluble human VEGF receptor KDR-5N-Fc A vector for expressing) was prepared by the following procedure. 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.
pUC-KDR-Xbの EcoRI/HincII (1.9 kbp) 断片及び配列番号 6及び配列番号 7 の塩基配列を有する合成リンカーをベクター pBluescriptll SK (-)の EcoRI/Notl咅 [5 位に挿入し、 pBS-KDR- 5Nを構築した。 pBS- KDR- 5Nの Xbal/SnaBl (1.9 kbp) 断片及び pAMoPRFc (抗原の調製 (1)参照） 上のヒト抗体の Fc領域をコードする SnaBI/Notl (0.7 kbp) をバキュロウィルス組み換え pVL1393プラスミドのポリ ヘドリン (Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位 に組み込み、可溶性ヒト VEGF受容体 KDR-5Nとヒト抗体 Fc領域との融合遺伝 子発現べクタ一 pVし- KDR-5N- Fcを構築した。 (4)可溶性ヒト VEGF受容体 KDR-4Nとヒト抗体 Fc領域との融合遺伝子発現べ クタ一の構築 The EcoRI / HincII (1.9 kbp) fragment of pUC-KDR-Xb and a synthetic linker having the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7 were inserted into EcoRI / Notl 咅 [position 5 of vector pBluescriptll SK (-), and pBS- KDR-5N was constructed. 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 (see Preparation of Antigen (1)) 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
ヒ卜 VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒ卜 VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜393番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片、 2アミノ酸 残基からなるリンカ一 （リンカ一 # 2) 及びヒト抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR - 4N - Fcと 称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 KDR-4N-FCは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 4個のィムノグロブリン様部位及び 2アミノ酸残基からなるリンカ一 （リ ンカ一 # 2) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。  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 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.
配列番号 8および配列番号 9に示した塩基配列を有するプライマー 10 pmoK pUC-KDR-Xb DNA 10 ng、 及び、 10 mM デォキシヌクレオチド三リン酸 Primer having the nucleotide sequence shown in SEQ ID NO: 8 and SEQ ID NO: 9 10 ng of pmoK pUC-KDR-Xb DNA 10 ng and 10 mM deoxynucleotide triphosphate
(deoxynucleotide triphosphates) を含む 10 mM MgCl2、 0.001% (W/V)ゼラチン溶 液 100 1に 2.5 units Taqポリメラーゼを加えた。 反応は 95°Cで 5分間の前処 理した後に、 95°Cで 90秒間、 50°Cで 90秒間、 最後に 72°Cで 90秒間のポリメラ ーゼ'チェイン 'リアクション （PCR) を 30回繰り返し、 DNA断片を回収した。 この DNA断片を Hindlllと Kpnlにより切断し、 520 bpの Hindin-Kpnl DNA断片 を得た。 本 DNA 断片と、 pAMoPRFc 上のヒト抗体の Fc 領域をコードする Kpnl/Notl (0.7 kbp) 断片とをベクター pAMoPRFc (抗原の調製 (1)参照） の Hindlll/Notl部位に挿入し、 pAMo- 4N- Fcを構築した。 pAMo_4N- Fcの Hindlll/Notl2.5 units Taq polymerase was added to 100 mM 10% MgCl 2 , 0.001% (W / V) gelatin solution containing (deoxynucleotide triphosphates). The reaction after previous treatment of 5 minutes at 95 ° C, 9 0 seconds 9 5 ° C, 90 seconds at 50 ° C, finally 72 ° C for 90 seconds polymerase peptidase 'chain' reaction (PCR) of Was repeated 30 times to recover a DNA fragment. This DNA fragment was digested with Hindlll and Kpnl to obtain a 520 bp Hindin-Kpnl DNA fragment. This DNA fragment and the Kpnl / Notl (0.7 kbp) fragment encoding the Fc region of the human antibody on pAMoPRFc were inserted into the Hindlll / Notl site of the vector pAMoPRFc (see (1) Preparation of antigen) to obtain pAMo-4N- Fc was constructed. Hindlll / Notl of pAMo_4N- Fc
(1 kbp) 及び pUC- KDR- Xbの Xbal/Hindlll (0.7 kbp) をバキュロウィルス組み 換え pVL1393プラスミドのポリヘドリン （Polyhedrin) 遺伝子の転写開始点の下 流 5'側 Xbai及び 3'側 Notl部位に組み込み、 可溶性ヒ卜 VEGF受容体 KDR - 4N とヒト抗体 Fc領域との融合遺伝子発現ベクター pVL- KDR-4N - Fcを構築した。 (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.
(5)可溶性ヒト VEGF受容体 KDR-3Nとヒト抗体 Fc領域との融合遺伝子発現べ クタ一の構築 (5) Expression of fusion gene between soluble human VEGF receptor KDR-3N and human antibody Fc region Construction of Kuta
ヒト VEGF受容体 KDRのシグナルぺプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜294番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片、 6アミノ酸 残基からなるリンカ一 （リンカ一 # 1) 及びヒト抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR-3N-FCと 称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒ卜 VEGF 受容体 KDR- 3N-Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 3個のィムノグロブリン様部位及び 6アミノ酸残基からなるリンカ一 （リ ンカ一 # 1) 及びヒ卜抗体 Fc領域から成る融合タンパク質に相当する。  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.
pUC-KDR-Xb (抗原の調製 (1)参照） の EcoRI/EcoT14I (1.2 kbp) 断片及び配列 番号 10及び配列番号 11の塩基配列を有する合成リンカーを pBluescriptll SK (-) の EcoRl/Notl 部位に挿入し、 pBS - KDR- 3N を構築した。 pBS- KDR- 3N の Xbal/SnaBI ( 1.2 kbp) 断片及び pAMoPRFc (抗原の調製 (1)参照） 上のヒト抗体 の Fc領域をコードする SnaBI/Notl (0.7 kbp) 断片をバキュロウィルス組み換え PVL1393プラスミドのポリヘドリン （Polyhedrin) 遺伝子の転写開始点の下流 5' 側 Xbal及び 3'側 Notl部位に組み込み、可溶性ヒ卜 VEGF受容体 KDR- 3Nとヒト 抗体 Fc領域との融合遺伝子発現べク夕一 pVL-KDR - 3N- Fcを構築した。  An EcoRI / EcoT14I (1.2 kbp) fragment of pUC-KDR-Xb (see Preparation of Antigen (1)) and a synthetic linker having the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 11 were added to the EcoRl / Notl site of pBluescriptll SK (-). After insertion, pBS-KDR-3N was constructed. 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 (see Preparation of Antigen (1)) were ligated to the baculovirus recombinant PVL1393 plasmid. A fusion gene expression of soluble human VEGF receptor KDR-3N and human antibody Fc region, integrated into the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the polyhedrin gene (pVL-KDR) -Constructed 3N-Fc.
(6)可溶性ヒト VEGF受容体 KDR-2Nとヒト抗体 Fc領域との融合遺伝子発現べ クタ一の構築  (6) Construction of a fusion gene expression vector of soluble human VEGF receptor KDR-2N and human antibody Fc region
ヒト VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜194番目に相当する可溶性ヒト VEGF受容体 KDR断片、 6アミノ酸 残基からなるリンカ一 （リンカ一 # 1) 及びヒ卜抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR - 2N-Fcと 称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 KDR-2N- Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 2個のィムノグロブリン様部位及び 6アミノ酸残基からなるリンカ一 （リ ンカー # 1 ) 及びヒ卜抗体 Fc領域から成る融合タンパク質に相当する。 A 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 194 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) Was prepared by the following procedure. 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.
pUC- DR-Xb (抗原の調製 (1)参照） の EcoRI/VspI (0.9 kbp) 断片及び配列番 号 12及び配列番号 13の塩基配列を有する合成リンカ一を pBluescriptll SK (-)の EcoRI/Notl部位に挿入し、 pBS- KDR- 2Nを構築した。 pBS- KDR- 2Nの Xbal/SnaBl An EcoRI / VspI (0.9 kbp) fragment of pUC-DR-Xb (see Preparation of Antigen (1)) and a synthetic linker having the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13 were digested with EcoRI / Notl of pBluescriptll SK (-). This was inserted into the site to construct pBS-KDR-2N. Xbal / SnaBl of pBS-KDR-2N
(0.9 kbp) 断片及び pAMoPRFc (抗原の調製 (1)参照） 上のヒト抗体の Fc領域を コードする SnaBl/Notl (0.7 kbp) をバキュロウィルス組み換え pVL1393プラス ミドのポリヘドリン （Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3' 側 Notl部位に組み込み、 可溶性ヒト VEGF受容体 KDR-2Nとヒト抗体 Fc領域 との融合遺伝子発現べクタ一 pVL-KDR-2N-Fcを構築した。 (0.9 kbp) Fragment and pAMoPRFc (see Preparation of Antigen (1)) SnaBl / Notl (0.7 kbp) encoding the Fc region of human antibody on baculovirus recombinant pVL1393 plasmid Polyhedrin (Polyhedrin) gene transcription start point And a fusion gene expression vector, pVL-KDR-2N-Fc, of the soluble human VEGF receptor KDR-2N and the human antibody Fc region was constructed by incorporation into the downstream 5 ′ Xbal and 3 ′ Notl sites.
(7)可溶性ヒト VEGF受容体 KDR-1Nとヒト抗体 Fc領域との融合遺伝子発現べ クタ一の構築  (7) Construction of fusion gene expression vector of soluble human VEGF receptor KDR-1N and human antibody Fc region
ヒト VEGF受容体 KDRのシグナルぺプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜104番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片、 6アミノ酸 残基からなるリンカ一 （リンカ一 # 1) 及びヒト抗体 Fc領域を構成する 227ァ ミノ酸から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR-IN-Fcと 称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 KDR- IN- Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 1個のィムノグロブリン様部位及び 6 アミノ酸残基からなるリンカ一 （リ ンカー # 1 ) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。  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.
PBS- DR-2 (抗原の調製 (6)参照) の Bglll/ otl (2.8 kbp) 断片に、配列番号 14及び配列番号 15の塩基配列を有する合成リンカ一を連結し、 pBS- KDR- 1 Nを 構築した。 pBS- KDR- の Xbal/SnaBI (0.4 kbp) 断片及び pAMoAPRFc (抗原の 調製 (1)参照） 上のヒ卜抗体の Fc領域をコードする SrmBl/Notl (0.7 kbp) をパキ ュロウィルス組み換え pVL 1393プラスミドのポリヘドリン （Polyhedrin) 遺伝子 の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込み、 可溶性ヒ卜 VEGF 受容体 KDR- 1 N とヒト抗体 Fc領域との融合遺伝子発現べクタ一 pVL- KDR- 1N- Fcを構築した。 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. Recombinant expression of fusion gene between soluble human VEGF receptor KDR-1N and human antibody Fc region by integrating into the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the polyhedrin (Polyhedrin) gene of the pulo1393 plasmid recombinant with the urovirus The vector pVL-KDR-1N-Fc was constructed.
(8)可溶性ヒ卜 VEGF受容体 KDR-7D 1 Nとヒト抗体 Fc領域との融合遺伝子発 現ベクターの構築  (8) Construction of fusion gene expression vector of soluble human VEGF receptor KDR-7D1N and human antibody Fc region
可溶性ヒト VEGF受容体 KDR- 7N- Fc (抗原の調製 (1)参照） から、 N末端側か ら 1番目のィムノグロプリン様部位を形成する 31番目のアミノ酸から 102番目 のァミノ酸までの計 72個のァミノ酸を欠失させた KDR断片、 6ァミノ酸残基か らなるリンカ一 （リンカ一 # 1 ) 及びヒト抗体 Fc領域を構成する 227アミノ酸 から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR-7 Δ 1Ν- Fcと称 す） を発現するためのベクタ一を以下の手順で作製した。 可溶性ヒト VEGF受 容体 KDR- 7D 1 N- Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側 から 2〜7番目のィムノグロプリン様部位及び 6ァミノ酸残基からなるリンカ一 (リンカー # 1 ) 及びヒト抗体 Fc領域から成る融合タンパク質に相当すろ。 配列番号 16及び配列番号 17に示した塩基配列を有するプライマー 10 pmoK PVL- DR-7N (抗原の調製 (14)参照） DNA 10 ng、 及び、 10 mM デォキシヌクレ ォチド三リン酸（deoxynucleotide triphosphates)を含む 10 mM MgCl2、 0.001% (W/V) ゼラチン溶液 100 H 1に 2.5 units Taqポリメラ一ゼを加えた。 反応は 95°Cで 5 分間の前処理した後に、 95°Cで 90秒間、 50°Cで 90秒間、 最後に 72°Cで 90秒間 のポリメラーゼ ·チェイン · リアクション （PCR) を 30回繰り返し、 DNA断片 を回収した。 本 DNA断片を Xbalと Bglllで切断し、 0.8 kbpの Xbal/Bglll断片を 得た。本 D A断片及び pVL-KDR-5N (抗原の調製 ( 17)参照)の Bglll/Notl ( 1 .6 kbp) 断片を pBluescriptll SK (-)の Xbal/Notlに挿入し、 pBS- KDR- 5 Δ 1 Νを作製した。 pBS-KDR-5 Δ 1 Νの Xbal/Hincn ( l .6kbp) 断片及び pVL- KDR- 7N_Fc (抗原の調 製 (1)参照） の Hincll/Notl ( 1.2 kbp) をバキュロウィルス組み換え pVL1393プラ スミドのポリヘドリン （Polyhedrin)遺伝子の転写開始点の下流 5'側 Xbal及び 3' 側 Notl部位に組み込み、 可溶性ヒト VEGF受容体 KDR- 7D 1Nとヒト抗体 Fc領 域との融合遺伝子発現べクタ一 pVL- KDR- 7 Δ ΙΝ-Fcを構築した。 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 -7Δ1Ν-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. 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 Ν was prepared. The Xbal / Hincn (1.6 kbp) fragment of pBS-KDR-5 Δ1Ν and the Hincll / Notl (1.2 kbp) of pVL-KDR-7N_Fc (see Antigen Preparation (1)) 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 Δ ΙΝ-Fc.
(9)可溶性ヒト VEGF受容体 KDR- 5 ΔΐΝとヒト抗体 Fc領域との融合遺伝子発 現ベクターの構築  (9) Construction of fusion gene expression vector of soluble human VEGF receptor KDR-5ΔΐΝ and human antibody Fc region
可溶性ヒト VEGF受容体 KDR-5N- Fc (抗原の調製 (3)参照） から、 N末端側か ら 1番目のィムノグロブリン様部位を形成する 31番目のアミノ酸から 102番目 のアミノ酸までの計 72個のアミノ酸を欠失させた KDR断片、 6アミノ酸残基か らなるリンカ一 （リンカ一 # 1) 及びヒト抗体 Fc領域を構成する 227アミノ酸 から成る融合タンパク質 （以下、 可溶性ヒト VEGF受容体 KDR- 5 ΔΐΝ-Fcと称 す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF受 容体 KDR- 5 ΔΙΝ-Fcは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端 側から 2〜5番目のィムノグロプリン様部位及び 6ァミノ酸残基からなるリンカ 一 （リンカ一 # 1) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。  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 ΔΐΝ-Fc) was prepared by the following procedure. Soluble human VEGF receptor KDR-5 ΔΙΝ-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.
PBS-KDR-5D1N (抗原の調製 (8)参照） の Xbal/Notl ( 1.4 kbp ) 断片及び pAMoAPRFc (抗原の調製 (1)参照） 上のヒト抗体の Fc 領域をコードする SnaBl/Notl (0.7 kbp) をバキュロウィルス組み換え pVL1393プラスミドのポリ ヘドリン （Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位 に組み込み、可溶性ヒト VEGF受容体 KDR- 5 Δ ΐΝとヒト抗体 Fc領域との融合 遺伝子発現ベクター pVL- KDR- 5 ΔΙ -Fcを構築した。  SnaBl / Notl (0.7 kbp) encoding the Xbal / Notl (1.4 kbp) fragment of PBS-KDR-5D1N (see Antigen Preparation (8)) and the Fc region of the human antibody on pAMoAPRFc (see Antigen Preparation (1)) ) Was inserted into the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the polyhedrin (Polyhedrin) gene of the baculovirus recombinant pVL1393 plasmid, and the soluble human VEGF receptor KDR-5ΔΐΝ and the human antibody Fc region were A fusion gene expression vector pVL-KDR-5ΔΙ-Fc was constructed.
(10)可溶性ヒト VEGF受容体 KDR- 4 Δ ΐΝとヒト抗体 Fc領域との融合遺伝子 発現べクタ一の構築  (10) Construction of fusion gene expression vector of soluble human VEGF receptor KDR-4Δ Δ and human antibody Fc region
可溶性ヒト VEGF受容体 KDR-4N- Fc (抗原の調製 (4)参照） から、 ヽ :末端側か ら 1番目のィムノグロプリン様部位を形成する 31番目のアミノ酸から 102番目 のアミノ酸までの計 72個のアミノ酸を欠失させた KDR断片、 6アミノ酸残基か らなるリンカ一 （リンカ一 # 2) 及びヒ卜抗体 Fc領域を構成する 227アミノ酸 から成る融合夕ンパク質（以下、可溶性ヒ卜 VEGF受容体 KDR-4D 1N-FCと称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF受容体 DR-4 ΔΐΝ-Fcは、可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側から 2〜4番目のィムノグロブリン様部位及び 2ァミノ酸残基からなるリンカ一 （リ ンカ一 # 2) 及びヒト抗体 Fc領域から成る融合タンパク質に相当する。 From the soluble human VEGF receptor KDR-4N-Fc (see Antigen Preparation (4)), ヽ: 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 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 ΔΐΝ-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.
抗原の調製 (8)で回収した Xbal/Bglll- PCR断片（0.8 kbp)及び pVL- KDR- 4N (抗 原の調製 (18)参照） の Bglll/Notl (0.9 kbp) 断片を pBluescriptll SK (-)の Xbal/Notl に挿入し、 pBS- KDR- 4 △ INを作製した。 pBS- KDR - 4 ΔΐΝの Xbal/Kpnl (1.0 kbp) 断片及び pAMoAPRFc (抗原の調製 (1)参照） 上のヒト抗体の Fc領域をコードす る SnaBI/Notl (0.7 kbp) をバキュロウィルス組み換え pVL1393プラスミドのポ リヘドリン (Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3，側 Notl部 位に組み込み、可溶性ヒ卜 VEGF受容体 KDR-4 ΔΐΝとヒト抗体 Fc領域との融 合遺伝子発現ベクター pVL-KDR- 4 ΔΙΝ-Fcを構築した。  Preparation of Antigen The Xbal / Bglll-PCR fragment (0.8 kbp) recovered in (8) and the Bglll / Notl (0.9 kbp) fragment of pVL-KDR-4N (see Preparation of antigen (18)) were converted into pBluescriptll SK (-). Into Xbal / Notl of pBS-KDR-4ΔIN. Baculovirus recombinant pVL1393 plasmid with Xbal / Kpnl (1.0 kbp) fragment of pBS-KDR-4ΔΐΝ 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-4ΔΐΝ 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 ΔΙΝ-Fc was constructed.
(11)可溶性ヒト VEGF受容体 KDR- 7N発現ベクターの構築  (11) Construction of soluble human VEGF receptor KDR-7N expression vector
ヒ卜 VEGF受容体 KDRのシグナルぺプチドを構成する配列番号 27記載の 19 ァミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜738番目に相当する可溶性ヒト VEGF受容体 KDR断片 （以下、 可溶 性ヒ卜 VEGF受容体 KDR-7Nと称す） 及びリンカ一由来の 2アミノ酸残基を発 現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 DR-7Nは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側から Ί個の ィムノグロプリン様部位に相当する。  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 Ί immunoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR.
pBS- DR-Xb-S (抗原の調製 (1)参照） を SnaBl/BamHI で切断し、 終止コドン と Notl部位とを含む合成リンカー （配列番号 18及び配列番号 19) を組み込み、 pBS- KDR(Xb)- S- Nを作製した。 pBS- KDR- Xb- S- Nの Xba卜 N。U (2.3 kb) 断片を パキュロウィルス組み換え PVL1393プラスミドのポリヘドリン （Polyhedrin) 遺 伝子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込み、 可溶性ヒ卜 VEGF受容体 KDR- 7N発現ベクター pVL- KDR- 7Nを作製した。 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. XbaN of pBS-KDR-Xb-SN. 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.
(12)可溶性ヒ卜 VEGF受容体 KDR- 7N ' 発現ベクターの構築  (12) Construction of expression vector for soluble human VEGF receptor KDR-7N '
ヒト VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒ卜 VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1 ~714番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片 （以下、 可溶 性ヒ卜 VEGF受容体 KDR- 7N ' と称す） を発現するためのベクターを以下の手順 で作製した。 可溶性ヒト VEGF受容体 KDR- 7N ' は、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側から 7偭目のィムノグロプリン様部位の約 2/3ま でに相当する。  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を Stul及び Sphlで切断し、終止コドン及び Notl部位を含む合成 リンカ一 （SB列番号 23及び配列番号 21) を挿入した。 Xba卜 Notl (2.2 kbp) 断 片をバキュロウィルス組み換え pVL1393プラスミドのポリヘドリン（Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込み、可溶性ヒト VEGF受容体 KDR- 7N ' 発現べクタ一 pVL- KDR- 7N ' を作製した。  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.
(13)可溶性ヒ卜 VEGF受容体 KDR-5N発現ベクターの構築  (13) Construction of soluble human VEGF receptor KDR-5N expression vector
ヒト VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜518番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片 （以下、 可溶 性ヒ卜 VEGF受容体 KDR- 5N と称す） を発現するためのベクターを以下の手順 で作製した。可溶性ヒト VEGF受容体 KDR- 5Nは、可溶性ヒト VEGF受容体 KDR の細胞外領域の N末端側から 5個のィムノグロプリン様部位に相当する。  Soluble human VEGF receptor corresponding to the 19 amino acids described in SEQ ID NO: 27 and the mature human VEGF receptor KDR that constitute the signal peptide of human VEGF receptor KDR, and the amino acid sequence described in SEQ ID NO: 26 corresponding to positions 1 to 518 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.
pUC-KDR-Xb ( EcoRl-HincII ( 1.9 kb) 断片及び SnaBl部位、 終止コドン、 otl 部位を含む合成 D A (配列番号 22 及び配列番号 23) を pBluescriptll SK (-)の EcoRI/Notl部位に挿入し、 pBS-KDR- 5Nを作製した。 pBS- KDR- 5Nの Xba卜 Notl ( 1.6 kb)断片をバキュロウィルス組み換え pVL1393プラスミドのポリへドリン (Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込 み、 可溶性ヒト VEGF受容体 KDR- 5N発現ベクター pVし- KDR-5Nを作製した。 Insert pUC-KDR-Xb (EcoRl-HincII (1.9 kb) fragment and synthetic DA (SEQ ID NO: 22 and SEQ ID NO: 23) containing SnaBl site, stop codon, and otl site) into the EcoRI / Notl site of pBluescriptll SK (-). The Xbanot Notl (1.6 kb) fragment of pBS-KDR-5N was ligated to the baculovirus recombinant pVL1393 plasmid at the 5'-end Xbal and 3 'downstream of the transcription start point of the polyhedrin (Polyhedrin) gene. 'Side Notl site Thus, a soluble human VEGF receptor KDR-5N expression vector pV-KDR-5N was prepared.
(14)可溶性ヒ卜 VEGF受容体 KDR-4N発現べクタ一の構築  (14) Construction of vector expressing soluble human VEGF receptor KDR-4N
ヒ卜 VEGF受容体 KDRのシグナルべプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒ卜 VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜393番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片 （以下、 可溶 性ヒト VEGF受容体 KDR-4Nと称す） 及びリンカ一由来の 2アミノ酸残基を発 現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 DR-4 は、 可溶性ヒ卜 VEGF受容体 KDRの細胞外領域の N末端側から 4個の ィムノグロプリン様部位に相当する。  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.
pAMo-4N-Fc (抗原の調製 (4)参照） の Xbal/Kpnl (1.2 kb) 断片及び配列番号 24及び配列番号 25の塩基配列を有する合成リンカーをバキュロウィルス組み換 え pVL1393プラスミドのポリヘドリン （Polyhedrin) 遺伝子の転写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込み、 可溶性ヒ卜 VEGF受容体 KDR- 4N発現 ベクター pVL_KDR- 4Nを作製した。  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.
(15)可溶性ヒ卜 VEGF受容体 KDR - 3N発現ベクターの構築  (15) Construction of soluble human VEGF receptor KDR-3N expression vector
ヒ卜 VEGF受容体 KDRのシグナルぺプチドを構成する配列番号 27記載の 19 アミノ酸及び成熟体ヒト VEGF受容体 KDRである、 配列番号 26記載のァミノ 酸配列 1〜294番目に相当する可溶性ヒ卜 VEGF受容体 KDR断片 （以下、 可溶 性ヒト VEGF受容体 KDR- 3Nと称す） 及びリンカ一由来の 2アミノ酸残基を発 現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF 受容体 DR-3Nは、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側から 3個の ィムノグロプリン様部位に相当する。  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.
PBS- DR-3 (抗原の調製 (5)参照) の Xbal/SnaBl ( 1.2 kb) 断片、 配列番号 18 及び配列番号 19の塩基配列を有する合成リンカ一をバキュロウィルス組み換え PVL1393プラスミドのポリヘドリン （Polyhedrin) 遺伝子の転写開始点の下流 5' 側 Xbal及び 3'側 Bglll部位に組み込み、 可溶性ヒ卜 VEGF受容体 KDR - 3N発現 ベクタ一 pVL-KDR- 3Nを作製した。 Xbal / SnaBl (1.2 kb) fragment of PBS-DR-3 (Preparation of antigen (5)), a synthetic linker having the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19 was baculovirus-recombinant. Polyhedrin of PVL1393 plasmid Integrates into the 5 'Xbal and 3' Bglll sites downstream of the gene transcription start site, and expresses soluble human VEGF receptor KDR-3N The vector pVL-KDR-3N was prepared.
(16)可溶性ヒ卜 VEGF受容体 KDR- 7 Δ ΐΝ発現べクタ一の構築  (16) Construction of soluble human VEGF receptor KDR-7ΔΐΝ expression vector
可溶性ヒ卜 VEGF受容体 KDR-7ヽ' （抗原の調製 (14)参照） から、 ヽ末端側から 1番目のィムノグロブリン様部位を形成する 31番目のアミノ酸から 102番目の アミノ酸までの計 72個のアミノ酸を欠失させた KDR断片、 6アミノ酸残基から なるリンカ一 （リンカ一 # 1 ) 及びヒト抗体 Fc領域を構成する 227アミノ酸か ら成る融合タンパク質 （以下、 可溶性ヒ卜 VEGF受容体 KDR- 7 Δ1Ν と称す） を発現するためのベクターを以下の手順で作製した。 可溶性ヒト VEGF受容体 KDR-7 Δ 1 は、 可溶性ヒト VEGF受容体 KDRの細胞外領域の N末端側から 2 〜7番目のィムノグロプリン搽部位及び 6アミノ酸残基からなるリンカー（リン カー # 1) から成る融合タンパク質に相当する。  From the soluble human VEGF receptor KDR-7 ヽ '(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 ヽ -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 Δ1Ν) was prepared by the following procedure. The soluble human VEGF receptor KDR-7Δ1 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.
pBS-KDR-δ Δ 1Ν (抗原の調製 (9)参照） の Xbal/Hincll ( 1.6 kbp) 断片及び PVL-KDR-7 (抗原の調製 (14)参照） の HincII/Notl (0.67 kbp) 断片をバキュ口 ウィルス組み換え PVL1393プラスミドのポリヘドリン （Polyhedrin) 遗伝子の転 写開始点の下流 5'側 Xbal及び 3'側 Notl部位に組み込み、可溶性ヒト VEGF受容 体 KDR- 7D 1\発現べクタ一 pVし- KDR- 7D 1Nを作製した。  The Xbal / Hincll (1.6 kbp) fragment of pBS-KDR-δΔ1Ν (see antigen preparation (9)) and the HincII / Notl (0.67 kbp) fragment of PVL-KDR-7 (see antigen preparation (14)) Bacchus virus recombinant Polyhedrin of the PVL1393 plasmid (Polyhedrin) 下流 Integrates into the 5 'Xbal and 3' Notl sites downstream of the transcription start site of the gene and expresses the soluble human VEGF receptor KDR-7D1 \ -KDR-7D 1N was prepared.
(17)昆虫細胞による可溶性ヒト VEGF受容体 KDR発現を行うための組み換え ウィルスの作製  (17) Production of recombinant virus for expression of soluble human VEGF receptor KDR by insect cells
昆虫細胞による蛋白質の生産には目的遗伝子を組み込んだ組み換えウィルス の作製が必要であるが、 その作製にはトランスファーベクターと呼ばれる目的 蛋白質をコードする cDXA を特殊なプラスミドに組み込む過程と野生型ウィル スとトランスファーベクタ一を昆虫細胞にコトランスフエクシヨンし、 相同組 み換えにより組み換えウィルスを取得する過程を経る。 以上の過程についてフ ァーミンジェン社製バキュロゴ一ルドスター夕一キッ ト （製品番号 PM- 21001 K ) を用いてそのマニュアルに従い以下の手順で行った。  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).
TMヽ- FH インセクトメディウ厶 （ファーミンジェン社製） にて培養した昆虫 細胞 Sf9 (ファーミンジェン社製） に線状バキュロウィルス DNA [バキュロゴ 一ルド ·バキュロウィルス DNA(BaculoGold baculovirus DNA) 、 ファーミンジェ ン社製] および作製したトランスファーベクタ一DNA をリポフエクチン法にて 導入すること [蛋白質核酸酵素、 ^ , 2701 (1992)] により行い組み換えバキュ口 ウィルスを以下のように作製した。 Insects cultured on TM ヽ -FH Insect Medium (Pharmingen) Transfection of linear baculovirus DNA (BaculoGold baculovirus DNA, Pharmingen) and the prepared transfer vector DNA into cells Sf9 (Pharmingen) by the lipofectin method. [Protein Nucleic Acid Enzyme, ^, 2701 (1992)] to produce a recombinant baculovirus as follows.
(1)で作製した発現べクタ一の 1 H gと線状バキュロウィルス DNA の 20ngと を 12 / 1の蒸留水に溶解し、 さらにリポフエクチン 6 1と蒸留水 6 1とを混 和したものを加え室温で 15分間放置した。 一方 Sf9 細胞 1 X 106個を 2ml の S1 00-11培地 [ギブコ (Gibco) 社製] に懸濁し、 直径 35mmの細胞培養用プラス チックシャーレに入れた。 ここに上記のプラスミド DNA、 線状バキュ口ウィル ス DNA およびリポフエクチン混和溶液全量を加え 27°Cで 3 日間培養後、 組み 換えウィルスを含む培養上清 1mlを採取した。シャーレには新たに Sf900-II培地 lmlを加え、さらに 27°Cで 3日間培養し組み換えウィルスを含む培養上清をさら に 1.5ml得た。 さらに、 （2)〜(16)で作製した発現ベクターを用い同様の操作を行 つた。 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. On the other hand, 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. To the Petri dish, 1 ml of Sf900-II medium was newly added, and the mixture was further cultured at 27 ° C for 3 days to obtain 1.5 ml of a culture supernatant containing the recombinant virus. Further, the same operation was performed using the expression vectors prepared in (2) to (16).
次に蛋白質発現に用いるために得られた組み換えウィルスを各々、 以下の手 順で増殖させた。  Next, each of the recombinant viruses obtained for use in protein expression was propagated by the following procedure.
Sf9細胞 2 X 107個を 10mlの Sf900- II培地に懸濁し、 175cm2フラスコ （グライナ 一社製） に入れて室温で 1 時間放置して細胞をフラスコに付着させた。 放置後 上清を除き新たに 15mlの TMN- FHインセクトメディゥムと上記の組み換えウイ ルスを含む培養上清のうち lml を加え 27°Cで 3 日間培養した。 培養後上清を l，500 X gで 10分間遠心分離して細胞を除き、 蛋白質発現に使用する組み換えゥ ィルス溶液を得た。 2 × 10 7 Sf9 cells were suspended in 10 ml of Sf900-II medium, placed in a 175 cm 2 flask (manufactured by Grina) and allowed to stand at room temperature for 1 hour to allow the cells to adhere to the flask. After standing, the supernatant was removed, and 1 ml of a new culture supernatant containing 15 ml of TMN-FH Insect Medium and the above recombinant virus was added, followed by culturing at 27 ° C for 3 days. After the culture, the supernatant was centrifuged at 1,500 × g for 10 minutes to remove the cells, thereby obtaining a recombinant virus solution used for protein expression.
得られた組み換えウィルス溶液についてウィルスの力価をバキュ口ゴールド スター夕一キット 'マニュアル （ファーミンジェン社製） に記載の方法で算定 した。 Sf9細胞 6 X 10s個を 4mlの Sf900-Ii培地に懸濁し、 直径 60mmの細胞培養用プ ラスチックシャーレに入れ、 室温で 1 時間放置して細胞をシャーレに付着させ た。次に上清を除き新たに Sf900- Π培地 400 II 1と Sf900- 11培地で 1000倍に希釈 した上記組み換えウィルス溶液を加え室温で 1 時間放置した後、培地を除き 5ml の 1%低融点ァガロース [ァガープラーク ·ァガロース (Agarplaque Agarose), フ ァ一ミンジェン社製] を含む培地 [滅菌した lml の 5%ァガ一プラークプラス - ァガロース水溶液と 4mlの TMN- FHィンセクトメディゥムを混和し、 42°Cに保 温したもの] を該シャーレに流し込んだ。 室温で 15分間放置した後、 乾燥を防 ぐためビニルテープをシャーレにまき、 密閉可能なプラスチック製容器に該シ ャ一レを入れ、 27°Cで 6 日間培養した。 該シャーレに 0.01% ニュートラルレツ ドを含む PBS lmlを加えさらに 1 日培養した後、 出現したプラークの数を数え た。 以上の操作より該組み換えウィルス溶液はいずれも約 1 X 107プラークフォ 一ミングユニット （以下、 PFU と称す） /ml のウィルスを含んでいることがわ かった。 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.
(18) 昆虫細胞における可溶性ヒト VEGF受容体 KDR- Fc各種誘導体、 およ び、 可溶性ヒト VEGF受容体 KDR各種誘導体の発現、 精製  (18) Expression and purification of various soluble human VEGF receptor KDR-Fc derivatives and various soluble human VEGF receptor KDR derivatives in insect cells
1 (1) 〜 （16) で示した可溶性ヒト VEGF受容体 KDR-Fc各種誘導体、 およ び、可溶性ヒト VEGF受容体 KDR各種誘導体は以下のようにして得た。 High Five 細胞 4 X 個を 175cm2フラスコ（グライナ一社製）中の EX- CELL™400培地 (JRH Bioscience社製） 30mlに懸濁し、 室温で 1時間放置し、 フラスコに付着させた。 1 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 ™ 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.
(1) 〜 （16) で得られたトランスファーベクター由来の組み換えウィルスを約 1 〜3 X 108PFU /ml の濃度で含む溶液を lml加え、 室温で 2時間感染させた。 培養 上清を除き新たに 30mlの EX- CELL™400培地 30mlを加え 27°Cにて 3〜4日間培 養した。 培養終了後、 培養上清を回収し 1，500 X gで 10分間遠心分離を行い上 清を得た。 1 ml of a solution containing the recombinant virus derived from the transfer vector obtained in (1) to (16) at a concentration of about 1 to 3 × 10 8 PFU / ml was added, and the cells were infected at room temperature for 2 hours. The culture supernatant was removed and a fresh 30 ml of EX-CELL ™ 400 medium was added, followed by culturing at 27 ° C for 3 to 4 days. After completion of the culture, the culture supernatant was recovered and centrifuged at 1,500 X g for 10 minutes to obtain a supernatant.
可溶性ヒ卜 VEGF受容体 KDR- Fc各種誘導体については、 プロセップ Aカラ ムを用いて以下のように精製した。 For soluble human VEGF receptor KDR-Fc derivatives, Prosep A Purification was carried out as follows using a system.
カラムに約 lmlのプロセップ A [Bioprocessing社製] を充填し、 10mlの 20 mM リン酸ナトリウム緩衝液 （pH7.2) を用いて lml/分の流速でカラムを洗浄した。 洗浄後、 上記のように調整した可溶性ヒ卜 VEGF受容体 KDRを含む培養液 500 〜1000mlを 100ml/時の流速でプロセップ Aカラムに通塔した。 さらに 10mlの 20 m リン酸ナトリゥム緩衝液 （pH7.2) を用いて lml/分の流速で洗浄した後、 50mMクェン酸緩衝液 （pH3) を 7ml通塔し、 プロセップ Aカラムに吸着した蛋 白質の溶出を行った。 各分画に含まれる蛋白を SDSポリアクリルアミドゲル電 気泳動 (SD S-PAGE)にて解析した。  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. After washing with 10 ml of 20 m sodium phosphate buffer (pH 7.2) at a flow rate of 1 ml / min, 7 ml of 50 mM citrate buffer (pH 3) was passed, and the protein adsorbed on the Prosep A column was washed. Was eluted. The protein contained in each fraction was analyzed by SDS polyacrylamide gel electrophoresis (SD S-PAGE).
可溶性ヒト VEGF受容体 KDR各種誘導体については、 以下のように精製した。  Various derivatives of the soluble human VEGF receptor KDR were purified as follows.
50mlの DEAE-Sepharose CL-6B(Pharmacia Biotech社製)を充填した力ラムが液 の入口側に、 40mlの Heparin Sepharose CL-6B (Pharmacia Biotech社製)を充填した カラムが出口側になるように直列に接続し、 300mlの 20mMりん酸ナトリウム緩 衝液 (pH8)で洗浄した。 洗浄後、 可溶性ヒ卜 VEGF受容体 KDRを含む培養液 400 〜800mlを 50〜 100ml/時の流速で通塔した。 更に、 300mlの 20mMりん酸ナトリ ゥム緩衝液 (pH8)で洗浄した後、 Heparin Sepharose CL-6Bカラムのみに 400mlの 0〜1M NaCl/20mMりん酸ナトリゥム緩衝液にて連続濃度勾配をかけ、 吸着蛋白 質の溶出を行った。 溶出液は 7mlずつ分画し、 各分画に含まれる蛋白質を SDS- PAGEにて解析し、可溶性ヒト VEGF受容体 KDRを含む分画を 60〜80ml回収し た。 回収した精製画分はセントリブレップ 10 (アミコン社製） を用いて濃縮し、 可溶性ヒト KDR3N、 KDR4N、 KDR5N、 KDR7 ' および KDR7Nを溶液としてそ れぞれ 2.8ml, 8ml、 5.5mK 4mlおよび 4.8ml (蛋白質濃度/純度は 345.5 ll g/ml/30%, 264 g/ml/50〜60%、 380.5 fl g/ml/70 , 1.59mg/ml/60%および 815 g/ml/70 -80%) 得た。  The force ram filled with 50 ml of DEAE-Sepharose CL-6B (Pharmacia Biotech) is on the inlet side of the solution, and the column filled with 40 ml of Heparin Sepharose CL-6B (Pharmacia Biotech) is on the outlet side. They were connected in series and washed with 300 ml of a 20 mM sodium phosphate buffer solution (pH 8). After washing, 400 to 800 ml of a culture solution containing soluble human VEGF receptor KDR was passed at a flow rate of 50 to 100 ml / hour. Furthermore, after washing with 300 ml of 20 mM sodium phosphate buffer (pH 8), a continuous concentration gradient is applied only to the Heparin Sepharose CL-6B column with 400 ml of 0 to 1 M NaCl / 20 mM sodium phosphate buffer, followed by adsorption. The protein was eluted. The eluate was fractionated by 7 ml, and the protein contained in each fraction was analyzed by SDS-PAGE, and 60 to 80 ml of the fraction containing the soluble human VEGF receptor KDR was recovered. 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.
取得した可溶性ヒ卜 VEGF受容体 KDR-Fc各種誘導体、 および、 可溶性ヒト VEGF受容体 KDR各種誘導体の模式図は第 2図および第 3図に示した。 (19) 可溶性ヒト VEGF受容体 KDR の純度の確認 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
精製可溶性ヒト VEGF受容体 KDR-Fcの純度を SDS- PAGEを用いて確認した。 SDS-PAGEは文献記載の方法 [Anticancer Research, 12, 1121 (1992)] に従つた。 ゲルには 5〜20%グラジェントゲル （ァ 1 ^一社製） を用い、 還元条件下でレーン あたりのタンパク量として 2 11 gの KDR-Fcそれぞれ泳動し、クーマシーブリリ アントブルーにて染色した。 第 4図に結果を示した。 KDR- 7N- Fc、 KDR- 5N- Fc、 KDR - 4N- Fc、 KDR-3N- Fc、 KDR- 2N- Fc、 KDR- IN- Fc、 KDR- 5Δ1Ν- Fc、 KDR - 4 ΔΙΝ-Fcの純度は 95 %以上であった。  The purity of the purified soluble human VEGF receptor KDR-Fc was confirmed using SDS-PAGE. SDS-PAGE followed the method described in the literature [Anticancer Research, 12, 1121 (1992)]. A 5-20% gradient gel (manufactured by A1 ^ 1) was used as the gel, and 211 g of KDR-Fc as a protein amount per lane was electrophoresed under reducing conditions, and stained with Coomassie brilliant blue. Figure 4 shows the results. Purity of KDR-7N-Fc, KDR-5N-Fc, KDR-4N-Fc, KDR-3N-Fc, KDR-2N-Fc, KDR-IN-Fc, KDR-5Δ1c-Fc, KDR-4ΔΙΝ-Fc Was over 95%.
(20) 対照抗原蛋白の精製  (20) Purification of control antigen protein
対照抗原蛋白は以下のようにして得た。 High Five細胞 4 X 107個を 175cm2フラ スコ （グライナ一社製） 中の EX-CELL™400培地 （JRH Bioscience社製） 30ml に懸濁し、 室温で 1時間放置し、 フラスコに付着させ、 27°Cにて 3〜4日間培養 した。 培養終了後、 培養上清を回収し 1，500 X g で 10分間遠心分離を行い上清 を得た。 カラムにへパリンーセファロース CL-6Bゲル （Pharmacia Biotech AB 社製） 約 20mlを充填し、 200mlの 20mM 卜リス—塩酸 (PH7.5)緩衝液で 0.5ml/分 の流速で洗浄した。洗浄後、上記のように調製した High Five細胞の培養液 500ml を 0.5ml/分の流速でへパリンーセファロース CL- 6Bカラムに通塔した。 さらに 200mlの 0.2M NaClを含む 20mM 卜リスー塩酸 (pH7.5)で 0.5ml/分の流速で洗浄し た後、 1M NaClを含む 20mM トリス—塩酸 (pH7.5)からなる緩衝液を 200 ml通塔 し、 へパリン—セファロ一スに吸着した蛋白質を溶出した。 lM NaCl溶出画分を セントリブレップ 10 (アミコン社製） を用いて濃縮し対照抗原蛋白を蛋白濃度 867 u g/mlの溶液として 7 ml得た。 The control antigen protein was obtained as follows. 4 × 10 7 High Five cells were suspended in 30 ml of EX-CELL ™ 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. Filled with heparin over Sepharose CL-6B gel (Pharmacia Biotech AB, Inc.) of about 20ml to the column, 20 mM Bok squirrel 200 ml - was washed at a flow rate of 0.5 ml / min with hydrochloric acid (P H7.5) buffer. After washing, 500 ml of the culture solution of High Five cells prepared as described above was passed through a Heparin-Sepharose CL-6B column at a flow rate of 0.5 ml / min. After washing with 200 ml of 20 mM Tris-HCl (pH 7.5) containing 0.2 M NaCl at a flow rate of 0.5 ml / min, 200 mL of buffer containing 20 mM Tris-HCl (pH 7.5) containing 1 M NaCl was added to 200 ml After passing through the column, the protein adsorbed on the heparin-sepharose was eluted. The 1M NaCl-eluted fraction was concentrated using CentriBrep 10 (manufactured by Amicon) to obtain 7 ml of a control antigen protein as a solution having a protein concentration of 867 ug / ml.
(21) 可溶性ヒ卜 VEGF受容体 KDR- Fc各種誘導体のヒト VEGF結合活性の  (21) Soluble human VEGF receptor KDR-Fc
(18)で取得した可溶性ヒト VEGF 受容体 KDR- Fc 各種誘導体 （KDR- 7N-Fc， KDR-5N-Fc, DR-4N-Fc, KDR— 3N— Fc， KDR—2N— Fc, KDR - IN— Fc， KDR - 5Δ1Ν - Fc， KDR- 4A1N-Fc, KDR- 2Δ 1Ν - Fc) のヒト VEGF結合活性を以下の （2卜 1) VEGF 結合阻害試験、 （21-2) VEGF結合試験により確認した。 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-5Δ1Ν-Fc, The human VEGF binding activity of KDR-4A1N-Fc and KDR-2Δ1Ν-Fc) was confirmed by the following (2) 1) VEGF binding inhibition test and (21-2) VEGF binding test.
(21-1) VEGF結合阻害試験  (21-1) VEGF binding inhibition test
96 ゥエル ·ィムオビロン ™— Pフィル卜レ一ション · プレート （96- well Immobilon™-P Filtration Plate ； ミリポア社製） にメタノールを 100 μ. 1/ゥエル で分注し、 プレート底部の PVDF膜を親水化した。 水で洗浄後、 PBS 希釈 4 /Χ g/ml可溶性ヒ卜 KDR- 7N-Fcを 50 1/ゥエルで分注し、 4 °Cでー晚放置して吸 着させた。 洗浄後、 1 %牛血清アルブミン (BSA) を含む PBSを 200 1 / ゥェ ル加え、 室温 30分間反応させて残っている活性基をブロックした。 PBSで洗浄 後、 （18) で取得した精製可溶性ヒト VEGF 受容体 KDR-Fc 各種誘導体 Dispense methanol into 96-well Immobilon ™ -P Filtration Plate (96-well Immobilon ™ -P Filtration Plate; manufactured by Millipore) at 100 μl / l-well and hydrophilicize the PVDF membrane at the bottom of the plate. It has become. After washing with water, PBS-diluted 4 / Χg / ml soluble human KDR-7N-Fc was dispensed at 501 / well, and allowed to stand at 4 ° C for adsorption. After washing, 200 1 / well of PBS containing 1% bovine serum albumin (BSA) was added and reacted at room temperature for 30 minutes to block the remaining active groups. After washing with PBS, purified soluble human VEGF receptor obtained in (18) KDR-Fc Various derivatives
(KDR-7N-Fc, KDR-5N-Fc, KDR— 4N— Fc, KDR— 3N— Fc， KDR— 2N— Fc, KDR— IN— Fc， KDR - 5A1N-Fc， KDR- 4Δ 1Ν- Fc, KDR- 2A1N-Fc) を 50 1/ゥエルで分注し （最 終濃度 0.05〜6.25 I g/ml) 、 さらに、 1251標識ヒ卜 VEGF (最終濃度 4ng/ml： アマシャム社製） を 50 1 / ゥエル加え、 室温で 1.5 時間反応させた。 0.05% tween-PBS で洗浄後、 50°Cにてゥエルを乾燥させ、 マイクロシンチ -0 (パッカ —ド社製） を 10 1/ゥエル加え、 トップカウント （パッカード社製） を用いて、 各ゥエルに結合した 1251標識ヒト VEGFの放射活性を測定した。 (KDR-7N-Fc, KDR-5N-Fc, KDR— 4N— Fc, KDR— 3N— Fc, KDR— 2N— Fc, KDR— IN— Fc, KDR-5A1N-Fc, KDR-4Δ1Ν-Fc, KDR- 2A1N-Fc) and dispensed at 50 1 / Ueru (final concentration .05-6.25 I g / ml), further 125 1-labeled human Bok VEGF (final concentration 4 ng / ml: Amersham) 50 1 The reaction was carried out at room temperature for 1.5 hours. After washing with 0.05% tween-PBS, dry the wells at 50 ° C, add 10 1 / well of Microscint-0 (manufactured by Packard), and use each well with a TopCount (manufactured by Packard). the radioactivity of 125 1-labeled human VEGF bound to the measured.
結果を第 5A図に示す。 KDR— 7N— Fc, KDR- 5Δ1Ν- Fc, KDR— 5N—Fc, KDR-4A 1N-Fc, KDR-4N-Fcは濃度依存的に 1251標識ヒ卜 VEGFの可溶性ヒ卜 KDR7N- Fc への結合を阻害することが示された。 一方、 KDR-3N- Fc, KDR-2N-FC, KDR- Ι -Fc, KDR-2A 1N- Fcは全く結合阻害活性を示さなかった。 結合阻害活性の強 さは、 KDR— 7N— Fc > KDR— 5 Δ 1Ν— Fc > DR-5i -Fc > DR-4 A lN-Fc > KDR- 4N-Fcの順番であった。 従って、 VEGFの KDRへの結合には、 少なくとも N末から 1番目、 6番目、 7番目の lg様ドメインは関与しないことが示された。 また、 N末から 2、 3、 4番目の lg様ドメイン （N末端アミノ酸から 103〜393番 目） があれば VEGFに結合できることが示された。 (21-2) VEGF結合試験 The results are shown in FIG. 5A. KDR- 7N- Fc, KDR- 5Δ1Ν- Fc, KDR- 5N-Fc, KDR-4A 1N-Fc, KDR-4N-Fc concentration dependent manner 125 1-labeled human Bok VEGF to soluble human Bok KDR7N- Fc It has been shown to inhibit binding. On the other hand, KDR-3N-Fc, KDR-2N-FC, KDR-Ι-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-5Δ1Ν-Fc>DR-5i-Fc>DR-4AlN-Fc> KDR-4N-Fc. Therefore, it was shown that at least the first, sixth, and seventh lg-like domains from the N-terminus were not involved in the binding of VEGF to KDR. It was also shown that the presence of the second, third and fourth lg-like domains (103-393 from the N-terminal amino acid) from the N-terminal can bind to VEGF. (21-2) VEGF binding test
96 ゥエル ·ィムオビロン ™— Pフィル卜レーション · プレート （96- well Immobilon™-P Filtration Plate ； ミリポア社製） にメタノールを 100 X 1/ゥエル で分注し、 プレート底部の PVDF膜を親水化した。 水で洗浄後、 PBS 希釈した 0.1〜12.5 II g/mlの （18) で取得した精製可溶性ヒト VEGF受容体 KDR- Fc各種 誘導体 （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— 2Δ1Ν— Fc) を 50 β 1/ゥエルで分 注し、 4 °Cで一晩放置して吸着させた。 洗浄後、 1 %牛血清アルブミン (BSA) を 含む PBSを 200 /i 1 /ゥエル加え、 室温 3分間反応させて残っている活性基をブ ロックした。 PBSで洗浄後、 1251標識ヒト VEGF (最終濃度 4ng/ml：アマシャ ム社製） を 50 ゥエル加え、 室温で 1.5 時間反応させた。 0.05%tween- PBS で洗浄後、 50°Cにてゥエルを乾燥させ、 マイクロシンチ- 0 (パッカード社製） を 10 1/ゥエル加え、 トップカウント ひ \°ッ力一ド社製） を用いて、 各ゥエル に結合した 1251標識ヒト VEGFの放射活性を測定した。 Methanol was dispensed into a 96-well Immobilon ™ -P Filtration Plate (96-well Immobilon ™ -P Filtration Plate; manufactured by Millipore) at 100 × 1 / well to hydrophilize the PVDF membrane at the bottom of the plate. After washing with water, PBS-diluted 0.1-12.5 II g / ml of purified soluble human VEGF receptor obtained in (18) 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—2Δ1Ν-Fc) are dispensed at 50 β1 / Fell and 4 ° C was allowed to stand overnight for adsorption. After washing, PBS containing 1% bovine serum albumin (BSA) was added at 200 / i1 / well, and reacted at room temperature for 3 minutes to block remaining active groups. After washing with PBS, 125 1-labeled human VEGF (final concentration 4 ng / ml: manufactured by Amersham) was added in 50 μl, and the mixture was reacted at room temperature for 1.5 hours. After washing with 0.05% 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.
結果を第 5B図に示す。 KDR- 7N - Fc, DR-5AlN-Fc, KDR- 5N_Fc, KDR-4A IN - Fc, DR-4N-Fcは濃度依存的に 1251標識ヒト VEGFに結合することが示され た。 一方、 KDR- 3N - Fc， KDR - 2N— Fc, KDR - IN- Fc， KDR - 2A1N-FCは全く結合活 性を示さなかった。 結合活性の強さは、 KDR-7N-Fc > KDR-5A1N-Fc = DR- 5N-Fc > DR-4A 1N-Fc > KDR-4N- Fc の順番であった。 従って、 VEGF の KDRへの結合には、 少なくとも N末から 1番目、 6番目、 7番目の Ig様ドメイ ンは関与しないことが示された。 また、 N末から 2、 3、 4番目の lg様ドメインThe results are shown in FIG. 5B. 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. On the other hand, KDR-3N-Fc, KDR-2N-Fc, KDR-IN-Fc, and KDR-2A1N-FC showed no binding activity. The binding activity was in the order of KDR-7N-Fc> KDR-5A1N-Fc = DR-5N-Fc>DR-4A1N-Fc> KDR-4N-Fc. Therefore, it was shown that the binding of VEGF to KDR does not involve at least the first, sixth and seventh Ig-like domains from the N-terminal. Also, the second, third and fourth lg-like domains from the end of N
(N末端アミノ酸から 103から 393番目） があれば VEGFに結合できることが 示された。 (103rd to 393th from the N-terminal amino acid) was shown to be able to bind to VEGF.
(22) 昆虫細胞におけるヒト VEGFの発現  (22) Expression of human VEGF in insect cells
ヒト VEGFは以下のようにして得た。 High Five細胞 4 X 107個を 175cm2フラ スコ (グライナ一社製) に EX- CEし L™400培地 (JRH Bioscience社製) 30mlに懸 濁し、 室温で 1 時間放置し、 フラスコに付着させた。 文献 [セル .グロース · アンド 'ディファレンシェ一ション (Cell Growth & Differentiation), 7, 213,(1996)] 記載の方法により得られたヒト VEGF組み換えバキュロウィルス溶液を約 1 〜3 X 108PFU/mlの濃度で含む溶液を iml 加え、 室温で 2時間感染させた。 培養 上清を除き新たに 30mlの EX-CELL™400培地 30mlを加え 27°Cにて 3〜4日間培 養した。 培養終了後、 培養上清を回収し 1,500 X g で 10分間遠心分離を行い上 清を得た。 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 ™ 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 ™ 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.
カラムに約 40mlのへパリン―セファロース CL- 6Bゲル [フアルマシア ·バイ ォテック (Pharmacia Biotech) AB社製] を充填し、 400mlの 20mM トリス—塩酸 (pH7.5) からなる緩衝液を用いて 0.5ml/分の流速で洗浄した。 洗浄後、 上記のよ うに調製したヒ卜 VEGFを含む培養液 1500mlを 0.5ml/分の流速でへパリン―セ ファロース CL-6Bカラムに通塔した。さらに 400mlの 20mMトリス—塩酸 (pH7.5) を用いて 0.5ml/分の流速で洗浄した後、 0.2M、 0.5Mおよび 1Mの NaCl含有 20mM トリス—塩酸 (PH7.5)からなる緩衝液各 120 mlを順次通塔し、 へパリン—セファ ロースに吸着した蛋白質を段階的に溶出を行うと共に 8ml ずつ溶出液を分画し た。 各分画に含まれる蛋白質を SDSポリアクリルアミドゲル電気泳動にて解析 し、 ヒト VEGFを含む分画 （0.5〜1M NaCl画分） を 120ml回収した。 セントリ プレップ- 10 (アミコン社製） で濃縮後、 ヒト VEGFを溶液として 4ml (蛋白濃 度 1.2 mg/ml) 得た。  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. After washing with 400 ml of 20 mM Tris-HCl (pH 7.5) at a flow rate of 0.5 ml / min, a buffer solution containing 20 mM Tris-HCl (PH7.5) containing 0.2 M, 0.5 M and 1 M NaCl was added. The protein adsorbed on heparin-sepharose was eluted in a stepwise manner, and the eluate was fractionated in 8 ml portions. The protein contained in each fraction was analyzed by SDS polyacrylamide gel electrophoresis, and 120 ml of a fraction containing human VEGF (0.5-1 M NaCl fraction) was collected. After concentration with Centriprep-10 (manufactured by Amicon), 4 ml of human VEGF was obtained as a solution (protein concentration: 1.2 mg / ml).
2 . 動物の免疫と抗体産生細胞の調製  2. Animal immunization and preparation of antibody-producing cells
1(18)で得られた各種抗原 10〜50 I g をそれぞれアルミニウムゲル 2mg およ び百日咳ワクチン（千葉県血清研究所製） 1 X 109細胞とともに 5週令雌 BALB/c (日本 S L C社製） 、 B6C3F1マウス （日本チャールズリバ一社製） あるいは雌 SDラット （日本 S L C社製） に投与し、 2 週間後より 10〜50 X gの蛋白質を 1 週間に 1 回、 計 4 回投与した。 また、 N1H3T3- KDR細胞 1 X 107個を 5週令雌 BALB/c (日本 S L C社製） 3匹に投与し、 計 6回投与した。 眼底静脈叢、 心臓、 あるいは、 尾静脈より採血し、 その血清抗体価を以下に示す酵素免疫測定法で 調べ、 3.で示す酵素免疫測定法により十分な抗体価を示したマウスあるいはラッ 卜から最終免疫 3 日後に脾臓を摘出した。なお、 NIH3T3- KDR細胞を投与した 5 週令雌 BALBんでは免疫がかからず、 可溶性 KDRに対する抗体価は上昇しなか つた。 5-week-old female BALB / c (Japan SLC, Inc.) together with 2 mg of aluminum gel and 1 × 10 9 cells of pertussis vaccine (Chiba Prefectural Serum Institute) ) And B6C3F1 mice (Charles River Japan, Inc.) or female SD rats (SLC Japan), and after 2 weeks, 10-50 X g of protein was administered once a week, a total of four times a week. . In addition, 1 × 10 7 N1H3T3-KDR cells were administered to three 5-week-old female BALB / c (manufactured by SLC Japan), for a total of 6 administrations. 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.
脾臓を MEM 培地 （日水製薬社製） 中で細断し、 ピンセッ卜でほぐし、 遠心分 離 （l，200rpm、 5 分間） した後、 上清を捨て、 トリスー塩化アンモニゥム緩衝液 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.
(PH7.65) で 1 〜2 分間処理し赤血球を除去し、 MEM 培地で 3 回洗浄し、 細 胞融合に用いた。 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.
3 . 酵素免疫測定法 3. Enzyme immunoassay
1(18)で得られた可溶性ヒト VEGF受容体 KDR-Fc各種誘導体、 KDR各種誘導 体を免疫したマウスあるいはラッ卜に由来する抗血清およびハイプリドーマの 培養上清の測定に関しては、 抗原として、 1 (18)の昆虫細胞培養上清より得られ た可溶性ヒト VEGF受容体 KDR- Fc各種誘導体、 KDR各種誘導体を用いた。 96 ゥエルの EIA 用プレート （グライナ一社製） に、 PBS希釈 1 〜10 g/ml可溶 性ヒト VEGF受容体 KDR- Fc各種誘導体、 KDR各種誘導体および対照抗原とし て 1 (20) で得られた High Five細胞培養上清のへパリンカラム吸着画分、 ある いは、 抗 GD3 マウスヒ トキメラ抗体 KM871 [ Cancer Immunology and Immunotherapy, 36, 373(1993) ] をそれぞれ 50 1/ゥエルで分注し、 4 °Cでー晚 放置して吸着させた。 洗浄後、 1 %牛血清アルブミン (BSA)を含む PBSを 100 fi For the measurement of antiserum and hybridoma culture supernatants derived from mice or rats immunized with the soluble human VEGF receptor KDR-Fc various derivatives and KDR various derivatives obtained in 1 (18), 1 Various soluble human VEGF receptor KDR-Fc derivatives and various KDR derivatives obtained from the insect cell culture supernatant of (18) were used. 96-well plates for EIA (Glyna) were diluted with PBS at 1 to 10 g / ml. 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 / ゥ, and 4 ° C. It was adsorbed by standing. After washing, add 100% PBS containing 1% bovine serum albumin (BSA).
1 / ゥエル加え、 室温 1 時間反応させて残っている活性基をブロックした。 1 / Pell was added, and the mixture was reacted at room temperature for 1 hour to block the remaining active groups.
1%BSA- PBS を捨て、 被免疫マウスあるいは被免疫ラット抗血清およびハイプリ ドーマの培養上清を 50 1/ゥエルで分注し 2 時間反応させた。 0.05 % tween- PBS で洗浄後、 ペルォキシダーゼ標識ゥサギ抗マウスィムノグロブリンあるい はペルォキシダーゼ標識ゥサギ抗ラットイムノグロブリン（ともに DAKO社製） を 50 /X 1/ゥエルで加えて室温、 1 時間反応させ、 0.05% tween- PBS で洗浄後 ABTS 基質液 [2.2-アジノビス （3-ェチルベンゾチアゾール- 6- スルホン酸） アンモニ ゥム] を用いて発色させ OD415nmの吸光度 E max [モレキュラー .デバイシ一 ズ （Molecular Devices)社製] を測定した。 1% BSA-PBS was discarded, and antiserum of immunized mice or immunized rats and culture supernatants of hybridomas were dispensed at 50 1 / well and allowed to react for 2 hours. After washing with 0.05% tween-PBS, peroxidase-labeled heron anti-mouse immunoglobulin or peroxidase-labeled heron anti-rat immunoglobulin (both from DAKO) were added at 50 / X 1 / well, and reacted at room temperature for 1 hour. ABTS after washing with 0.05% tween -PBS Color was developed using a substrate solution [2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium], and the absorbance E max at OD415 nm (Molecular Devices) was measured.
4 . マウス骨髄腫細胞の調製  4. Preparation of mouse myeloma cells
8 -ァザグァニン耐性マウス骨髄腫細胞株 P3-U 1 を正常培地で培養し、 細胞融 合時に 2 X 107以上の細胞を確保し、 細胞融合に親株として供した。 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.
5 . 八イブリドーマの作製  5. Production of eight hybridomas
2.で得られたマウス脾細胞あるいはラット脾細胞と 4.で得られた骨髄腫細胞 とを 10:1 になるよう混合し、 遠心分離 （l，200rpm、 5 分間） した後、 上清を捨 て、 沈澱した細胞群をよくほぐした後、 攪拌しながら、 37でで、 ポリエチレン グライコール— 1000(PEG- 1000)2g、MEM 培地 2ml および DMSO 0.7rnlの混液 0.2 〜lml/108マウス脾細胞を加え、 1 〜2 分間毎に MEM 培地 1 〜2ml を数回加え た後、 MEM培地を加えて全量が 50mlになるようにした。遠心分離（900rpm、 5 分 間） 後、 上清を捨て、 ゆるやかに細胞をほぐした後、 メスピペットによる吸込 み、 吸出しでゆるやかに細胞を HAT 培地 100ml中に懸濁した。 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.
6. Binding EL1SAによるハイブリドーマスクリーニング  6. Hybridoma screening with Binding EL1SA
5.で得られた懸濁液を 96ゥエル培養用プレートに 100 β 1/ゥエルずつ分注し、 5%C〇2インキュべ一夕一中、 37°Cで 10〜14日間 5%C〇2下で培養した。 この培養 上清を実施例 1の 3 に記載した酵素免疫測定法で調べ、 1(18)で得られた可溶性 ヒト VEGF受容体 KDR- Fc各種誘導体、 KDR各種誘導体に特異的に反応し、 か つ 1 (20) で得られた対照抗原に反応しないゥエルを選び、 さらに HT培地と正 常培地に換え、 2回クローニングを繰り返して、 抗ヒト VEGF受容体 KDRモノ クロ一ナル抗体を生産するハイプリ ドーマ株を確立した。 以下にその結果を示 す。 動物 匹数 免疫原 スクリーニング原 スクリ一ニンク'法 スクリ一ニング 確立したハイブり ドーマ数 Dispense the suspension obtained in step 5 into a 96-well culture plate at 100 β 1 / well, and incubate at 5 ° C in a 2 % incubator overnight at 37 ° C for 10-14 days. Cultured under 2 . The culture supernatant was examined by the enzyme immunoassay described in Example 1-3, and specifically reacted with the soluble human VEGF receptor KDR-Fc various derivatives obtained in 1 (18), KDR various derivatives. Select a well that does not react with the control antigen obtained in step 1 (20), replace it with HT medium and normal medium, and repeat cloning twice to obtain a hybrid antibody that produces anti-human VEGF receptor KDR monoclonal antibody. A dorma strain was established. The results are shown below. Animals Number of immunogens Screening source Screening 'method Screening Established number of domes
したゥエル数  Number
SDラッ 卜 3 KDR(7N') KDR(7N') Binding ELISA 3024 4 (KM1660-1663)  SD Rat 3 KDR (7N ') KDR (7N') Binding ELISA 3024 4 (KM1660-1663)
SDラッ 卜 2 KDR(7N') KDR(7N) 2016 1 (KM1667)  SD Rat 2 KDR (7N ') KDR (7N) 2016 1 (KM1667)
Balb/cマウス 1 KDR(2N)-Fc KDR(7N) 420 7 (KM1859-1865)  Balb / c mouse 1 KDR (2N) -Fc KDR (7N) 420 7 (KM1859-1865)
Balb/cマウス 1 KDR/NIH3T3 cell KDR(7N') 504 1 (KM1659)  Balb / c mouse 1 KDR / NIH3T3 cell KDR (7N ') 504 1 (KM1659)
Balb/cマウス 1 KDR(7N') KDR(7N') 420 1 (KM1664)  Balb / c mouse 1 KDR (7N ') KDR (7N') 420 1 (KM1664)
Balb/cマウス 1 KDR(7N') KDR(7N)-Fc 420 2 (K 1665.1666)  Balb / c mouse 1 KDR (7N ') KDR (7N) -Fc 420 2 (K 1665.1666)
Balb/cマウス 2 KDR(7N') KDR(2N)-Fc 840 1 (KM1668)  Balb / c mouse 2 KDR (7N ') KDR (2N) -Fc 840 1 (KM1668)
Balb/cマウス 2 KDR(2N)-Fc KDR(7N) 840 1 (KM1768)  Balb / c mouse 2 KDR (2N) -Fc KDR (7N) 840 1 (KM1768)
Balb/cマウス 2 KDR(7N) KDR(3N)-Fc 840 2 (KM1825.1826)  Balb / c mouse 2 KDR (7N) KDR (3N) -Fc 840 2 (KM1825.1826)
Balb/cマウス 2 KDR(7N) KDR{5N)-Fc 840 4 (ΚΜ1Θ27-1830)  Balb / c mouse 2 KDR (7N) KDR {5N) -Fc 840 4 (ΚΜ1Θ27-1830)
Balb/cマウス 2 KDR(7N) KDR(5N) 840 14 (Κ 1Β31-1Θ38, 1853-1858) Balb / c mouse 2 KDR (7N) KDR (5N) 840 14 (Κ 1Β31-1Θ38, 1853-1858)
Balb/cマウス 4 KDR(5N) KDR(5N) 1680 10 (K 1943-1950, 1932、 1933) 'Balb / c mouse 4 KDR (5N) KDR (5N) 1680 10 (K 1943-1950, 1932, 1933) ''
Balb/cマウス 1 KDR{7N) KDR(7N') 420 3 (KM1778〜1780) Balb / c mouse 1 KDR (7N) KDR (7N ') 420 3 (KM1778 ~ 1780)
Balb/cマウス 1 KDR(7N)-Fc KDR(5N) 504 3 (K 1987-1989)  Balb / c mouse 1 KDR (7N) -Fc KDR (5N) 504 3 (K 1987-1989)
Balb/cマウス 1 KDR(5N)-Fc KDR(5N) 420 1 (KM1942)  Balb / c mouse 1 KDR (5N) -Fc KDR (5N) 420 1 (KM1942)
Balb/cマウス 3 KDR(5N) KDR(5N) 1260 8 (KM1943-1950)  Balb / c mouse 3 KDR (5N) KDR (5N) 1260 8 (KM1943-1950)
8alb/cマウス 3 KDR(7N)-Fc DR(5A1N)-Fc 1260 11 (K 1965-1975)  8alb / c mouse 3 KDR (7N) -Fc DR (5A1N) -Fc 1260 11 (K 1965-1975)
B6C3F1マウス 1 KDR(7N)-Fc KDR(5厶 1N)-Fc/m VEG KDR結合阻害試験 420 0  B6C3F1 mouse 1 KDR (7N) -Fc KDR (5m 1N) -Fc / m VEG KDR binding inhibition test 420 0
BGC3F1マウス 1 KDR(5厶 1N)-Fc KDR(7N)-Fc/RI 420 7 (KM1991-1997) BGC3F1 mouse 1 KDR (5m 1N) -Fc KDR (7N) -Fc / RI 420 7 (KM1991-1997)
1(18)で得られた可溶性ヒ卜 VEGF受容体 KDR - Fc各種誘導体、 KDR各種誘導 体および KDR- NIH3T3細胞を免疫した Balb/cマウス、 B6C3F1マウス、 あるい は SDラット計 32匹から得られたハイプリドーマを約 16548ウェルスクリ一二 ングし、 1 (18)で得られた可溶性ヒ卜 VEGF受容体 KDR- Fc各種誘導体、 KDR各 種誘導体に特異的に反応し、 かつ 1 (20) で得られた対照抗原あるいは KM871 に反応しない計 74クローンの抗ヒ卜 VEGF受容体 KDRモノクローナル抗体を 取得し、 それぞれ表 1のように命名した。 これらの抗ヒト VEGF受容体 KDRモ ノクローナル抗体の中で、 40個のモノクローナル抗体 （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) が 15.で示す免疫細胞染色法により細胞表面上の KDR に反応することが 示された。 しかし、 血管内皮細胞の VEGF刺激による増殖促進活性を阻害する 活性を示すような KDRの生物活性を阻害するモノクローナル抗体は取得できな かった。 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. Among these anti-human 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. However, 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.
7 . モノクローナル抗体のェピトープ解析  7. Epitope analysis of monoclonal antibodies
6.で述べた抗ヒト VEGF受容体 KDRモノク口一ナル抗体の特異性をハイブリ ドーマ培養上清を用いて 3.に記載した酵素免疫測定法を用いて確認した。  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.
代表的な結果を第 6図に、 まとめた結果を第 7図に示す。 上記 74種のモノク ローナル抗体のうち、 KM 1668他 32種が 1番目の Ig様ドメイン （1〜104ァミノ 酸に対応） に反応し、 KM1987他 3種が 1番目の lg様ドメイン （1〜104 ァミノ 酸に対応） と 2番目の Ig様ドメイン （105〜194アミノ酸に対応） の間に反応し、 KM 1855他 5 種が 2番目の Ig様ドメイン （105〜194アミノ酸に対応） に反応し、 KM1858他 2種が 3番目の Ig様ドメイン （195〜294アミノ酸に対応） に反応し、 KM 1854他 3種が 4番目の Ig様ドメイン （295〜 393アミノ酸に対応） に反応し、 KM1832他 14種が 5番目の Ig様ドメイン （394〜518アミノ酸に対応） に反応し、 KM1665他 2種が 6〜7番目の g様ドメイン （519〜738アミノ酸に対応） に反応 した。 従って、 1番目の lg様ドメインに対する免疫原性の高いモノクローナル 抗体の 43%が 1番目の lg様ドメインに反応した。 （21) で示したように KDR の 1番目の lg様ドメインは VEGFの結合活性に関与しないが、 免疫原性が高い ため、 ELISAスクリ一ニングでは中和活性を示すモノクローナル抗体の作製が困 難であると推定された。 Fig. 6 shows typical results, and Fig. 7 summarizes the results. Among the 74 kinds of monoclonal antibodies, 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). Thus, 43% of the highly immunogenic monoclonal antibody against the first l g-like domain in response to the first lg-like domain. As shown in (21), 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.
8. [125I] VEGF- KDR結合阻害アツセィによる抗体価の測定 8. Measurement of antibody titer using [ 125 I] VEGF-KDR binding inhibition assay
免疫原性が高く、 中和活性に関係のない 1番目の lg様ドメインに対するモノ クローナル抗体を排除するため、 1 (18) で得られた KDR-5A1N- Fcをマウスに 免疫した。マウス抗血清のヒト VEGFとヒト VEGF受容体 KDR の結合阻害活性 を以下の手順に従い評価した。  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.
96ゥエル 'マルチスクリーン一 I Pプレ一ト （96- well MultiScreen- IP Plate； ミリポア社製） にメタノールを 100 1/ゥエルで分注し、 プレート底部の PVDF 膜を親水化した。 水で洗浄後、 PBSで 4 / g/mlの濃度に希釈した可溶性ヒト VEGF受容体 KDR- 7N- Fcを 50 1/ゥエルで分注し、 4 °Cでー晚放置して吸着さ せた。 洗浄後、 1 %牛血清アルブミン (BSA)含有 PBSを 200 1 / ゥエル加え、 室温 30 分間反応させて残っている活性基をブロックした。 PBSで洗浄後、 1%BSA- PBS溶液で 100、 1000、 10000倍に希釈した抗血清、 1%BSA- PBS溶液で 希釈した精製モノクローナル抗体 （0.01〜25 g/ml) 、 あるいは、 ハイプリド 一マの培養上清を 50 1/ゥエルで分注し、さらに、 4 ng/mlの1251標識ヒ卜 VEGF (アマシャム社製） を 50 1 /ゥエル加え室温で 1.5時間反応させた。 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. After washing with water, 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. . After washing, 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. After washing with PBS, antiserum diluted with 1% BSA-PBS solution 100, 1000, and 10000 times, purified monoclonal antibody (0.01 to 25 g / ml) diluted with 1% BSA-PBS solution, or hybridoma dispensed in the culture supernatant at 50 1 / Ueru min were further 4 ng / ml of 125 1-labeled human Bok VEGF (manufactured by Amersham) 50 1 / Ueru added and reacted at room temperature for 1.5 hours.
0.05%tween- PBSで洗浄後、 50°Cにてゥエルを乾燥させ、マイクロシンチ - 0 (パ ッカード社製） を 10 1/ゥエル加え、 トップカウント （パッカード社製） を用 いて、 各ゥエルに結合した 1251標識ヒ卜 VEGFの放射活性を測定した。 After washing with 0.05% Tween-PBS, dry the wells at 50 ° C, add 10 1 / well of Microscint-0 (manufactured by Packard), and use a top count (manufactured by Packard) to add each well to each well. the radioactivity of the bound 125 1-labeled human Bok VEGF was measured.
ハイプリド一マの培養上清の活性を検討した結果を表 2に示した。 表 2 Table 2 shows the results of examining the activity of the culture supernatant of the hybridoma. Table 2
Figure imgf000067_0001
Figure imgf000067_0001
KDR- 5Δ1Ν- Fc免疫したマウス 3匹の抗血清は全て 100倍希釈において 50 % 以上の結合阻害活性を示し、 3匹中 1匹の抗血清は 1000倍希釈において 34.3 % と最も強い結合阻害活性を示した。 KDR-7N- Fc, KDR- 5N_Fc免疫マウスそれぞ れ 3匹、 2匹の抗血清は 100倍希釈において 50 %以上の結合阻害活性を示した。 従って、 結合阻害活性が最も強く、 さらに、 免疫原性の強い 1番目の lg様ドメ ィンを含まない KDR- δΔ ΐΝ-Fcが免疫原として適することが示された。 KDR-5 5Δ1Ν- 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. The antisera from three and two KDR-7N-Fc and KDR-5N_Fc immunized mice, respectively, showed a binding inhibitory activity of 50% or more at 100-fold dilution. Thus, binding inhibition activity most strongly, further do not contain a strong first l g-like domain fin immunogenic KDR- δΔ ΐΝ-Fc was shown to be suitable as an immunogen.
9. [125I] VEGF-KDR結合阻害アツセィによるハイプリドーマのスクリーニング KDR - 5N-Fc免疫マウス 1匹よりハイブリドーマを作製し、 得られた約 672ゥ エルの培養上清を用いて 8.で示した [1251]VEGF-KDR結合阻害アツセィでスクリ 一二ングしたところ、 培養上清では 90.1、 66.7、 59.0 , 85.7、 86.8、 78.0、 91.2 % の結合阻害活性を示す 7 クローンのモノクローナル抗体産生ハイプリドーマを 得、 これらをそれぞれ KM 1991〜1997と命名した （表 1 ) 。 9. Screening of hybridoma by [ 125I ] 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 µl 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).
10. モノクローナル抗体 KM1991〜1997のェピ I ^一プ解析  10. Epitope analysis of monoclonal antibodies KM1991-1997
9.で述べた抗ヒ卜 VEGF受容体 KDRモノクローナル抗体の特異性を精製抗体 5 g/iPlを用いて 3に記載した酵素免疫測定法を用いて確認した。  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.
代表的な結果を第 8図に、 まとめた結果を第 7図に示す。 KM 1992、 K 1995 に代表される上記 7種のモノク口一ナル抗体は全て 4番目の Ig様ドメイン（295 〜393アミノ酸に対応） に反応した。 従って、 KDRの N末から 4番目の Ig様ド メイン （295〜393アミノ酸に対応） が VEGFとの結合に特に重要であることが 示された。 特に、 KM1991、 M 1992 , M1993. KM1994および KM1995は、 13. に示した VEGF受容体 KDRの自己リン酸化阻害活性、 あるいは、 14.に示した VEGF依存的血管内皮細胞の増殖阻害活性を示し、 KDR の生物活性を阻害する 中和モノクローナル抗体であることが示された。 6.で取得された計 74クローン の抗ヒト VEGF受容体 KDRモノクローナル抗体では、 DRの生物活性を阻害す る中和モノクロ一ナル抗体を取得することができなかったのに対し、 9.では中和 モノクローナル抗体が取得できたことから、 免疫原性の強い 1番目の lg様ドメ インを含まない KDR- 5Δ 1 Ν- Fcが免疫原として適すること、 [125I]VEGF-KDR結 合阻害ァッセィがハイプリ ドーマスクリ一ニング系として適することが明らか となった。 Fig. 8 shows typical results, and 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. In particular, KM1991, M1992, M1993. 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. A total of 74 clones of the anti-human VEGF receptor KDR monoclonal antibody obtained in 6. could not obtain a neutralizing monoclonal antibody that inhibits the biological activity of DR, whereas in 9. Since a monoclonal antibody was obtained, KDR-5Δ1Ν-Fc, which does not contain the first lg-like domain with strong immunogenicity, is suitable as an immunogen. [ 125 I] VEGF-KDR binding inhibition assay Was found to be suitable as a hybrid masking system.
モノクローナル抗体の抗体クラスを決めるためにサブクラスタイピングキッ 卜 [ザィメット （Zymed ) 社製] を用いた酵素免疫測定法を行った。 その結果 を以下の表 3に示す。 表 3Subcluster typing kit to determine antibody class of monoclonal antibody An enzyme-linked immunosorbent assay was carried out using a mouse [Zymed]. The results are shown in Table 3 below. Table 3
Figure imgf000069_0001
Figure imgf000069_0001
本発明で確立したモノクローナル抗体は IgMである KM1659、 KM 1942, IgAで ある KM1664、 IgEである KM1991、 KM1996、 KM1997を除き、 すべて IgGクラ スであった。 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.
11. モノクローナル抗体の精製  11. Purification of monoclonal antibodies
プリスタン処理した 8 週令ヌード雌マウス （Balb/c) に 6、 9で得られたハイ プリドーマ株を 5 〜10 X 106細胞 Z匹それぞれ腹腔内に注射した。 10〜21 日後に、 ハイプリドーマは腹水癌化した。 腹水のたまったマウスから、 腹水を採取 （1〜 8ml/匹） し、 遠心分離 （3,000rpm、 20 分間） して固形分を除去した後力プリル 酸沈殿法 （アンチボディーズ ·ァ · ラボラトリ一 ·マニュアル） により精製し、 精製モノクロ一ナル抗体とした。 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 .
12. 抗 KDRモノクローナル抗体によるヒ卜 VEGFとヒ卜 VEGF受容体 KDRの 結合阻害活性の確認  12. Confirmation of anti-KDR monoclonal antibody binding inhibitory activity between human VEGF and human VEGF receptor KDR
9 .で述べた抗ヒト VEGF受容体 KDR モノクローナル抗体のヒト VEGFとヒト VEGF受容体 KDR の結合阻害活性を 8.に示した手順に従い確認した。  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.
対照として、抗ヒト VEGF受容体 KDR モノクローナル抗体のヒト VEGFとヒ ト VEGF受容体 Fit- 1の結合阻害活性を以下の手順に従い確認した。  As a control, 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.
96ゥエル ·マルチスクリーン— I Pプレート （96- well MultiScreen-IP Plate； ミリポア社製）にメタノールを 100 1/ゥエルで分注し、 プレート底部の PVDF 膜を親水化した。 水で洗浄後、 PBS で 1.6 g/mlの濃度に希釈した可溶性ヒト VEGF受容体 Fit - 1 7Nを 50 /2 1/ゥエルで分注し、 4 °Cでー晚放置して吸着させ た。 洗浄後、 1 %牛血清アルブミン (BSA) 含有 PBS を 50 w 1 ,/ ゥエル加え、 室 温 1 時間反応させて残っている活性基をブロックした。 PBS で洗浄後、 ハイブ リドーマの培養上清あるいは 0.5M NaCl を含む 1%BSA- PBS溶液で希釈した精 製モノクローナル抗体 （0.01〜7.29 / g/ml) を 50 1/ゥエルで分注し、 さらに、 3ng/mlの 1251標識ヒト VEGF (アマシャム社製） を 50 μ 1 / ゥエル加え室温で 1.5 時間反応させた。 0.05%tween-PBSで洗浄後、 50°Cにてゥエルを乾燥させ、 マ イク口シンチ- 0 (パッカード社製） を 30 l/ゥエル加え、 トップカウント （パ ッカ一ド社製） を用いて、 各ゥエルに結合した1251標識ヒト VEGFの放射活性を 測定した。 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. After washing with water, 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. After washing with PBS, 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. After washing with 0.05% 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.
結果を第 8図に示す。 KM 1992、 1993、 1994、 1995は濃度依存的にヒト VEGF とヒト VEGF受容体 KDRの結合を阻害した。 ヒ卜 VEGFとヒ卜 VEGF受容体 KDRの結合の 50 %阻害を示す KM 1992、 1993、 1994、 1995の濃度 （IC5。） は 2.26、 2.43、 0.74, 1.95 n g/mlであった。 一方、 コントロールとして使用したマウス IgGlクラスである抗シァリルルイス Aモノクローナル抗体 KM231 [アンチキヤ ンサー · リサーチ (Anticancer Research), ， 1579(1990) ] は全く阻害活性を示さ なかった。 さらに、 コントロール実験系として用いたヒト VEGF とヒ卜 VEGF 受容体 Flt-1 の結合を KM1992、 1993、 1994、 1995は全く阻害せず、 KM1992、 1993、 1994、 1995は KDR特異的な結合阻害剤であることが示された。 The results are shown in FIG. KM 1992, 1993, 1994 and 1995 inhibited the binding of human VEGF to the human VEGF receptor KDR in a concentration-dependent manner. KM 1992 showing a 50% inhibition of binding of human Bok VEGF and human Bok VEGF receptor KDR, 1993, 1994, 1995 of the concentration (IC 5.) Is 2.26, 2.43, 0.74, was 1.95 ng / ml. On the other hand, the mouse used as a control The IgGl class anti-Sialyl Lewis A monoclonal antibody KM231 [Anticancer Research,, 1579 (1990)] showed no inhibitory activity. Furthermore, 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.
13.抗 KDRモノク口一ナル抗体による VEGF受容体 KDRの自己リン酸化阻害13. Inhibition of VEGF receptor KDR autophosphorylation by anti-KDR monoclonal antibody
9 .で述べた抗ヒ卜 VEGF受容体 KDR モノクローナル抗体によるヒト VEGF の生物活性の阻害を以下の手順により確認した。 The inhibition of the biological activity of human VEGF by the anti-human VEGF receptor KDR monoclonal antibody described in 9 was confirmed by the following procedure.
ヒ卜 VEGF受容体 KDR発現 NIH3T3細胞 (NIH3T3- KDR)を 10 %FCS - DMEM培 地 20mlを用いて l75cm2フラスコにて 5〜10 X 106cells/フラスコになるまで培養 した。 培養後、 0.1 mMのオル卜バナジン酸 （V) ナトリウムを含む 0.1 %FCS_ DMEMlOmlに交換し、 抗 VEGF受容体 KDR抗体を 10 g/mlとなるように添加 して氷上で 30分間前処理を行った。前処理後、ヒ卜 VEGF (R&D社製)を 50 ng/ml となるように添加し、 氷上で 45分間刺激を行った。 刺激後、 フラスコから培地 を除去し、細胞破砕緩衝液 [20 mM Hepes (pH7.4), 150 mM NaCl, 0.2% TritonX-100, 10% Glycerol, 2 mM Na3V〇4, 10 mM Na4P2〇7, 5 mM EDTA, 50 mM NaF, 1.5 mM MgCl2, 1 mM PMSF, 10 n g/ml aprotinin, 5 (i g/ml leupeptin] を 2ml加え、 細胞を破砕し、 細胞破碎液を得た。 細胞破砕液を 15,000 X gで 10分間遠心分離を行い、 上清に ャギ抗マウス lgG(H+L)セファロース 4B (Zymed Laboratories社製)を 100 1添加 し 4°Cで i時間穏やかに混合した。 5，000 X gで 1分間遠心分離後、 上清にャギ 抗マウス lgG(H+L)セファロース 4Bを 30 /2 K マウス抗 KDRモノクローナル抗 体 KM1668を 10 μ g添加し、 4°Cで 1晚穏やかに混合し、免疫沈降を行った。 5，000 X gで 1分間遠心分離を行い、 セファロ一スを回収し、 回収したセファロ一スを lmlの細胞破砕緩衝液で洗浄した。 洗浄操作は繰り返して 6回行った。 洗浄後、 回収したセファロ一スから 30 1の 2 -メルカプトエタノールを含む SDSポリア クリルアミドゲル電気泳動用サンプルバッファ一 （2倍濃度） にて吸着タンパク 質を溶出し、 全量を SDSボリアクリルアミドゲル電気泳動、 ウエスタンブロッ ティングに供した。プロッティング後の PVDF膜は 1%BSA- PBSに室温で 30分間 反応させブロッキング操作を行い、 ゥサギ抗リン酸化チロシン抗体 （2 / g/ml) (Upstate Biotechnology Incorpotated 社製）を 4°Cにて 1 晚反応させた。 0.05% Tween-PBSで洗浄し、 ペルォキシダーゼ標識したブタ抗ゥサギ IgG (1000倍希 釈： Dako 社製） を室温で 1 時間反応させた。 0.05% Tween-PBS で洗浄し、 ECL™Western blotting detection reagents (アマシャム社製） を用いて、 ゥサギ抗 リン酸化チロシン抗体が結合したバンドを検出した。 Human Bok VEGF receptor KDR expressing NIH3T3 cells (NIH3T3-KDR) and 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. After the stimulation, remove the medium from the flask, and add cell disruption buffer ( 20 mM Hepes (pH 7.4), 150 mM NaCl, 0.2% TritonX-100, 10% Glycerol, 2 mM Na 3 V〇 4 , 10 mM Na 4 P 2 〇 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碎液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. It was mixed. after centrifugation for 1 minute at 5,000 X g, the supernatant catcher formic anti-mouse lgG (H + L) Sepharose 4B and 30/2 K mouse anti-KDR monoclonal antibody KM1668 was added 10 mu g to, The mixture was gently mixed for 1 晚 at 4 ° C, and immunoprecipitation was performed.Centrifugation was performed at 5,000 × g for 1 minute to collect the cepharose. Washing was repeated 6 . Adsorbed proteins by SDS polyacrylamide gel electrophoresis sample buffer one containing mercaptoethanol (double concentration) - went washed, recovered cephalometric Ichisu from 30 1 2 The substance was eluted, and the whole amount was subjected to SDS polyacrylamide gel electrophoresis and western blotting. After the plotting, the PVDF membrane was reacted with 1% BSA-PBS at room temperature for 30 minutes to perform a blocking operation, and ゥ a heron anti-phosphorylated tyrosine antibody (2 / g / ml) (Upstate Biotechnology Incorpotated) at 4 ° C. 1 晚 reacted. After washing with 0.05% Tween-PBS, peroxidase-labeled porcine anti-Peacock IgG (1000-fold dilution: manufactured by Dako) was reacted at room temperature for 1 hour. After washing with 0.05% Tween-PBS, a band bound to a heron anti-phosphorylated tyrosine antibody was detected using ECL ™ Western blotting detection reagents (Amersham).
結果を第 10図に示す。 VEGF添加により VEGF受容体 KDRの自己リン酸化が 検出されたが、 マウス抗 KDRモノクローナル抗体 KM1991, 1992, 1994, 1995添 加により KDRの自己リン酸化が抑制された。  The results are shown in FIG. Autophosphorylation of VEGF receptor KDR was detected by the addition of VEGF, but autophosphorylation of KDR was suppressed by the addition of the mouse anti-KDR monoclonal antibody KM1991, 1992, 1994, 1995.
14.抗 KDRモノクローナル抗体による VEGF依存的血管内皮細胞の増殖阻害  14. Inhibition of VEGF-dependent endothelial cell proliferation by anti-KDR monoclonal antibody
9 . で述べた抗ヒ卜. VEGF受容体 KDR モノクローナル抗体によるヒト VEGF の生物活性の阻害を以下の手順により確認した。  The inhibition of the biological activity of human VEGF by the anti-human VEGF receptor KDR monoclonal antibody described in 9 was confirmed by the following procedure.
48ウェルマイク口タイ夕一プレートに E- BM培地に 5 %ゥシ胎児血清（FBS)、 ヒ卜組み換え型上皮成長因子 （hEGF) 10ng/ml 、 ハイドロコーチゾン 1 pi g/mK ゲン夕マイシン 50 i g/mU アンファテリシン B 50ng/ml を添加した培地 （クラ ボウ社製）に浮遊させたヒト皮膚由来微小血管内皮細胞 HMVEC (クラボウ社製） を 4000個/ 800 11 1/ゥエルになるように加えた。 次に、 上記培地で希釈した精製 抗 VEGF受容体 KDR モノクローナル抗体 （終濃度 2 fi g/mlおよび 20 g/ml) 100 PL 1/ゥエルで添加し、さらに、上記培地で希釈したヒト VEGF (R&D社製） を 100 /2 1/ゥエル （終濃度 lng/ml) で添加し、 37°C C02インキュベータ一中で 5 日間培養した。 培養後、 培養上清を除去し、 200 fi 1 の 10 % FCS 添加のフエノ ールレッド除去 RPMI1640 (二ッスィ社製） を各ゥエルに加え、 さらに、 20 /i 1 の細胞発色用試薬 (Cell Counting Kit；同仁化学社製） を加え、 37°Cで 1 〜2 時間 培養した。 培養終了後、 150 1 を ΕΙΑ 用 96ウェルマイク口タイ夕一プレート に移し、 OD450nm の吸光度を測定した。 1 ng/ml の VEGF添カロ時の HMVEC の 増殖を 100 %、 VEGF非添加時の HMVEC の増殖を 0 %とした相対的な細胞増殖 活性を示した。 In 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. Was. Then added in the medium was diluted with purified anti-VEGF receptor KDR monoclonal antibodies (final concentration 2 fi g / ml and 20 g / ml) 100 PL 1 / Ueru further human VEGF (R & D diluted in the culture medium the company Ltd.) was added at 100/2 1 / Ueru (final concentration lng / ml), were cultured for 5 days in 37 ° C C0 2 incubator scratch. After culturing, remove the culture supernatant, add 200 fi 1 of 10% FCS-added phenol red-removed RPMI1640 (manufactured by Nissi) to each well, and add a 20 / i 1 Cell Counting Kit (Cell Counting Kit). ; Dojin Chemical Co., Ltd.) and cultured at 37 ° C. for 1-2 hours. After culturing, use 150 1 for 96 96 well microphone mouth And measured the absorbance at OD450nm. The relative cell growth activity was shown assuming that the proliferation of HMVEC at 1 ng / ml of VEGF added calo was 100% and that of HMVEC without VEGF added was 0%.
結果を第 U図に示す。 抗 VEGF受容体 KDR モノクローナル抗体 KM1992お よび KM1995は濃度依存的に VEGF依存的な HMVEC の増殖を阻害した。 2 μ. g/ml添加時の KM1992および KM1995の増殖阻害活性は、それぞれ 74.3 %、 70.9 %、 20 g/ml添加時の KM1992および KM1995の増殖阻害活性は、それぞれ 108.7 %、 103.0 %であった。 一方、 コントロールとして使用した KM231および抗体非添 加群は阻害活性を示さなかった。  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 μg / 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. . On the other hand, the KM231 and antibody-free groups used as controls did not show any inhibitory activity.
15. モノクローナル抗体のヒト VEGF受容体 KDR発現細胞との反応性の確認 15. Confirmation of reactivity of monoclonal antibody with human VEGF receptor KDR-expressing cells
6および 9で述べた抗ヒト VEGF受容体 KDRモノクローナル抗体の特異性を 免疫細胞染色を用いて以下の手順に従い確認した。 The specificity of the anti-human VEGF receptor KDR monoclonal antibody described in 6 and 9 was confirmed according to the following procedure using immunocytochemistry.
ヒト VEGF受容体 KDR発現 NIH3T3細胞 (N1H3T3- KDR)、コントロール NIH3T3 細胞 (NIH3T3- Neo)、 ヒト皮膚由来微小血管内皮細胞 HMVEC (クラボウ社製） 、 ヒ卜さい帯静脈由来血管内皮細胞 HUVEC (クラボウ社製） 2〜5 X 105個を丸底 96ゥエルプレートに免疫細胞染色用緩衝液 （1% BSA 、 0.02% EDTA 、 0.05% ァ ジ化ナトリウムを含む PBS ) 100 II 1に懸濁して分注した。 4 °C、 350 X gで 1 分間遠心分離後、 上清を除き、 6、 9で述べた精製抗体 （10 β g/ml) 50 1を加 えて 4 °Cで 30分間反応させた。 反応後、 200 1の免疫細胞染色用緩衝液を各 ゥエルに加え 4 。じ、 350 X g で 1 分間遠心分離後、 上清を除き細胞の洗浄を行 つた。 この洗浄操作をさらに 2 回行った後、 FITC標識抗マウスィムノグロプリ ン抗体あるいは F1TC標識抗ラットイムノグロブリン抗体 （和光純薬社製） を 1 II g/mlの濃度で含む免疫細胞染色用緩衝液 50 1 を加えて J °Cで 30分間反応 させた。 反応後、 上記と同様の洗浄操作を 3 回行った後フローサイ卜メーター (コール夕一社製） を用いて解析を行った。 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. After centrifugation at 350 × g for 1 minute at 4 ° C, the supernatant was removed, and the purified antibody (10 βg / 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. After performing this washing operation two more times, 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).
結果を第 12図に示す。抗ヒト VEGF受容体 KDRモノクローナル抗体 KM1668 はコン卜ロール細胞には反応せず KDR発現細胞に特異的に顕著に反応した（A)。 また、 ヒト血管内皮細胞 HMVECおよび HUVEC に反応し、 血管内皮細胞上の DRを検出可能性あることが示された。 The results are shown in FIG. Anti-human VEGF receptor KDR monoclonal antibody KM1668 Did not react with the control cells but specifically reacted with the KDR-expressing cells (A). In addition, in response to human vascular endothelial cells HMVEC and HUVEC, it was shown that DR on vascular endothelial cells could be detected.
また、 9.で示した [125I]VEGF- KDR結合阻害ァッセィにより選択された KM1992、 KM1993、 KM1994、 KM1995は、 ヒ卜血管内皮細胞 HMVECおよび HUVECに反 応し、 血管内皮細胞上の KDRを検出可能性あることが示された （第 13図） 。 産業上の利用可能性 In addition, 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
本発明により、 ヒ卜血管新生部の血管内皮細胞上に特異的に発現されている ヒト VEGF受容体 KDRに特異的に結合するモノクローナル抗体が提供される。 本発明のモノクローナル抗体は免疫細胞染色におけるヒト血管新生部の免疫学 的検出、 KDR の作用の中和による固形腫瘍の増殖、 転移形成、 慢性関節リュウ マチにおける関節炎、 糖尿病性網膜症、 未熟児網膜症、 乾鮮など異常な血管新 生により病態が進行する疾患の診断あるレ ^は治療に有用である。 配列表フリーテキス卜  According to the present invention, there is provided 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. Sequence Listing Free Text
配列番号 1一人工配列の説明：合成 DNA SEQ ID NO: 1 Description of Artificial Sequence: Synthetic DNA
配列番号 2—人工配列の説明：合成 DNA SEQ ID NO: 2—Description of Artificial Sequence: Synthetic DNA
配列番号 3—人工配列の説明：合成 DNA SEQ ID NO: 3—Description of artificial sequence: Synthetic DNA
配列番号 4一人工配列の説明：合成 DNA SEQ ID NO: 4 Description of Artificial Sequence: Synthetic DNA
配列番号 5—人工配列の説明：合成 DNA SEQ ID NO: 5—Description of artificial sequence: Synthetic DNA
配列番号 6—人工配列の説明：合成 DNA SEQ ID NO: 6—Description of artificial sequence: Synthetic DNA
配列番号 7 -人工配列の説明：合成 DNA SEQ ID NO: 7-Description of artificial sequence: Synthetic DNA
配列番号 8—人工配列の説明：合成 DNA SEQ ID NO: 8—Description of Artificial Sequence: Synthetic DNA
配列番号 9一人工配列の説明：合成 DNA  SEQ ID NO: 9 Description of Artificial Sequence: Synthetic DNA
配列番号 1 0—人工配列の説明：合成 DNA  SEQ ID NO: 10—Description of artificial sequence: Synthetic DNA
配列番号 1 1一人工配列の説明：合成 DNA 配列番号 1 2—人工配列の説明：合成 DNA 配列番号 1 3 -人工配列の説明：合成 DNA 配列番号 1 4一人工配列の説明：合成 DNA 配列番号 1 5 -人工配列の説明：合成 DNA 配列番号 1 6 -人工配列の説明：合成 DNA 配列番号 1 7 -人工配列の説明：合成 DNA 配列番号 1 8—人工配列の説明：合成 DNA 配列番号 1 9一人工配列の説明：合成 DNA 配列番号 2 0一人工配列の説明：合成 DNA 配列番号 2 1 一人工配列の説明：合成 DNA 配列番号 2 2一人工配列の説明：合成 DNA 配列番号 2 3一人工配列の説明：合成 DNA 配列番号 2 4—人工配列の説明：合成 DNA 配列番号 2 5一人工配列の説明：合成 DNA SEQ ID NO: 1 Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 12—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 13-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 14 Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 15-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 16-Description of Artificial Sequence: Synthetic DNA SEQ ID No. 17-Description of Artificial Sequence: Synthetic DNA SEQ ID No. 18-Description of Artificial Sequence: Synthetic DNA SEQ ID No. 19-Description of Artificial Sequence: Synthetic DNA SEQ ID No. 20 Description of One Artificial Sequence: Synthetic DNA SEQ ID No. 2 1 Description of One Artificial Sequence: Synthetic DNA SEQ ID No. 22 Description of One Artificial Sequence: Synthetic DNA SEQ ID No. 2 3 Description of One Artificial Sequence: Synthetic DNA SEQ ID No. 24—Artificial Sequence description: synthetic DNA SEQ ID NO: 25 Artificial sequence description: synthetic DNA

Claims

請求の範囲 The scope of the claims
1. ヒ卜 VEGF受容体 KDR に反応するが、 ヒ卜 VEGF受容体 Fit- 1 には反 応しないモノクローナル抗体。  1. A monoclonal antibody that reacts with human VEGF receptor KDR but does not react with human VEGF receptor Fit-1.
2. モノクローナル抗体が、 ヒ卜 VEGF受容体 KDRの細胞外領域に特異的 に結合するモノクローナル抗体である、 請求項 1記載のモノク口一ナル抗体。  2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody that specifically binds to an extracellular region of human VEGF receptor KDR.
3. モノクロ一ナル抗体が、 ヒ卜 VEGFのヒト VEGF受容体 KDRへの結合 を阻害し、 かつ KDRの作用を中和するモノクローナル抗体である、 請求項 1記 載のモノクローナル抗体。  3. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody that inhibits binding of human VEGF to human VEGF receptor KDR and neutralizes the action of KDR.
4. モノクローナル抗体が、 ヒ卜 VEGF受容体 KDRの、 配列番号 26に記載 されたアミノ酸配列 1〜518 番目の領域にあるェピトープを認識するモノクロ ーナル抗体である、 請求項 1〜3のいずれか 1項に記載のモノクローナル抗体。  4. The monoclonal antibody according to any one of claims 1 to 3, wherein the monoclonal antibody is a monoclonal antibody that recognizes an epitope in the region of the amino acid sequence 1 to 518 of the human VEGF receptor KDR shown in SEQ ID NO: 26. The monoclonal antibody according to the item.
5. モノクローナル抗体が、 ヒ卜 VEGF受容体 KDRの、 配列番号 26に記載 されたアミノ酸配列 1〜104、 1〜194、 105〜393、 295〜393および 394〜518 番 目から選ばれるアミノ酸配列の領域にあるェピトープを認識するモノクロ一ナ ル抗体である、 請求項 1〜 3のいずれか 1項に記載のモノクローナル抗体。  5. The monoclonal antibody has an amino acid sequence selected from the amino acid sequences of amino acids 1 to 104, 1 to 194, 105 to 393, 295 to 393, and 394 to 518 of human VEGF receptor KDR as set forth in SEQ ID NO: 26. The monoclonal antibody according to any one of claims 1 to 3, which is a monoclonal antibody that recognizes an epitope in a region.
6. モノクローナル抗体が、 マウス IgGlサブクラスまたはマウス IgG2bサブ クラスに属する、 請求項 1〜 5のいずれか 1項に記載のモノクローナル抗体。 6. monoclonal antibody belongs to the mouse IgGl subclass or mouse I g G2b subclass, monoclonal antibody according to any one of claims 1-5.
7. モノクローナル抗体が、 モノクローナル抗体 KM1668、 KM1992 および KM1995から選ばれる、 請求項 1〜 6のいずれか 1項に記載のモノクローナル抗 体。  7. The monoclonal antibody according to any one of claims 1 to 6, wherein the monoclonal antibody is selected from monoclonal antibodies KM1668, KM1992 and KM1995.
8. 請求項 1〜7 のいずれか 1項に記載のモノクローナル抗体を生産するハ イブリドーマ。  8. A hybridoma that produces the monoclonal antibody according to any one of claims 1 to 7.
9. ハイブリドーマが KM1668(FERM BP - 6216)、 K 1992(FERM BP - 6217)ま たは KM1995(FERM BP - 6218)である、 請求項 8記載のハイブリドーマ。  9. The hybridoma according to claim 8, wherein the hybridoma is KM1668 (FERM BP-6216), K 1992 (FERM BP-6217) or KM1995 (FERM BP-6218).
10. モノクローナル抗体が、 遺伝子組換え抗体である、 請求項 1〜7のいず れか 1項に記載のモノクローナル抗体。 10. The monoclonal antibody according to any one of claims 1 to 7, wherein the monoclonal antibody is a recombinant antibody.
11. 遺伝子組換え抗体が、 ヒト化抗体、 一本鎖抗体およびジスルフィ ド安定 化抗体から選ばれるモノクローナル抗体である、 請求項 10記載のモノクロ一ナ ル抗体。 11. The monoclonal antibody according to claim 10, wherein the recombinant antibody is a monoclonal antibody selected from a humanized antibody, a single-chain antibody, and a disulfide-stabilized antibody.
12. ヒト化抗体がヒ卜型キメラ抗体である請求項 11記載の抗体。  12. The antibody according to claim 11, wherein the humanized antibody is a human chimeric antibody.
13. ヒト型キメラ抗体が、 請求項 1〜6 のいずれかに記載のモノクローナル 抗体の抗体重鎖 （H鎖） 可変領域 (V領域)および抗体軽鎖 (L鎖) V領域と、 ヒト抗 体の H鎖定常領域 (C領域)および L鎖 C領域とからなるキメラ抗体である請求 項 12記載のヒ卜型キメラ抗体。  13. A human-type chimeric antibody comprising the antibody heavy chain (H chain) variable region (V region) and the antibody light chain (L chain) V region of the monoclonal antibody according to any one of claims 1 to 6, and a human antibody. 13. The human chimeric antibody according to claim 12, which is a chimeric antibody comprising the H chain constant region (C region) and the L chain C region.
14. H鎖 V領域およびし鎖 V領域のアミノ酸配列が、 モノクローナル抗体 KM1668(FERM BP- 6216)、 モノクローナル抗体 KM1992(FERM BP- 6217)またはモ ノクローナル抗体 KM1995(FERM BP - 6218)から選ばれるモノクローナル抗体の H鎖 V領域およびし鎖 V領域のアミノ酸配列と同じアミノ酸配列を有する、 請 求項 13記載のヒト型キメラ抗体。  14. The amino acid sequence of the H chain V region and the chain V region is selected from the monoclonal antibody KM1668 (FERM BP-6216), the monoclonal antibody KM1992 (FERM BP-6217), or the monoclonal antibody KM1995 (FERM BP-6218). 14. The human chimeric antibody according to claim 13, which has the same amino acid sequence as the H chain V region and the V chain region of the antibody.
15. ヒト化抗体が CDR (相補性決定領域） 移植抗体である請求項 11記載の 抗体。  15. The antibody according to claim 11, wherein the humanized antibody is a CDR (complementarity determining region) -grafted antibody.
16. CDR移植抗体が請求項 1〜6のいずれかに記載のモノクロ一ナル抗体の H鎖および L鎖の V領域相補性決定領域と、 ヒ卜抗体の H鎖および L鎖の C領 域および V領域フレームワーク領域とからなる抗体である請求項 15記載の CDR 移植抗体。  16. The CDR-grafted antibody comprises the V region complementarity-determining regions of the H chain and L chain of the monoclonal antibody according to any one of claims 1 to 6, and the C region of the H chain and L chain of the human antibody. 16. The CDR-grafted antibody according to claim 15, which is an antibody comprising a V region framework region.
17. H鎖 V領域および L鎖 V領域の相補性決定領域のアミノ酸配列が、 モノ クローナル抗体 KVU668(FERM BP- 6216)、 モノクローナル抗体 KM1992(FERM BP-6217)またはモノクローナル抗体 KM1995(FERM BP- 6218)から選ばれるモノ クローナル抗体の H鎖 V領域および L鎖 V領域の相補性決定領域のアミノ酸配 列と同じアミノ酸配列を有する、 請求項 16記載の CDR移植抗体。  17. The amino acid sequence of the complementarity determining region of the H chain V region and L chain V region is determined by the monoclonal antibody KVU668 (FERM BP-6216), the monoclonal antibody KM1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM BP-6218). 17. The CDR-grafted antibody according to claim 16, 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 a monoclonal antibody selected from the group consisting of:
18. —本鎖抗体が、 抗体の H鎖 V領域およびし鎖 V領域を含む、 請求項 1 1 記載の一本鎖抗体。 18. The single-chain antibody according to claim 11, wherein the single-chain antibody comprises the H chain V region and the V chain region of the antibody.
19. 一本鎖抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列が、請求項 1 〜 6から選ばれるモノクローナル抗体の H鎖 V領域および L鎖 V領域のアミノ 酸配列と同じアミノ酸配列を有する、 請求項 18記載の一本鎖抗体。 19. The amino acid sequence of the H chain V region and L chain V region of the single chain antibody is the same as the amino acid sequence of the H chain V region and L chain V region of the monoclonal antibody selected from claims 1 to 6. 19. The single-chain antibody of claim 18,
20. 一本鎖抗体の H鎖 V領域およびし鎖 V領域の相補性決定領域のアミノ酸 配列が、 請求項 1〜 6から選ばれるモノクローナル抗体の H鎖 V領域および L 鎖 V領域の相補性決定領域のアミノ酸配列と同じアミノ酸配列を有する、 請求 項 18記載の一本鎖抗体。  20. The complementarity determination of the H chain V region and L chain V region of the monoclonal antibody, wherein the amino acid sequence of the complementarity determining region of the H chain V region and the chain V region of the single chain antibody is selected from claims 1 to 6. 19. The single-chain antibody according to claim 18, which has the same amino acid sequence as that of the region.
21. —本鎖抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列が、モノクロ ーナル抗体 KM1668(FERM BP - 6216)、 モノクローナル抗体 KM1992(FERM BP- 6217)またはモノク口一ナル抗体 KM1995(FERM BP- 6218)から選ばれるモノク口 ーナル抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列と同じアミノ酸配列 を有する、 請求項 19記載の一本鎖抗体。  21. The amino acid sequence of the H chain V region and L chain V region of the main chain antibody is the monoclonal antibody KM1668 (FERM BP-6216), the monoclonal antibody KM1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM 20. The single-chain antibody having the same amino acid sequence as the H chain V region and L chain V region of a monoclonal antibody selected from BP-6218).
22. 一本鎖抗体の H鎖および L鎖の V領域の相補性決定領域のアミノ酸配列 が、 モノクローナル抗体 KM1668(FERM BP - 6216)、 モノクローナル抗体 KM1992(FERM BP- 6217)またはモノクローナル抗体 KM1995(FERM BP-6218)から 選ばれるモノクローナル抗体の H鎖 V領域および L鎖 V領域の相補性決定領域 のァミノ酸配列と同じアミノ酸配列を有する、 請求項 20記載の一本鎖抗体。  22. The amino acid sequence of the complementarity determining region of the V region of the H chain and L chain of the single chain antibody is the monoclonal antibody KM1668 (FERM BP-6216), the monoclonal antibody KM1992 (FERM BP-6217) or the monoclonal antibody KM1995 (FERM 21. The single-chain antibody according to claim 20, 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 a monoclonal antibody selected from BP-6218).
23. ジスルフィ ド安定化抗体が、抗体の H鎖 V領域およびし鎖 V領域を含む、 請求項 11記載のジスルフィ ド安定化抗体。  23. The disulfide-stabilized antibody according to claim 11, wherein the disulfide-stabilized antibody comprises an H chain V region and a chain V region of the antibody.
24. ジスルフィ ド安定化抗体の H鎖 V領域およびし鎖 V領域のアミノ酸配列 が、 請求項 1〜 6から選ばれるモノクローナル抗体の H鎖 V領域およびし鎖 V 領域のアミノ酸配列と同じアミノ酸配列を有する、 請求項 23記載のジスルフィ ド安定化抗体。  24. The amino acid sequence of the H chain V region and the V chain region of the disulfide stabilized antibody is the same as the amino acid sequence of the H chain V region and the V chain region of the monoclonal antibody selected from claims 1 to 6. 24. The disulfide stabilized antibody according to claim 23, which has
25. ジスルフィ ド安定化抗体の H鎖 V領域およびし鎖 V領域の相補性決定領 域のアミノ酸配列が、 請求項 1〜 6から選ばれるモノクローナル抗体の H鎖 V 領域およびし鎖 V領域の相補性決定領域のアミノ酸配列と同じアミノ酸配列を 有する、 請求項 23記載のジスルフイ ド安定化抗体。 25. Complementation of the H chain V region and the V chain region of the monoclonal antibody selected from claims 1 to 6, wherein the amino acid sequence of the complementarity determining region of the H chain V region and the V chain region of the disulfide stabilized antibody is selected from claims 1 to 6. The same amino acid sequence as that of the sex determining region 24. The disulfide stabilized antibody according to claim 23, which has
26. ジスルフィ ド安定化抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列 力 モノクローナル抗体 KM1668(FERM BP- 6216)、 モノクローナル抗体 M1992(FERM BP-6217)またはモノクローナル抗体 KM1995(FERM BP- 6218)から 選ばれるモノク口一ナル抗体の H鎖 V領域および L鎖 V領域のアミノ酸配列と 同じアミノ酸配列を有する、 請求項 24記載のジスルフィ ド安定化抗体。  26. Amino acid sequence of H chain V region and L chain V region of disulfide stabilized antibody Monoclonal antibody KM1668 (FERM BP-6216), monoclonal antibody M1992 (FERM BP-6217) or monoclonal antibody KM1995 (FERM BP-6218) 25. The disulfide-stabilized antibody according to claim 24, which has the same amino acid sequence as the H chain V region and L chain V region of a monoclonal antibody selected from:
27. ジスルフィ ド安定化抗体の H鎖および L鎖の V領域の相補性決定領域の アミノ酸配列が、 モノクローナル抗体 KM1668(FERM BP- 6216)、 モノクローナル 抗体 KM1992(FERM BP-6217)またはモノク口一ナル抗体 KM1995(FERM BP- 6218) , から選ばれるモノクローナル抗体の H鎖 V領域および L鎖 V領域の相補性決定 領域のアミノ酸配列と同じアミノ酸配列を有する、 請求項 25記載のジスルフィ ド安定化抗体。  27. The amino acid sequence of the complementarity determining region of the V region of the H-chain and L-chain of the disulfide-stabilized antibody is either monoclonal antibody KM1668 (FERM BP-6216), monoclonal antibody KM1992 (FERM BP-6217), or 26. The disulfide-stabilized antibody according to claim 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 a monoclonal antibody selected from antibody KM1995 (FERM BP-6218).
28. 請求項 1〜7、 10〜27のいずれか 1項に記載のモノクローナル抗体を用 いてヒト VEGF受容体 KDRを免疫学的に検出する方法。  28. A method for immunologically detecting human VEGF receptor KDR using the monoclonal antibody according to any one of claims 1 to 7, 10 to 27.
29. 請求項 1〜7、 10〜27のいずれか 1項に記載のモノクローナル抗体を用 いてヒト VEGF受容体 KDRを免疫学的に定量する方法。  29. A method for immunologically quantifying human VEGF receptor KDR using the monoclonal antibody according to any one of claims 1 to 7 and 10 to 27.
30. 請求項 1〜7、 10〜27のいずれか 1項に記載のモノクローナル抗体を用 いてヒト VEGF受容体 KDRを細胞表面に発現した細胞を免疫学的に検出する方 法。  30. A method for immunologically detecting cells expressing human VEGF receptor KDR on the cell surface using the monoclonal antibody according to any one of claims 1 to 7 and 10 to 27.
31. 請求項 1〜7、 10〜27 のいずれか 1項に記載のモノクロ一ナル抗体を用 いてヒ卜 VEGF受容体 KDRを細胞表面に発現した細胞を免疫学的に定量する方 法。  31. A method for immunologically quantifying cells expressing human VEGF receptor KDR on the cell surface, using the monoclonal antibody according to any one of claims 1 to 7 and 10 to 27.
32. 請求項 1〜7、 10〜27 のいずれか 1項に記載のモノクロ一ナル抗体を用 いてヒト VEGFとヒト VEGF受容体 KDRとの結合を阻害する方法。  32. A method for inhibiting the binding between human VEGF and human VEGF receptor KDR using the monoclonal antibody according to any one of claims 1 to 7, 10 to 27.
33. 請求項 1〜7、 10〜27 のいずれか 1項に記載のモノクローナル抗体を用 いてヒト VEGF受容体 KDRを中和する方法。 33. A method for neutralizing human VEGF receptor KDR using the monoclonal antibody according to any one of claims 1 to 7, 10 to 27.
34. 請求項 1〜7、 10〜27のいずれか 1項に記載のモノクローナル抗体を 用いる、 血管新生異常疾患の診断方法。 34. A method for diagnosing an angiogenesis disorder, using the monoclonal antibody according to any one of claims 1 to 7 and 10 to 27.
35. 請求項 1〜7、 10〜27のいずれか 1項に記載のモノクローナル抗体を 用いる、 血管新生異常疾患の治療方法。  35. A method for treating an angiogenesis disorder, using the monoclonal antibody according to any one of claims 1 to 7 and 10 to 27.
36. 請求項 1〜7、 10〜27のいずれか 1項に記載のモノクローナル抗体を 有効成分とする、 血管新生異常疾患の診断薬。  36. A diagnostic agent for an angiogenesis disorder, comprising the monoclonal antibody according to any one of claims 1 to 7 and 10 to 27 as an active ingredient.
37. 請求項 1〜7、 10〜2ァのいずれか 1項に記載のモノクローナル抗体を 有効成分とする、 血管新生異常疾患の治療薬。  37. A therapeutic agent for an angiogenesis disorder, comprising the monoclonal antibody according to any one of claims 1 to 7 and 10 to 2a as an active ingredient.
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