WO2000035956A1 - Antihuman vegf monoclonal antibody - Google Patents

Abstract

An antihuman VEGF monoclonal antibody which inhibits binding of human VEGF to two receptors KDR and Flt-1 corresponding thereto, thereby inhibiting the functions of KDR and Flt-1 and, moreover, inhibiting various biological activities of human VEGD.

Description

 Details

 Anti-human VEGF monoclonal antibody

Technical field

 The present invention provides a monoclonal antibody which specifically binds to human VEGF and inhibits binding of human VEGF to two kinds of receptors, KDR and Flt-1, a hybridoma producing the antibody, The present invention relates to a method for immunologically detecting human VEGF using the monoclonal antibody, and a diagnostic method, a diagnostic agent and a therapeutic agent using the monoclonal antibody.

Background art

 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. Furthermore, pathological conditions include solid tumor growth or metastasis, diabetic retinopathy, and angiogenesis are deeply involved in the pathogenesis and promotion of 'hidden rheumatoid arthritis' [J. Folkman et al .; J. Biol. Chem., 267, 10931 (1992)].

Angiogenesis is triggered by the secretion of angiogenic factors, which secrete proteases from endothelial cells of existing blood vessels in the vicinity, destroy the basement membrane and stroma, and subsequently migrate and proliferate vascular endothelial cells. [J. Folkman et al .; J. Biol. Chem., 267, 10931 (1992)] Vascular permeability is a factor that induces angiogenesis. factor (VPF) / Vascular endothelial growth factor (VEGF) is known as the most important factor in angiogenesis in the above-mentioned developmental stages and angiogenesis in pathological conditions [M. Shibuya; Advances in Cancer Research, Vol. 67 , 281 (1995)]. YPF / VEGF is a homodimer protein with a molecular weight of about 40,000. In 1983, as a vascular permeability factor (VPF) [DR Senger 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)], and cDNA cloning revealed that they were the same substance [DW Leung et al .; Science, 246, 1306 (1989); PJ Keck et al .; Science, 246, 1309 (1989)]. (Hereinafter referred to as VEGF).

 As for the activity of VEGF, a proliferation promoting activity (ED50 = 2-3 pM) on vascular endothelial cells has been reported [N. Ferrara et al., Biochem. Biophys. Res. Comm.,, 851 (1989)], Activity [AE Koch et al .; J. Immunology, 152, 4149 (1994)], Meta-oral protease secretion promoting activity [EN Unemori et al., J. Cell Physiol.,! 153, 557 (1992)], Secretion of perokinase and tPA Promoting activity [MS Pepper et al .; Biochem. Biophys. Res. Comm., M, 902 (1991)], Expression promoting activity of transcription factor ETS-i [C. Iwasaka et al .; J. Cell. Physiol., 169, 522 ( 1996)], the activity of increasing the expression of integrin 3 [DR Senger et al .; American J. Pathology, 149, 293 (1996)] and the like, and an angiogenesis promoting activity in vivo [T. Asahara et al .; Circulation, 92 sup j Π, 365, (1995)], and vascular permeability promoting activity [DR Senger et al .; Science, m 983, (1983)].

 VEGF has been reported to be a growth factor with extremely high specificity for vascular endothelial cells [N. Ferrara et al .; Biochem. Biophys. Res. Com, 161.851 (1989)]. It has been reported that VEGF has four types of proteins by alternative splicing [KA Houck et al .; J. Biol. Chem., 267, 26031 (1991)].

 Among diseases associated with angiogenesis, it has been reported that VEGF is deeply involved in the growth or metastasis of solid tumors, diabetic retinopathy, and pathogenesis of rheumatoid arthritis.

For solid tumors, kidney cancer [A. Takahashi; Cancer Research, 54, 4233 (1994)], breast cancer [LF Brown et al .; Human Pathology, 26, 86 (1995)], encephaloma Ryu. A. Berkman J. Clinical Investigation, 91, 153 (1993)], Gastrointestinal cancer It is reported that VEGF is produced in many human tumor tissues such as [LF Brown et al., Cancer Research, 53, 4727 (1993)], ovarian cancer [TA Olson et al., Cancer Research, 54, 276 (1994)]. Have been. Examination of the relationship between VEGF and the prognosis of patients with breast cancer revealed that tumors with high VEGF expression had more active tumor neovascularization and lower patient survival rates than tumors with low expression. [M. Toi et al .; Japanese J. Cancer Research, 85, 1045 (1994)]. In a xenograft model experimental system in which human tumors were subcutaneously implanted into nude mice, it has been reported that the anti-VEGF monoclonal antibody A4.6.1 has a tumor growth inhibitory effect [JK Kim et al .; Nature, 362, 841 (1993)]. Furthermore, it has been reported that an anti-VEGF monoclonal antibody A4.6.1 can suppress cancer metastasis in a metastatic cancer model of human tumor in a nude mouse [O. Melnyk et al .; Cancer Research, 56, 921, (1996)]. Therefore, if VEGF activity can be suppressed, it is expected that tumor growth and metastasis formation 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 and ascites retention [S. Kondo et al .; Biochimica et Biophysica Acta, 1221]. , 211 (1994)], and blocking VEGF is also expected to prevent the accumulation of cancerous pleural effusion and ascites.

 In diabetic retinopathy, abnormal angiogenesis leads to blindness through 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. [LP 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 by suppressing the VEGF activity by intraocular administration of the anti-VEGF neutralizing monoclonal antibody A4.6.1 [AP Adamis et al .; Arch Opthalmol., Lli, 66 (1996)]. Therefore, it is expected that angiogenesis in diabetic retinopathy can be suppressed by suppressing excessively produced VEGF activity.

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 VEGF at high concentrations. It has been reported that macrophages in joints produce VEGF [AE Koch et al .; Journal of Immunology, J_52, 4149 (1994); RA Fava et al .;

Experimental Medicine, Sato, 341 (1994)].

 It is expected that suppressing an excessively produced VEGF activity can suppress angiogenesis in arthritis.

 Fit-l (fms-like tyrosine kinase), the first human VEGF receptor belonging to the receptor tyrosine kinase family, has been described so far [M. Shibuya et al .; Oncogene, 5, 519 (1990); C. Vries et al .; Science, 255, 989 (1992)] and the second receptor, KDR (kinase insert domain-containing receptor) [BI Terman et al .; W092 / 14748, Priolity Feb. 22, 1991; BI Terman et al. Biochem. Biophys. Res. Comm., 187, 1579 (1992)]. A mouse homolog of the human VEGF receptor KDR is Flk-1 [W. Mat thews et al .; Proc. Natl. Acad. Science, USA, 88, 9026 (1991); A. Ul Uch et al .; W094 / 11499 Priolity Nov. 13, 1992; B. Millauer et al .; Cell, 72, 835 (1993)]. The extracellular domains of Flt-1 and KDR / Flk-1 consist of seven immoglobulin-like domains, and the intracellular domain consists of a membrane protein with a molecular weight of 180-200 kilodaltons containing a tyrosine kinase domain. VEGF specifically binds to Flt-1 and KDR / Flk-1 with KD values of 20 pM and 75 pM, respectively. FU-1 and KDR / Flk-1 have been reported to be specifically expressed in vascular endothelial cells [TP Quinn et al .; Proc. Natl. Acad. Sci. USA, 90, 7533 (1993); RL Kendall Natl. Acad. Sci. USA, 90, 8915 (1993)].

Regarding the expression of Flt-1 in various diseases, tumor vascular endothelial cells of human glioblastoma tissue [Nature, 359, 845 (1992)] and tumor vascular endothelial cells of human gastrointestinal cancer tissue [Cancer Research, 53, 4727] (1993)] reported that flt-1 mRNA expression was higher than that of vascular endothelial cells in normal tissues. In addition, the expression of fit-1 mRNA was also observed in the vascular endothelial cells of the joints of patients with rheumatoid arthritis by in situ hybridization. [J. Experimental Medicine, 180, 341 (1994)]. These results strongly suggest that the VEGF-VEGF receptor Yuichi Fit-1 system plays an important role in tumor angiogenesis. F11-1 has been reported to bind VEGF and autophosphorylate the intracellular domain [Science, 255, 989 (1992)], but its detailed function is unknown. However, in the fit-1 knockout mouse in which the fit-1 gene has been disrupted, in the early development of blood islands and subsequent angiogenesis, abnormal vascular architecture was caused by abnormal vascular endothelial cell morphology, and the embryo died at 8.5 to 9.5 days of age. Therefore, Flt-1 has been presumed to play an essential function in vascular endothelial cell lumen formation during angiogenesis [Nature, 376, 66 (1995)]. On the other hand, the expression of KDR in various human diseases has been studied in tumor vascular endothelial cells of human brain tumor tissue [E. Hatva et al .; American J. Pathology, H6, 368 (1995)], and in human gastrointestinal cancer tissues. In tumor vascular endothelial cells [LF Brown et al .; Cancer Research, 53, 4727, (1993)], it has been reported that the expression of KDR mRNA levels is increased 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.

 Furthermore, it has been reported that the expression of KDR mRNA is also observed in vascular endothelial cells of the joints of patients with rheumatoid arthritis by in situ hybridization [RA Fava et al .; J. Experimental Medicine, 180, 341 (1994)], VEGF -VEGF receptor Yuichi suggests the importance of the KDR system.

 Regarding the function of the VEGF receptor KDR / Flk-1, the expression of KDR in the vascular endothelial cells of the bushing artery causes the vascular endothelial cells to proliferate and migrate in response to VEGF. Has been reported to be involved in the proliferation and migration of vascular endothelial cells [J. Waltenberger et al .; J. Biol. Chem., 269, 26988 (1994)].

Furthermore, flk-1 knockout mice in which the mouse-type flk-1 gene was disrupted did not show any mature vascular endothelial cells, did not form yolk sac blood islands, and died in the uterus. Thus, it has been reported that KDR / flk-1 is involved in the proliferation and differentiation of vascular endothelial cells even in animal individuals [F. Shalby et al .; Nature, 376, 62 (1995)].

 As described above, among the various functions of VEGF, it is presumed that proliferation and migration of vascular endothelial cells are carried out by KDR, and luminal formation of vascular endothelial cells is carried out by Fit-1. It is presumed that the activity of other VEGFs, such as the enhancement of protein expression and the promotion of protease production, is related to any receptor, but the details of the mediation are unknown.

 Because the angiogenesis abnormalities observed in Flt-1 knockout mice and ilk-1 knockout mice are completely different, angiogenesis can be more effectively inhibited by blocking two types of receptors simultaneously. It is estimated to be.

 Anti-KDR / Flk-1 U pozyme (Ribozyme) or anti-F11-1 ribozyme, which can suppress the expression of KDR / Flk-1 or Fit-1 in vascular endothelial cells, is used for human skin. Microvessel endothelial cells can inhibit VEGF-dependent proliferation of HMVEC, but each of them is a partial inhibition, so anti-KDR / F1k-1 lipozyme and anti-F11-1 lipozyme are added simultaneously to obtain two types of It has been reported that a stronger growth inhibitory effect was observed than simultaneous suppression of body expression (W097 / 15662).

 Mutations were introduced into the amino acids of VEGF, and the amino acid sites involved in the binding of the mutated VEGF to KDR and Fit-1 were examined. As a result, the binding of VEGF to KDR is VEGF Arg82, Lys84 and His86, and the binding of VEGF to Flt-1 is Asp63, Glu64 and G1u67 of VEGF, respectively, It has been shown that the binding amino acid sites of VEGF to KDR and Flt-1 are different [J. Biol. Chem., 2JI, 5638 (1996)]. Furthermore, it has been shown that the known anti-VEGF neutralizing monoclonal antibody A4.6.1 reacts with 82 to 92 amino acid residues including the KDR binding amino acid site, but does not react with the Flt-1 binding site [ J. Biol. Chem., 2U, 5638 (1996)].

Based on the above, the various biological activities of VEGF by binding to VEGF, inhibiting the binding of VEGF to two types of receptors, KDR and Fit-1, and inhibiting the functions of KDR and Flt-1 Antibodies that can inhibit the growth of solid tumors, metastasis, It is expected to be useful for the diagnosis or treatment of diseases in which the disease progresses due to abnormal angiogenesis such as arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness in rheumatism, but the antibody has not been established. .

The antibodies established so far include a mouse VEGF neutralizing monoclonal antibody, a mouse monoclonal antibody A4.6.1 [Growth Factors, 7, 53 (1992); Nature, 362, 844 (1993)], a mouse monoclonal antibody Antibodies MV101 and ¹ MV303 [Hybridoma, li, 475 (1995); Cancer Res., 55, 5296 (1993)], and human monoclonal antibodies VA01 and BL2 (ΕΡ0787742) are known. Antibodies also bind to VEGF and inhibit the function of either KDR or FU-1 by inhibiting the binding of VEGF to either of the two existing receptors, KDR or Fit-1 It is an anti-VEGF antibody.

Disclosure of the invention

 An object of the present invention is to diagnose or treat diseases in which the disease progresses due to abnormal vascular neoplasia such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, or dryness. Anti-VEGF that can inhibit the function of KDR and F11-1 by binding to VEGF and inhibiting the binding of VEGF to two receptors, KDR and Flt-1 To provide antibodies.

 The present inventors have prepared an anti-human VEGF monoclonal antibody that recognizes VEGF, and inhibit the binding of the monoclonal antibody to two kinds of receptors for VEGF, KDR and Flt-1. The present inventors have found that the function of t-1 can be inhibited, and that various biological activities of human VEGF can be inhibited, thereby completing the present invention.

 That is, the present invention relates to the following (1) to (12).

 (1) Monoclonal antibodies that bind to human VEGF and inhibit the binding of human VEGF to human VEGF receptors KDR and FU-1.

(2) The monoclonal antibody is a monoclonal antibody belonging to the mouse IgGl subclass. The monoclonal antibody according to (1), which is a monoclonal antibody.

 (3) The monoclonal antibody according to the above (2), wherein the monoclonal antibody is KM1544 or KM1548.

 (4) Eight hybridomas producing the monoclonal antibody according to any one of the above (1) to (3).

 (5) The hybridoma according to (4) above, wherein the hybridoma is hybridoma KM1544 (FERM BP-6555) or Λ hybridoma KM1548 (FERM BP-6556).

 (6) A method for inhibiting the binding of human VEGF to human VEGF receptor KDR and Fit-1 using the monoclonal antibody according to any one of (1) to (3).

 (7) A method for inhibiting the biological activity of human VEGF using the monoclonal antibody according to any one of the above (1) to (3).

 (8) A method for immunologically detecting human VEGF using the monoclonal antibody according to any one of the above (1) to (3).

 (9) A method for immunologically quantifying human VEGF using the monoclonal antibody according to any one of (1) to (3).

 (10) A method for diagnosing a disease based on the growth or metastasis of a solid tumor or a disease progressing due to abnormal angiogenesis using the monoclonal antibody according to any one of the above (1) to (3) .

 (11) A disease which is based on the growth or metastasis of a solid tumor or whose disease progresses due to abnormal angiogenesis, comprising the monoclonal antibody according to any one of the above (1) to (3) as an active ingredient. Therapeutic drugs.

 (12) A disease based on the growth or metastasis of a solid tumor, or a disease whose condition progresses due to abnormal angiogenesis, comprising the monoclonal antibody according to any one of the above (1) to (3) as an active ingredient. Diagnostics.

Diseases whose disease progresses due to abnormal angiogenesis include diseases based on solid tumor growth or metastasis, arthritis in rheumatoid arthritis, diabetic retinopathy, Examples include retinopathy of infants and freshness.

 The monoclonal antibody of the present invention binds to human VEGF, inhibits VEGF from binding to two types of receptors, KDR and Flt-1, and thereby inhibits the functions of KDR and Fit-1. Any substance can be used as long as it can inhibit various biological activities of human VEGF.

 The functions of the human VEGF receptors KDR and Flt-1 include secretion of protease from vascular endothelial cells, degradation of the basal membrane outside vascular endothelial cells, migration and proliferation of vascular endothelial cells, and tube formation Is shown.

 Inhibition of the biological activity of human VEGF refers to inhibition of the binding of human VEGF, a ligand, to KDR and Flt-1 as receptors, thereby inhibiting the function of KDR and Flt-1. Inhibits part or all of blood, resulting in the cessation of angiogenesis.

 Examples of the monoclonal antibody of the present invention include antibodies produced by hybridomas, humanized antibodies, and human antibodies.

 A hybridoma has a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing a mammal other than human with an antigen and myeoma cells derived from a mouse or the like. Means a cell producing the monoclonal antibody.

 Examples of the humanized antibody include a human chimeric antibody and a human homology determining region.

(complementarity determining region: hereinafter abbreviated as CDR).

The human chimeric antibody is composed of an antibody non-human animal heavy chain variable region (hereinafter, the heavy chain is referred to as an H chain, and the variable region is referred to as a HV or VH as a V region) and an antibody light chain variable region (hereinafter, referred to as a light chain). Represents the L chain as LV or VL), the heavy chain constant region of the human antibody (hereinafter, the constant region is also referred to as CH as the C region), and the light chain constant region of the human antibody (hereinafter also referred to as CL). Antibodies. Non-human animals can produce hybridomas such as mice, rats, hamsters, and rabbits. Anything that works can be used.

 The human chimeric antibody is obtained by obtaining cDNAs encoding VH and VL from a hybridoma producing the anti-human VEGF monoclonal antibody of the present invention, and preparing an animal having genes encoding human antibody CH and human antibody CL. A human-type chimeric antibody expression vector can be constructed by inserting it into an expression vector for cells, and can be expressed and produced by introducing it into animal cells.

 As the CH of the human chimeric antibody, any CH may be used as long as it belongs to human immunoglobulin (hereinafter, referred to as hlg). The CH of the hlgG class is suitable, and further, h IgGl belonging to the hlgG class, Any of the subclasses h IgG2, h IgG3, h IgG4 can be used. The CL of the human chimeric antibody may be any CL as long as it belongs to Mg, and a κ class or λ class CL can be used.

 The human CDR-grafted antibody means an antibody obtained by grafting the amino acid sequence of the CDR of VH and VL of an antibody of a non-human animal at an appropriate position of VH and VL of a human antibody.

 The human CDR-grafted antibody comprises a V region obtained by grafting the VH and VL CDR sequences of an anti-human VEGF monoclonal antibody derived from a non-human animal of the present invention into the VH and VL CDR sequences of any human antibody. A cDNA encoding the human antibody is inserted into an expression vector for animal cells having genes encoding the human antibody CH and the human antibody CL to construct a human CDR-grafted antibody expression vector. By introducing one into animal cells, a human CDR-grafted antibody can be expressed and produced.

 The CH of the humanized CDR-grafted antibody may be any CH as long as it belongs to hlg, but the hlgG class is preferable, and further subclasses such as h IgGl, h IgG2, h IgG3, h IgG4 belonging to the MgG class. Can be used. The CL of the human-type CDR-grafted antibody may be any CL as long as it belongs to hlg, and a CL class / λ class can be used.

Human antibodies originally mean antibodies naturally occurring in the human body. Also included are human antibody phage libraries produced by advances in child engineering, cell engineering, and developmental engineering techniques, and antibodies obtained from transgenic animals producing human antibodies.

 Antibodies present in the human body can be isolated, for example, by isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus and the like, and cloning the lymphocytes that produce the antibodies. The antibody can be purified.

 The human antibody phage library is a library in which antibody fragments such as Fab and single-chain antibodies are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. Phage expressing an antibody fragment having the desired antigen-binding activity can be recovered from the library using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted to a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.

 The human antibody-producing transgenic animal means an animal in which a human antibody gene has been integrated into cells. Specifically, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into an early embryo of another mouse, and then developing the embryo. Human antibodies can be produced from human antibody-producing transgenic animals by obtaining human antibody-producing hybridomas by the method of producing hybridomas used in mammals other than normal humans, and culturing the culture. Human antibodies can be produced and accumulated therein.

 Examples of the antibody fragment include Fab (abbreviation of —fragment tof-antigen—binding), Fab ′, F (a), I, single-chain antibody (single chain Fv: hereinafter abbreviated as scFv), Examples include disulfide-stabilized antibodies (disul fi de stabilized Fv; dsFv), peptides containing CDRs, and the like.

Fab is a fragment obtained by treating IgG with proteolytic enzyme papain (which is cleaved at the 224th amino acid residue of the H chain), and contains about half the amino acid at the N-terminal side of the H chain. And the entire L chain are linked by disulfide bonds, and are antibody fragments having a molecular weight of about 50,000 and having antigen-binding activity.

 Fab can be obtained by treating the anti-human VEGF monoclonal antibody of the present invention with the protease papain. Alternatively, it is possible to insert the DNA encoding the Fab of the antibody into a prokaryotic or eukaryotic expression vector and introduce the vector into a prokaryotic or eukaryotic organism to express the vector, thereby producing a Fab. it can.

F (at) ') ₂ is a fragment obtained by treating IgG with the protease pepsin (which is cleaved at the 234th amino acid residue in the H chain), and Fab is a disulfide bond in the hinge region. This is an antibody fragment having a molecular weight of about 100,000 and having an antigen-binding activity, which is slightly larger than that bound through the DNA.

F (ab ') ₂ can be obtained by treating the anti-human VEGF monoclonal antibody of the present invention with proteolytic enzyme pepsin. Alternatively, the following Fab ′ can be prepared by making a thioether bond or a disulfide bond.

Fab 'is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity, in which the disulfide bond in the hinge region of F (ab') ₂ is cleaved.

Fab ′ can be obtained by treating F (al) ′) ₂ that binds to human VEGF with a reducing agent dithiothreitol. Alternatively, DNA encoding the antibody Fa fragment is inserted into a prokaryotic or eukaryotic expression vector, and Fa is expressed by introducing the vector into a prokaryotic or eukaryotic organism. can do. scFv refers to a VH-P-VL or VL_P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter, referred to as P). As the VH and VL contained in the scFv of the present invention, any of the antibodies, humanized antibodies, and human antibodies produced by the hybridoma of the present invention can be used.

The scFv is obtained by obtaining cDNAs encoding the VH and VL of the anti-human VEGF monoclonal antibody of the present invention, constructing a DNA encoding the scFv, and using the DNA for expression in a prokaryotic expression vector or eukaryotic expression vector. And the expression vector is inserted into a prokaryotic organism. Alternatively, it can be expressed by introduction into eukaryotes to produce scFv. dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL has been substituted with a cysteine residue, which is linked via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. [Protein Engineering, 7, 697 (1994)]. As the VH and VL contained in the dsFv of the present invention, any of the antibodies, humanized antibodies, and human antibodies produced by the hybridoma of the present invention can be used.

 dsFv is obtained by obtaining cDNAs encoding the VH and VL of the anti-human VEGF monoclonal antibody of the present invention, constructing a DNA encoding the dsFv, and using the DNA for expression in a prokaryotic expression vector or eukaryotic expression vector. The expression vector can be expressed and produced by introducing it into a prokaryote or eukaryote.

 A peptide containing a CDR is composed of at least one region of a heavy chain or light chain CDR. A plurality of CDRs can be linked directly or via an appropriate peptide linker.

 The peptide containing the CDR is obtained by obtaining a cDNA encoding the VH and YL of the anti-human VEGF monoclonal antibody of the present invention, constructing a DNA encoding the CDR, and converting the DNA into a prokaryotic expression vector or a eukaryotic organism. The expression vector can be expressed and produced by introducing the expression vector into a prokaryote or eukaryote.

 Further, the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

An antibody derivative refers to an antibody produced by the hybridoma of the present invention, a humanized antibody, a human antibody or an antibody fragment thereof, which is bound to a radioisotope, protein or low-molecular compound. Antibody derivatives include the N-terminal or C-terminal of the H-chain or L-chain of the anti-human VEGF monoclonal antibody or antibody fragment of the present invention, an appropriate substituent or side chain in the antibody or antibody fragment, and the antibody or antibody. Manufactured by bonding radioisotopes, proteins, or low-molecular-weight compounds to the sugar chains in the fragments by a chemical method [Introduction to Antibody Engineering (Osamu Kanemitsu, 1994, Jinjinshokan Co., Ltd.)] be able to. Alternatively, DNA encoding the anti-human VEGF monoclonal antibody or antibody fragment of the present invention and DNA encoding the protein to be ligated are ligated and inserted into an expression vector, and the expression vector is transferred to host cells. It can also be produced by genetic engineering techniques to be introduced.

Examples of the radioisotope include ¹³¹ I, I and the like. For example, the radioisotope can be bound to the antibody by the chloramine T method or the like.

Low molecular drugs include alkylating agents such as nitrogen, mustard, and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexate, antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin, and doxorubicin; Anticancer drugs, such as plant alkyloids such as vincristine, vinblastine, and vindesine, and hormones such as evening moxifen and dexamethasone [Clinical Oncology (Japanese Society for Clinical Oncology, ed. 1996, Cancer and Chemotherapy Company)] Steroids such as oral cortisone and prednisone, non-steroids such as aspirin and indomethacin, immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, maleic acid Anti-inflammatory drugs such as anti-histamines such as lupheniramine and clemacitin [Inflammation and anti-inflammatory therapy 1977, Ito Denki Shuppan Co., Ltd.], etc. For example, as a method of binding daunomycin and antibody, daltar aldehyde And a method in which the amino group of daunomycin is bound to the amino group of the antibody via a water-soluble carbodiimide. As proteins, cytokines that activate immunocompetent cells are suitable. For example, human interleukin-12 (hereinafter abbreviated as ML-2), human granulocyte-mac ora fuzzy colony-stimulating factor (hereinafter abbreviated) , HGM-CSF), human macrophage colony-stimulating factor (hereinafter referred to as hM-CSF), human interleukin-12 (hereinafter referred to as ML-12) and the like. In addition, toxins such as ricin and diphtheria toxin can be used to directly damage cancer cells. For example, for a fusion antibody with a protein, a cDNA encoding the protein is ligated to cDNA encoding the antibody or antibody fragment, a DNA encoding the fusion antibody is constructed, and the DNA is used for prokaryotic or eukaryotic organisms. A fusion antibody can be produced by inserting the expression vector into an expression vector and introducing the expression vector into a prokaryote or eukaryote to cause expression. Hereinafter, a method for producing the antibody of the present invention will be described.

 That is, a human VEGF protein or a peptide chemically synthesized based on the amino acid sequence of human VEGF protein is prepared as an antigen, and an antigen-producing cell having antigen specificity is induced from an animal immunized with the antigen. The cells are fused with a myeloma cell line to prepare a hybridoma, and the hybridoma is cultured, or the hybridoma cells are administered to an animal to cause the animal to develop ascites tumor, and the culture solution or By separating and purifying ascites, an anti-human VEGF monoclonal antibody can be obtained.

 Examples of the antigen include a human VEGF protein, a human VEGF protein having a different amino acid length, and a fusion protein of the protein and another protein.

 The human VEGF protein can be produced in cells or in a culture supernatant by culturing cells that produce human VEGF protein in an appropriate medium. Examples of the cells include human tumor cell lines such as human gloma cells G55 and human colorectal cancer COLO205 [Nature. 362, 841 (1993)].

Furthermore, DNA encoding human VEGF is obtained from the above-described cells by genetic engineering techniques, and human VEGF protein, human VEGF protein having different amino acid lengths, and Alternatively, a fusion protein of the protein and the Fc portion of an antibody can be expressed to be used as an antigen. The method is described below.

 To obtain DNA encoding human VEGF, a cDNA described in the literature [J. Biol. Chen, 268, 22782 (1993)], or cells expressing the above-mentioned human VEGF intracellularly, can be obtained by a conventional method [ Molecular Cloning 2nd edition, Cold Spring Harbor Lab. Press New York (1989); hereafter abbreviated as “Molecula 1st Cloning 2nd Edition” and Current Protocols in Molecule. CDNA from “Role 'Biology, Supplements 1-38 (Current Protocols in Molecular Biology Supplement 1-38; hereinafter abbreviated as“ Current Protocols ”in“ Molecular ”Biology, Supplements 1-38)] Create a library.

 That is, RNA is extracted, and cDNA is synthesized from the RNA. A cDNA library is prepared by incorporating the obtained cDNA into a cloning vector and introducing it into host cells.

 By selecting a transformant containing the cDNA of interest from the library, DNA encoding human VEGF can be obtained.

 Methods for preparing total RNA from cells expressing human VEGF in cells include the guanidine Z cesium chloride method and the guanidine thiosinate method [Methods in EnzymoL, 154, 3 (1987)]. Examples of a method for preparing mRNA from total RNA include a column method or a batch method using oligo dT cellulose or the like. Alternatively, mRNA can be prepared using a kit such as Fast Track mRNA mRNA Isolation Kit (manufactured by Invitrogen) or Quick Prep mRNA mRNA Pyurifique Kit (manufactured by Pharmacia).

As a method for synthesizing cDNA from the RNA obtained above, the Okamaberg method [Mol. Cell. Biol.,, 161 (1982)] ゃ Gubler-Hoffman method [Gene, 25, 263 (1983)] ] And the like. CDNA can also be obtained using kits such as Superscript, Plasmid, System, Fortran, cDNA, Synthesis ♦ and Plasmid Cloning (Gibco BRL), Zap-cDNA, Synthesis' Kit (Stratagene). It can also be synthesized.

 Any phage vector or plasmid vector may be used as the cloning vector for incorporating the cDNA, as long as it can be replicated autonomously in the host cell and can stably maintain the cDNA. Specifically, ZAP Express [Stratagene, Strategies, 5, 58 (1992)], pBluescript II SK (10) [Nucleic Acids Research, Π, 9494 (1989)], Azap II (Strata Gene), AgtlO, Agtll [DNA cloning, A Practical Approach], 丄, 49 (1985)], ATriplEx (Clontech), AEXCell ( PT7T3 18U (Pharmacia), cD2 [Molecular and Cellular Biology (Mol. Cell. Biol.), 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], Ao [J. Biol. Chem., 268, 22782-22787 (1993), also known as pAoPRC3Sc (JP-A-05-336963)].

 As the host microorganism, any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XLI-Blue MRF '(Stratagene, Strategies,, 81 (1992)), Escherichia coli C6QQ (Genetics, 39, 440 (1954)), Escherichia coli YI088 [Science, 222, 778 (1983)], Escherichia col i YI090

(Science, 222, 778 (1983)), Escherichia coli NM522 (J. Mol. Biol., 166, 1 (1983)), Escherichia coli K802 (J. Mol. Biol., ^ 6, 118 (1966) 3, Escherichia coli JM105 [Gene, 38, 275 (1985)], Escherichia coli S0LR ™ Strain (commercially available from Stratagene), Escherichia coli LE392 (Molecular 'Cloning 2nd Edition) and the like are used.

Integrate the cDNA into the above-mentioned cloning vector, A cDNA library is prepared by introduction into a host cell.

 When the cloning vector is a plasmid, the vector is introduced into a host cell by an electroporation method or a calcium chloride method. When the cloning vector is a phage, it is introduced into a host cell by in vitro packaging or the like.

 From the cDNA library obtained above, transformed strains containing DNA encoding human VEGF are described, for example, in the literature [Science, 246, 1306 (1989); Science, 246, 1309 (1989)]. A probe is prepared based on the nucleotide sequence of the DNA encoding human VEGF, and the probe is labeled with a fluorescent substance, radiation, an enzyme, or the like, and the hybridization is performed using a black hybridization, a colony hybridization, By performing Southern hybridization or the like, a transformant that hybridizes can be selected.

 A recombinant vector obtained by inserting the full length or partial fragment cDNA encoding human VEGF obtained as described above [Science, 246, 1306 (1989); Science, 246, 1309 (1989)] downstream of the promoter of an appropriate vector The human VEGF-expressing cells obtained by constructing the cells and introducing them into host cells are cultured in an appropriate medium, so that the full-length or partial fragments of human VEGF can be directly or intracellularly or in the culture supernatant. It can be produced as a fusion protein.

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 coH and Bacillus subtilis, and those of the genus Bacillus. Examples of yeast include Saccharomyces cerevisiae, Schizosaccaromyces pombe, and the like. Examples of animal cells include Namalba cells, which are human cells, COS cells, which are monkey cells, and CHO cells, which are Chinese 'hamster cells. As insect cells, Sf9, Sf21 (Pharmingen), High Five (Invitrogen) and the like are exemplified.

 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 (Escherichia coH) is used as a host, an 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. Preferred are, for example, commercially available pGEX (Pharmacia), pET system (Novagen) and the like.

 As a method for introducing a recombinant vector into a bacterium, any method can be used as long as it is a method for introducing DNA into a bacterium. For example, a method using a calcium ion [Procedures-of-the- SA (Proc. Natl. Acad. Sci., USA), 69, 2110-2114 (1972)], the protoplast method (Japanese Unexamined Patent Publication No. 248394). When yeast is used as a host, for example, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like are used as expression vectors.

 As a method for introducing a recombinant vector into yeast, a method for introducing DNA into yeast includes, for example, an elect-mouth method [Methods. Enzymol., 194. 182-187]. (1990)], the Sueeloplast Act [Proceedings of the National Academy of Sciences. Obb Science 'of the United States. SA (Proc. Natl. Acad. Sci., USA), 84 , 1929-1933 (1978)] and the lithium acetate method [Journal of Bacteriol. (J. Bacteriol.), 153, 163-168 (1983)].

When animal cells are used as a host, expression vectors such as pA GE 107 [JP-A-3-22979; Cytotechnology, 3, 133 (1990)], AGE 103 [Journal of Biochemistry (J. Biochem.), Sato, 1307 (1987)], etc. Is used.

 As the promoter, any promoter can be used as long as it can be expressed in animal cells. For example, the promoter of the IE (i-ediate early) gene of cytomegalovirus (CMV), SV40 or Promoters and the like. The enhancer of the IE gene of human CMV may be used together with the promoter.

 Examples of a method for introducing a recombinant vector into animal cells include a method for introducing DNA into animal cells, for example, the electroporation method [Cytotechnology, 3 ^ 133 (1990)], the calcium phosphate method ( JP-A-2-227075), Lipoff-execution method [Proc. Natl. Acad. Sci., USA], 84 , 7413 (1987)].

 When insect cells are used as hosts, for example, Current Protocols in Molecular Biology Supplement 1-34, baculovirus' Expression Vectors, a. Q; Proteins can be expressed by methods described in 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 the recombinant virus is further infected into insect cells to express the protein. Obtain insect cells.

 As the gene transfer vector, for example, pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like are used.

The paculovirus is, for example, a virus that infects night moth insects. Autographer · Californi force · Nuclear 1 · Polyhedosis · Virus

(Au tographa cal i forni cannul ear polyhedros is virus) and the like are used. Methods for co-transferring the above-described recombinant gene into insect cells and the above baculovirus into insect cells for preparing a recombinant virus include, for example, calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), and Lipofexion method. [Procedin's' Ob 'The National' Academy of Sciences Op. The S.A. (Proc. Natl. Acad. Sc i., USA), 84, 7413 (1987 )] Is used.

 In addition, after producing a recombinant paculovirus using a Baculo Logo Stardust kit manufactured by Farmingen, etc., the recombinant virus is transferred to the insect cells such as Sf9, Sf21 or High Five described above. Can also be used to produce proteins by infecting [Biotechnology (Bio / Technology), 10, 457 (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, it can be carried out according to the method described in "Molecula-cloning 2nd edition". The proteins to be fused include i3-galactosidase, protein A, the IgG-binding region of protein A, chloramphenicol-acetyltransferase, poly (Arg), poly (Glu), protein G, maltose-binding protein, and Dalbin. Thione S-transferase, polyhistidine chain (His-tag), S peptide, DNA binding protein domain, Tac antigen, thioredoxin, green fluorescein protein, and any antibody epitope [ Akio Yamakawa Experimental Medicine, 13, 469-474 (1995)].

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 human VEGF as it is or as it is. It can be produced as a fusion protein. The above-described method of culturing the transformant in a medium is performed by the usual method used for culturing a host. It is done according to the method.

 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 it can be used (Molecular 'Cloning 2nd edition). Cultivation is usually carried out at 15 to 40 ° C for 16 to 96 hours under aerobic conditions such as shaking culture or deep aeration stirring culture. During the culture period, the pH is maintained at 3.0-9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like. During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed.

As a medium for culturing the transformant obtained using animal cells as a host, commonly used RPM11640 medium, Eag1e MEM medium, or a medium obtained by adding fetal calf serum or the like to such a medium can be used. Can be Cultivation is usually performed at 35 to 37 ° C. for 3 to 7 days in the presence of 5% C _2, and if necessary, antibiotics such as kanamycin and benicillin may be added to the medium during the cultivation.

 As a medium for culturing a transformant obtained by using an insect cell as a host, commonly used TNM-medium [Pharmingen], Sf90011 SFM [Life Technologies ( Life Technologies), ExCelHOO, ExCell405 [all manufactured by JRH Biosciences] and the like. The culture is performed for 25 to 3 days (TC for 1 to 4 days). During the culture, an antibiotic such as mycin mycin may be added to the medium as needed.

 In the above, when culture can be performed in a medium without serum added to the medium of animal cells and insect cells, the full-length or partial fragment of human VEGF can be used as it is or the fusion protein can be easily purified. It is preferable to use a culture medium without addition.

Human VEGF full length or partial fragment as it is or as fusion protein host If it accumulates in the cells, after the culture is completed, centrifuge the cells to obtain the cells. After suspending the cells in an aqueous buffer, the cells are disrupted by an ultrasonic method, a French press method or the like. The obtained cell lysate is centrifuged, and the protein is recovered from the supernatant.

 When an insoluble form of human VEGF is formed in cells, the insoluble form is solubilized with a protein denaturant, and then diluted to a solution that does not contain the protein denaturant or is diluted to such a concentration that the protein denaturant does not denature the protein Alternatively, the protein can be dialyzed to form a three-dimensional structure of the protein.

 When the full length or partial fragment of human VEGF 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.

 A polypeptide having a partial sequence of human VEGF can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarponyl method). Also, Kuwawa Trading (US Advanced ChemTech), Perkin-Elmer Japan (US Perkin-Elmer), Aroka (US Protein Technology Instrument), Kurabo (US Synthecel Vega), It can also be produced using a peptide synthesizer such as Nippon Perceptive, Limited (US PerSeptive) or Shimadzu Corporation.

Animals used for immunization are non-human animals such as mice, rats, hamsters, and rabbits, and any animal can be used as long as hybridomas can be produced. The non-human animal is immunized with the protein obtained above as an antigen, and antibody-producing cells are collected from spleen, lymph nodes, and peripheral blood of the animal. How to immunize May be administered subcutaneously, intravenously or intraperitoneally to an animal, but it is preferable to administer the antigen by binding a carrier protein with high antigenicity, or to administer the antigen together with an appropriate adjuvant. . Carrier proteins include keyhole limpet hemosinin, keyhole limpet hemosinin, bovine serum albumin, bovine thyroglobulin, etc. Adjuvants include Freund's complete adjuvant (Co immediately lete Freund's Adjuvant), hydroxylation Aluminum gel and B. pertussis vaccine.

 The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus or tail vein of the immunized animal 3 to 7 days after each administration, and the reactivity of the serum with the antigen is confirmed by an enzyme immunoassay or the like. [Enzyme immunoassay (ELISA)]: Published by the Medical Shoin (1976)], an immunized animal whose serum shows a sufficient antibody titer is used as a source of antibody-producing cells. Three to seven days after the last administration of the antigen, a method known from the immunized animal [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Manual, Cold Spring Harbor Laboratory, 1988 In the following, lymphocytes are excised and fused with lymphocytes and myeloma cells in accordance with the Antipodes. Laboratory Manual.

As myeloma cells, 8-azaguanine-resistant mice (derived from BALB / c) myeloma cell line P3-X63Ag8-Ul (P3-Ul), a cell line obtained from mice [G. Kohler et al. Pian Journal of Immunology (Europ. J. Immunol.), 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [M. Shulman et al .; Nature, 276, 269 ( 1978)], P3-X63-Ag8653 (653) [JF Kearney et al., Journal of Obiminology (J. I. 麵 1.), 123, 1548 (1979)], P3-X63-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 by known methods (antipodes laboratory manual. ) According to ensure 2 X 1 0 ⁷ or more cell numbers until cell fusion.

 After washing the antibody-producing cells and myeloma cells obtained above,

Add a cell-aggregating medium such as Glyco-Rue 1000 (PEG-1000) to fuse the cells and suspend them in the medium. For washing the cells, use MEM medium or PBS (1.83 g of sodium phosphate phosphate, 0.21 g of phosphate monophosphate, 7.65 g of salt, 1 liter of distilled water, H7.2) or the like. In addition, as a medium for suspending the fused cells, a HAT medium (normal medium [Dalmin (1.5 に), 2-mercapto) in RPMI-1640 medium, so that only the desired fused cells can be selectively obtained. ethanol ⁽⁵ X 10- 5 M), Jen evening mycin (10 g / ml) and fetal calf serum (FCS) (CSL Ltd., 10%) Hipokisa Nchin the added medium] (10- ⁴ M), thymidine (1. ⁵ X 10- 5 M) and aminopterin (4X 10- ⁷ M) was added medium) is used.

 After the culture, a part of the culture supernatant is taken, and a sample that reacts with the antigen protein and does not react with the non-antigen protein is selected by the following enzyme immunoassay. Next, cloning is performed by the limiting dilution method, and those with a stable and high antibody titer determined by enzyme immunoassay are selected as monoclonal antibody-producing hybridoma strains.

 Enzyme immunoassay

The antigen protein or cells expressing the antigen protein are coated on a plate, and the antiserum, hybridoma culture supernatant or the purified antibody obtained by the above method is reacted as the first antibody, and the second antibody is biotin, an enzyme. After reacting the immunoglobulin antibody against the animal species of the primary antibody labeled with a chemiluminescent substance or a radioactive compound, a reaction corresponding to the labeling substance is performed, and a monoclonal antibody that specifically reacts with the antigen is produced. Select as dormer. Specific examples of the hybridoma strain of the present invention include the hybridoma strains KM1544 and KM1548. The Hypridoma strains K1544 and KM1548 were registered on October 27, 1998 with the Institute of Biotechnology and Industrial Technology, Institute of Bio-Science and Technology (1-1-3 Tsukuba-Higashi, Ibaraki, Japan) and FERM BP-6555, respectively. Has been deposited as FERM BP-6556 You.

 Monoclonal antibodies were obtained by intraperitoneal administration of 5 ml of a culture solution obtained by culturing eight hybridoma cells or treated with pristane [2,6,104-tetratetramethylpentane (Pristane) O. 5 ml. The monoclonal antibody-producing hybridoma cells are intraperitoneally administered to the mouse or nude mouse of 8 to 10 weeks of age, and then separated and purified from ascites caused by ascites tumor.

 Methods for separating and purifying monoclonal antibodies include centrifugation, salting out with 40-50% ammonium sulfate, caprylic acid precipitation, DEAE-Sepharose column, anion exchange column, and protein A or G-column. Alternatively, there is a method in which chromatography using a gel filtration column or the like is performed alone or in combination. By 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.

 Antibody subclasses refer to isotypes within a class, including IgGl, IgG, IgG2b, and IgG3 in mice and IgGl, IgG2, IgG3, and IgG4 in humans, and particularly mouse IgGl, IgG2a, and human IgGl types. It has complement-dependent cytotoxic activity (hereinafter referred to as CDC activity) and antibody-dependent cytotoxic activity (hereinafter referred to as ADCC activity), and is useful for therapeutic applications.

 The present invention also relates to a method for immunologically detecting and quantifying human VEGF using the monoclonal antibody of the present invention.

Methods for immunologically detecting and quantifying human VEGF using the monoclonal antibody or the antibody fragment thereof of the present invention include a fluorescent antibody method, an immunoenzymatic antibody method (ELISA), and a radioactive substance labeled immunological antibody method (RIA). , Immunohistochemical staining, immunohistochemical staining such as immunocytostaining (ABC, CSA, etc.), Western blotting, Epidemiological sedimentation method, enzyme immunoassay method described above, sandwich ELISA method [Monoclonal Antibody Experiment Manual (Kodansha Scientific, 1987), Seismic Chemistry Laboratory Course 5 Immunobiochemical Research Method (Tokyo, Japan) Chemistry Doujinshi, 1986).

 The fluorescent antibody method is a reaction of the monoclonal antibody of the present invention or an antibody fragment thereof with isolated cells or tissues, and further, an anti-immunoglobulin antibody or a binding antibody labeled with a fluorescent substance such as fluorescin / isothiosinate (FITC). After reacting the fragments, a fluorescent dye is measured by a flow cytometer. The immunoenzymatic antibody method (ELISA) refers to the monoclonal antibody of the present invention isolated on cells or its lysate, tissue or its lysate, cell culture supernatant, serum, pleural effusion, ascites, eye fluid, etc. Alternatively, the antibody fragment is reacted with an anti-immunoglobulin antibody or binding fragment that has been labeled with an enzyme such as peroxidase or biotin, and then the colored dye is absorbed.

This is a method of measuring with a photometer.

 The radioactive substance-labeled immunoassay (RIA) refers to the isolated cells or their lysates, tissues or their lysates, cell culture supernatants, serum, pleural effusion, ascites, eye fluids, etc. This is a method in which an antibody or an antibody fragment thereof is reacted, and further reacted with a radiolabeled anti-immunoglobulin antibody or a binding fragment, followed by measurement using a scintillation counter or the like.

 The immune cell staining method and the immunohistochemical staining method are as follows: the isolated cells or tissues are reacted with the monoclonal antibody of the present invention or an antibody fragment thereof, and further, a fluorescent substance such as fluorescin / isothiocyanate (FITC); This is a method in which an anti-immunoglobulin antibody or a binding fragment labeled with an enzyme such as peroxidase or biotin is reacted, and then observed using a microscope.

The Western plotting method is based on SDS-polyacrylamide of cells or their lysates, tissues or their lysates, cell culture supernatants, serum, etc. After fractionation by gel electrophoresis and electrophoresis [Antibodies—A Laboratry Manual, Cold Spring Harbor Laboratory, 1988], the gel was plotted on a PVDF membrane or a nitrocellulose membrane, and the present invention was applied to the membrane. The monoclonal antibody or its fragment is reacted, and further reacted with a fluorescent substance such as FITC, an anti-mouse IgG antibody or a binding fragment labeled with an enzyme such as peroxidase or biotin, and then checked.

 The immunoprecipitation method refers to a method of reacting a cell or its disrupted solution, a tissue or its disrupted solution, a cell culture supernatant, a serum, or the like, separated from a living body with a monoclonal antibody or an antibody fragment thereof of the present invention, followed by protein G -An antigen-antibody complex is precipitated by adding a carrier having specific binding ability to immunoglobulin such as Sepharose.

 The sandwich ELISA method is a monoclonal antibody or an antibody fragment thereof of the present invention. Of two types of monoclonal antibodies having different antigen recognition sites, one of the monoclonal antibodies or antibody fragments is adsorbed on a plate in advance, and the other is monoclonal. Antibodies or antibody fragments should be labeled with a fluorescent substance such as FITC or an enzyme such as peroxidase or biotin. After reacting the antibody-adsorbed plate with cells or its lysate, tissue or its lysate, cell culture supernatant, serum, etc., separated from the living body, react the labeled monoclonal antibody or its antibody fragment with the labeled substance. This is a method for performing a corresponding reaction.

 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, human VEGF present in cells or tissues of a subject is detected by immunological detection as described above. Alternatively, a quantification method may be used. In addition, the monoclonal antibody or the antibody fragment thereof of the present invention can be used as a diagnostic drug 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 angiogenesis.

In addition, the activity of inhibiting the binding of human VEGF to human VEGF receptor KDR and the activity of human VEGF The activity of inhibiting the binding of VEGF receptor Fit-1 can be 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 and a VEGF-VEGF receptor Fit-1 binding inhibition test using a monoclonal antibody according to the method described in (1).

 That is, cells or tissues expressing Flt-1 or KDR are reacted with VEGF labeled with a radioactive substance, and VEGF bound to cells or tissues expressing F11-1 or KDR is treated with a scintillation counter or the like. It is a method of measuring. By reacting the antibody of the present invention simultaneously with VEGF labeled with a radioactive substance, the activity of inhibiting the binding of VEGF labeled with a radioactive substance to Flt-1 or KDR can be measured. It is.

 The autophosphorylation inhibitory activity of VEGF receptor KDR and VEGF receptor Fit-1 is measured by the method of measuring the autophosphorylation of growth factor receptor [Sequence Chemistry Laboratory Lecture Information transmission and cellular response (Tokyo Chemical Dojin, 1998 6 years)], and can be confirmed by performing an autophosphorylation inhibition test of VEGF-VEGF receptor KDR and VEGF-VEGF receptor Flt-1 using monoclonal antibodies .

 That is, VEGF is reacted with cells or tissues expressing Flt-1 or KDR, and the autophosphorylation of FU-1 or KDR, which is enhanced by the binding of VEGF, is immunoprecipitated and Western blotted. It is a method to detect by such as. By reacting the antibody of the present invention simultaneously with VEGF, it is possible to measure the activity of inhibiting the autophosphorylation of Flt-1 or KDR, which is enhanced by the binding of VEGF.

 The inhibitory activity of the biological activity of human VEGF on VEGF-dependent vascular endothelial cell proliferation, migration, and tube formation test. 10 Biochemical Experiment Lectures 10 Blood vessels (endothelium and smooth muscle) (Tokyo Kagaku Dojin, 1992 1 year)].

The VEGF-dependent vascular endothelial cell proliferation test is performed by reacting vascular endothelial cells with VEGF and measuring the number of vascular endothelial cell growth-promoting activities that are enhanced by the binding of VEGF. It is a method. By reacting the antibody of the present invention simultaneously with VEGF, it is possible to measure the activity of inhibiting the growth promoting activity of vascular endothelial cells promoted by VEGF.

 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 migration of vascular endothelial cells, which is enhanced by 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 enhanced by VEGF.

 The VEGF-dependent tube formation test of vascular endothelial cells is a method in which VEGF is reacted with vascular endothelial cells and the activity of promoting tube formation 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 enhanced by VEGF.

 Further, the monoclonal antibody of the present invention inhibits the functions of KDR and Fit-1 by inhibiting the binding of VEGF to two kinds of receptors, KDR and Fit-1, as described above. Since it can inhibit the various biological activities of human VEGF, it can be used as a therapeutic agent for diseases based on the growth or metastasis of solid tumors induced by excessive VEGF, or diseases in which the disease progresses due to abnormal vascular growth. it can.

BRIEF DESCRIPTION OF THE FIGURES

FIG. I shows the results of analyzing the binding activity of human VEGF monoclonal antibody to human VEGF.

FIG. 2 shows the result of analyzing the binding activity of human VEGF monoclonal antibody to human VEGF.

FIG. 3 shows the results of analyzing the binding activity of human VEGF monoclonal antibody to human VEGF.

Fig. 4 Study of human VEGF quantification system using human VEGF monoclonal antibody The results are shown.

FIG. 5 shows the results of analyzing the binding inhibitory effect of human VEGF monoclonal antibody between VEGF and soluble VEGF receptor KDR and soluble VEGF receptor F1 tol.

FIG. 6 shows the results of examining the KDR autophosphorylation inhibitory effect and the Fit-1 autophosphorylation inhibitory effect of a human VEGF monoclonal antibody.

FIG. 7 shows the results of examining VEGF-dependent vascular endothelial cell growth inhibitory activity of human VEGF monoclonal antibodies.

FIG. 8 shows the results of examining the in vivo antitumor effect of the human VEGF monoclonal antibody.

FIG. 9 shows the results of examining the in vivo antitumor effect of the human VEGF monoclonal antibody.

FIG. 10 shows the results of examining the in vivo antitumor effect of the human VEGF monoclonal antibody.

FIG. 11 shows the construction of a fusion gene expression vector Pv1-KDR-7N-Fc comprising a soluble human VEGF receptor KDR fragment and a human antibody Fc region.

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1

 1. Preparation of antigen

 (1) Expression and purification of human VEGF in insect cells

Human VEGF 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 (manufactured by Grainer), allowed to stand at room temperature for 1 hour, and allowed to adhere to the flask. A human VEGF recombinant paculovirus solution obtained by the method described in the literature [Cell Growth & Differentiation ,; ^ 213 (1996)] was used for about 1 to 3 × 10 ⁸ PFU. 1 ml of a solution containing a concentration of 1 ml / ml was added, and the cells were infected at room temperature for 2 hours. The culture supernatant was removed, and 30 ml of a fresh 30 ml EX-CELL ™ 400 medium was added, followed by culturing at 27 ° C for 3 to 4 days. End of culture After completion, the culture supernatant was collected and centrifuged at 1,500 X g for 10 minutes to obtain a supernatant.

 The column was filled with approximately 40 ml of heparin-Sepharose CL-6B gel [Pharmacia Biotech AB] and 0.5 ml / min with 400 ml of 20 mM Tris-HCl (ρ (7.5) buffer. Wash at flow rate. 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. Further, the column was washed with 20 ^ tris-hydrochloric acid 117.5) 4001111 at a flow rate of 0.5 ml / min. A 20 mM Tris-HCl (pH 7.5) buffer solution and a 20 mM Tris-HCl (pH 7.5) buffer solution containing 1 mM NaCl were passed successively in 120 ml portions to elute the protein adsorbed on heparin-sepharose stepwise. The eluate was fractionated in 8 ml portions. The protein contained in each fraction was analyzed by SDS-PAGE, and 120 ml of a fraction containing human VEGF (0.5-1 M NaCl fraction) was recovered. The fractions were concentrated with Centrrep-10 (manufactured by Amicon), and 4 ml (protein concentration: 1.2 mg / ml) of human VEGF was obtained as a solution.

Next, 480 ig of VEGF was dissolved in 6001 of a 0.1 M CH ₃ C00NH ₄ (pH 7) solution. To the VEGF solution 51, 120 g of mocyanin (KLH; manufactured by Calbiochem) was added to and dissolved in the keyhole limpet, and 30 l of 1% dal aldehyde was added, followed by stirring at room temperature for 5 hours. Dialysis was performed against PBS to obtain a VEGF-KLH fusion as an antigen for immunization.

 (2) Purification of human VEGF negative control antigen protein

As a soluble control VEGF negative control antigen protein, a heparin column-binding protein of the culture supernatant of High Five cells prepared as follows was used. 4 x 10 ⁷ High Five cells are suspended in 30 ml of EX-CELL ™ 400 medium (manufactured by JRH Bioscience) in a 175 cm ² flask (manufactured by Grainer), allowed to stand at room temperature for 1 hour, and allowed to adhere to the flask. The cells were cultured at H ° 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.

Heparin-Sepharose CL-6B gel on column [Pharmacia Biotech (Pharmacia Biotech, AB)] About 20 ml was packed, and the column was washed with 200 ml of a 20 mM Tris-hydrochloric acid (pH 7.5) buffer at a flow rate of 0.5 ml / min. After washing, 500 ml of the culture of High Five cells prepared as described above was passed through a heparin-cepharose 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, a buffer consisting of 20 mM Tris-HCl (pH 7.5) containing 1 M NaCl is used. The solution was passed through 200 ml, and the protein adsorbed on heparin-sepharose was eluted. The 1M NaCl-eluted fraction was concentrated using CentriBrep 10 (manufactured by Amicon) to obtain 7 ml (protein concentration: 867 g / ml) of a control antigen protein as a solution.

 2. Animal immunization and preparation of antibody-producing cells

1) 12.5 g of the VEGF-KLH fusion obtained from (2) was added to RIBI Adj uvant System (manufactured by RIBI ImmunoChei Research In) 0.1 ml or aluminum gel 2 mg and pertussis vaccine (manufactured by Chiba Prefectural Serum Institute) A 5-week-old female BALB / c (manufactured by SLC Japan) was administered together with 1 × 10 ⁹ cells, and the protein was administered once a week after 2 weeks, a total of 4 times.

 Blood was collected from the fundus venous plexus or the tail vein, and its serum antibody titer was examined by the enzyme immunoassay described below, and the spleen was excised 3 days after the last immunization from the mouse showing a sufficient antibody titer.

 The spleen was shredded in MEM medium (manufactured by Nissui Pharmaceutical), loosened with forceps, and centrifuged at 200 rpm for 5 minutes. Thereafter, the supernatant was discarded, and treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, washed three times with MEM medium, and used for cell fusion.

 3. Enzyme immunoassay

1 For the measurement of the antiserum derived from the mouse immunized with the VEGF-KLH fusion obtained in (2) and the culture supernatant of the hybridoma described below, the antigen was obtained from the insect cell culture supernatant of 1). Human VEGF was used. 96 ゥ L EIA plate (Dara Heparin column-adsorbed fraction of human VEGF and the High Five cell culture supernatant obtained in step 1 as a control antigen was diluted to 1 to 10 g / ml with PBS. The plate was left overnight at 4 ° C to adsorb the antigen. After washing the plate, PBS containing 1% bovine serum albumin (BSA) was dispensed at 100 1 / ゥ l and allowed to react at room temperature for 1 hour to block the remaining binding residues on the plate. Thereafter, 1% BSA-PBS was discarded, and the culture supernatants of the immunized mice and hybridomas were dispensed at 50 il / well and reacted for 2 hours. After washing with PBS containing 0.05% (V / V) tween20 (hereinafter abbreviated as tween-PBS), add peroxidase-labeled heron antimouse immunoglobulin (manufactured by DAK0) at 50 1 / well. Left for 1 hour at room temperature.

 After washing with 0.05-tween-PBS, 550 mg of ABTS substrate solution [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium] was added to 1 L of 0.1 M citrate buffer (pH 4.2). The solution was added with 1 l / ml of hydrogen peroxide immediately before use], and the absorbance at 0D415 nm E max [Molecular Devices] (Molecular Devices) was measured.

 4. Preparation of mouse myeloma cells

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. Preparation of hybridoma

The mouse spleen cells obtained in 2 and the myeloma cells obtained in 4 were mixed at a ratio of 10: 1, and centrifuged at 1,200-111 for 5 minutes. Discard the supernatant of the mixture, thoroughly dissolve the precipitated cell group, and mix with 2 g of polyethylene glycol 1000 (PEG-1000), 2 ml of ME medium and 0.7 ml of DMSO at 37 ° C with stirring. 0. 2~lml / (10 ⁸ mouse spleen cells) was added, and MEM medium is added several times l~2ml every 1-2 minutes, the total amount added MEM medium was made up to 50 ml. After centrifugation at 900 rpm for 5 minutes, the supernatant is discarded, the precipitated cell group is loosened gently, and then sucked and sucked with a female pipette. The cells are gently removed by HAT medium [RPMI 1640 medium containing 10% FCS ( Nissui Pharmaceutical Co., Ltd.) To which 100 nM hypoxanthine, 0.4 nM aminopterin, and 16 nM thymidine (manufactured by Behringer Mannheim) were added.

The suspension was dispensed by 100 A / Ueru the plate for 96 Ueru culture in 5% C0 ₂ incubator one, were cultured in 10 to 14 days at 37 ° C. The enzyme immunoassay described in Example 1-3 above was performed using each of the culture supernatants obtained by the culture. In the manner described above, human VEGF and the negative control antigen protein prepared with 1 (2) were adsorbed on a 96-well plate. Each cell culture supernatant was dispensed at 100/1 / well into the plate and left at room temperature for 2 hours.

 After the plate was washed with PBS, a peroxidase-labeled mouse heron anti-mouse immunoglobulin antibody was dispensed at 100 / X1 / well, and left at room temperature for 1 hour.

 As a result, a well of cell culture supernatant which reacted with human VEGF but did not react with the control antigen was selected. The hybridoma cells that produced the culture supernatant in the selection well were obtained by performing the cloning operation twice by the following limiting dilution method. Figure 1 shows the results.

 The cloning operation by the limiting dilution method involves re-rolling cells in a 96-well plate at 1 cell / well, culturing the hybridoma cells for 7 to 14 days, and then performing enzyme immunization on the cell culture supernatant. This is an operation of performing measurement, selecting a well of a cell culture supernatant that reacts with human VEGF and does not react with a control antigen, and obtains hybridoma cells that have produced culture supernatant in the selected well.

 Hybridomas were prepared from the three immunized mice of the above 2 to obtain a total of 12 kinds of anti-human VEGF monoclonal antibodies KM1537, intestine 38, TZ39, KM1540, KM154U KM1542, intestine 43, KM1544, KM1545, thigh 546, KM1547 and KM1548 Was.

The antibody class of the monoclonal antibody was determined by enzyme immunoassay using a subcluster typing kit (Zymed). The results are shown in Figure 1 below. KM1538 was of IgG3 class, KM1542 was of IgG2a class, and the remaining 10 monoclonal antibodies were all of IgGl class. 6. Purification of monoclonal antibodies

 Eight-week-old nude female mice (Balb / c) treated with pristane were intraperitoneally injected with each of the 5 to 20 X Z cells of the Hypridoma strain obtained in 5 above. After 10 to 21 days, the hybridoma became ascites carcinoma. Ascites was collected (1-8 ml / animal) from the mice that had accumulated ascites, and the solid content was removed by centrifugation at 3, OOO rpm for 5 minutes.

 The ascites fluid from which solids had been removed was purified by the force prillic acid precipitation method (Anti-Podise Laboratories' Manual) to obtain a purified monoclonal antibody.

 7.Examination of reactivity of monoclonal antibody to VEGF

 The reactivity of the anti-human VEGF monoclonal antibody obtained in 5 with VEGF was confirmed using the enzyme immunoassay described in 3.

 (1) Examination of VEGF concentration for plate coating

 Prepare a 4-fold dilution series (0.0078 to 8 g / ml) of VEGF obtained by the method described in 1 (1) above with PBS in a 96-well microplate for ELISA. The reaction was performed at 4 ° C for 1 晚. After the plate was washed with PBS, 1% 834-? 83 was reacted at 200/1 / well at room temperature for 30 minutes, and then washed with PBS. As a negative control antibody, a mouse monoclonal antibody (KM12570TO97 / 10354) that belongs to the mouse IgGl class and does not react with human VEGF but reacts with the human interleukin 5 (hereinafter abbreviated as IL-5) receptor chain is used. Was. The purified anti-VEGF monoclonal antibody obtained in 6 and the negative control antibody KM1257 were added at a concentration of 10 g / ml at a concentration of 501 / well, and reacted at room temperature for 2 hours. After washing with 0.05% Tween-PBS, a peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) diluted 400-fold with BSA-PBS was dispensed into 50 / l / well and left at room temperature for 1 hour. Reacted. After washing with 0.05% Tween-PBS, color was developed using ABTS substrate solution and the absorbance at 0D415nm was measured.

The result is shown in figure 2. As a result, it was revealed that 12 kinds of anti-VEGF monoclonal antibodies reacted with VEGF in a VEGF concentration-dependent manner, while the negative control antibody did not react with VEGF at all. VEGF detection limit of KM1538 is 2 ig / ml Although the sensitivity was poor, the VEGF detection limit of other anti-VEGF monoclonal antibodies was as good as 0.0078 to 0.125 // g / ml.

 (2) Examination of anti-VEGF monoclonal antibody concentration to be detected

 A 50 μl / well of 2 × g / ml VEGF diluted with PBS was added to a 96-well plate for ELISA using a 50 μl / well plate and reacted at 4 ° C. for 1 hour. After the plate was washed with PBS, 1% BSA-PBS was added at 200 1 / well, reacted at room temperature for 30 minutes, and washed with PBS. Purified anti-VEGF monoclonal antibody was prepared in a three-fold dilution series (0.0041 to lig / ml) with 1% BSA-PBS, and each was added at 50 l / well and allowed to react at room temperature for 2 hours. After washing with 0.05% Tween-PBS, a peroxidase-labeled anti-mouse immunoglobulin antibody diluted 400-fold with BSA-PBS was dispensed into 50 1 / well and allowed to react at room temperature for 1 hour. After washing with 0.05% Tween-PBS, color was developed using ABTS substrate solution and the absorbance at 0D415nm was measured.

 The results are shown in Figure 3. As a result, it was revealed that 11 kinds of anti-VEGF monoclonal antibodies except KM1538 react with VEGF in an antibody concentration-dependent manner. The concentration of anti-VEGF monoclonal antibody at the detection limit of VEGF was as high as 0.0041 to 0.0123 g / ml. 8. Epitope analysis of anti-VEGF monoclonal antibody using the binding inhibition experiment system

1 ig / ml human VEGF diluted with PBS was dispensed at 50 1 / well into a 96-well plate for EIA (manufactured by Grainer Co., Ltd.), and allowed to stand overnight at 4 ° C for adsorption. After washing the plate, 1% BSA-PBS was added at 100 a1 / well, and the mixture was reacted at room temperature for 1 hour to block the binding residues remaining on the plate. After that, discard the 1% BSA-PBS, add 50 l of purified anti-human VEGF monoclonal antibody diluted to 0.1 or lO g / ml with 1% BSA-PBS. : 1 g / mK for KM1539, KM1545 and KM1546 labeled with anti-human VEGF monoclonal antibody (Interdisciplinary Journal, 1985) 0.2 g for KM1537, KM1540, KM1541, KM1542, KM1543, KM1544, KM1547, KM1548 ig / ml) and reacted at room temperature for 2 hours. After washing with 0.05% Tween-PBS, avidin-labeled peroxidase (manufactured by VECTOR) diluted 4,000-fold with 1 BSA-PBS was added at 50 ^ 1 / Pell and reacted at room temperature for 1 hour. . 0.05% After washing with tween-PBS, color was developed using ABTS substrate solution, and the absorbance at 0D415 nm was measured. Table 1 shows the results. Addition of 1 / xg / ml purified monoclonal antibody has an activity that inhibits binding of pyotinylated antibody by 80% or more ++ 10.Addition of 1 g / ml purified monoclonal antibody reduces binding of pyotinylated antibody by 50-80. The activity that was inhibited by +++ and the activity that inhibited the binding of the biotinylated antibody by 50% or more by adding a purified monoclonal antibody at 10 g / ml was +.

 Table 1

Bisai Chin-Dani antibody 1539 1537 1540 1542 1543 154B 1548 1547 1544 1541 1545

 (KM No.) _ ―

 Inhibitory antibody

 KM 1538--one-one------

KM1537 +++++

 KM 1539 ++++

 KM 1542 ++ ++ +++ +

 KM 1540 11 ++

 K 1544 ++ +++ +++ ++ +

 KM 1548 + ++ ++ + +

 KM1547 ++++++++

 M1543 +++++++

 K 1546 + +

 K 1541 ++

+ 10 g / ml:> 50% inhibition

 ++ 1 / ig / ml:> 50% inhibition

 +++ 1 Aig / ml:> 80% inhibition

As a result, the biotinylated antibodies KM1539, KM1537, KM1540, and KM1542 were inhibited by the purified monoclonal antibody KM1542, and similarly, the biotinylated antibodies KM1543, KM1546 KM1548, KM1547, and KM1544 were inhibited by the purified monoclonal antibody KM1548. In addition, the biotinylated antibodies KM1541 and KM1545 were inhibited by the purified monoclonal antibody KM1541. From the above, KM1539, KM1537, KM1540, and KM1542 are each epi! It was found that the peptides were similar, and that KM1543, KM1546, KM1548, KM1547 and KM1544, and KM1541 and KM1545 had similar epitopes.

 9. Preparation of VEGF quantitative system

 Dispense 50 μl / well of anti-human VEGF monoclonal antibody KM1541 diluted to 10 / xg / ml with PBS into a 96-well EIA plate (manufactured by Grainer) at 4 ° C. Leave to adsorb. After washing the plate, 100% / well of 1% BSA-PBS was added, and the mixture was reacted at room temperature for 1 hour to block the binding residue remaining on the plate. The 1% BSA-PBS was discarded, and the human VEGF obtained in 1 (1) diluted with 1% BSA-PBS to a concentration of 1000-0.00035 ng / ml was reacted at 4 ° C. After washing with 0.05% tween-PBS, dilute 50% of biotin-labeled anti-human VEGF monoclonal antibody KM1544 or biotin-labeled anti-human VEGF monoclonal antibody KM1548 diluted to a concentration of 0.1 g / ml with 1% BSA-PBS. l / well were added and the reaction was carried out at room temperature for 2 hours.

 After washing with 0.05% teen-PBS, add avidin-labeled oxidase (manufactured by Vector) diluted 4,000-fold with 1% BSA-PBS at 50 / l / well and incubate at room temperature for 1 hour. Was. After washing with 0.05% Tween-PBS, color was developed using ABTS substrate solution and the absorbance of 0D415 was measured.

 Fig. 4 shows the results. The results clearly show that human VEGF can be measured at 50 pg / ml by using anti-human VEGF monoclonal antibody KM1541 and biotin-labeled anti-human VEGF monoclonal antibody KM1544 or biotin-labeled anti-human VEGF monoclonal antibody KM1548. It became.

 10.Preparation of soluble VEGF receptor KDR

(1) Construction of a fusion gene expression vector of soluble human VEGF receptor KDR fragment and human antibody Fc region A linker consisting of 19 amino acids constituting the signal peptide of human VEGF receptor KDR and a soluble human VEGF receptor KDR fragment corresponding to the 738th amino acid from the N-terminal amino acid of the mature form, a linker consisting of 6 amino acid residues (linker # 1) A vector for expressing a fusion protein comprising 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. The soluble human VEGF receptor KDR-7N corresponds to the seven imnoglobulin-like sites from the N-terminal side of the extracellular region of the soluble human VEGF receptor KDR.

 A cDNA clone encoding the full-length cDNA of human VEGF receptor KDR BCMGS-neo-KDR [A. Sawano et al., Cell Growth & Diferent ation 7, 213-221 (1996)] with EcoRI The pUC-KDR was prepared by cutting it and incorporating an approximately 2.8 kb fragment encoding the extracellular and membrane-bound regions of KDR into the ^ RI site of pUC18. pUC-KDR was cut with ^ I, and after Klenow treatment, pUC-KDR-Xb was prepared by inserting the ^ I linker shown in SEQ ID NO: 1. After inserting the XbaI-BamHI (2.3 kbp) fragment of pUC-KDR-Xb into the ^ site of pBluescript II KS (+), an I-lHI (5.2 kbp) fragment was prepared, ^ A synthetic linker containing a BI site (SEQ ID NO: 2 and SEQ ID NO: 3) was inserted to prepare pBS-KDR-Xb-S.

 Xbal / SnaBI (2.3 kbp) fragment of pBS-KDR-Xb-S, plasmid pAMoPRFc [T.Yago et al .; The Journal of Inunimology 158, 707-714, (1997)] Coding ^ BI / I (0.7 kbp) fragment into the baculovirus-recombinant pVL1393 plasmid at the 5 '^ I and 3' l sites downstream of the transcription start site of the polyhedrin (Polyhedrin) gene. A fusion gene expression vector, PVL-KDR-7N-FC, comprising a soluble human VEGF receptor KDR-7N and a human antibody Fc region was constructed (FIG. 11).

 (2) Preparation of recombinant virus for expression of soluble human VEGF receptor KDR by insect cells

The production of proteins by insect cells requires the production of a recombinant virus incorporating the target gene, which is called a transfer vector. The process involves the process of incorporating the cDNA encoding the protein into a special plasmid, the process of cotransfecting the wild-type virus and the transfer vector into insect cells, and obtaining the recombinant virus by homologous recombination. The above process was carried out using the Baculo logo starter kit (product number PM-21001K) manufactured by Farmingen in accordance with the manual and in the following procedure.

 Insect cells Sf9 (Pharmingen) cultured on TMN-FH Insect Medium (Pharmingen) were added to linear baculovirus DNA [Baculologil * Paculovirus 0¾ &(; 1] 1 (^ 01 ( 1 aculovirus DNA), manufactured by Pharmingen, Inc.] and the prepared transfer vector DNA were introduced by the lipofectin method [protein nucleic acid enzyme,, 2701 (1992)] to produce a recombinant baculovirus as follows.

Dissolve the expression vector lig prepared in 10 (1) and 20 ng of linear baculovirus DNA in 12 / x1 distilled water, add 6 l of lipofectin and 6 ml of distilled water, and add at room temperature. Let stand for minutes. On the other hand, 10 ⁶ IX Sf9 cells were suspended in 2 ml of Sf900-II medium (manufactured by Gibco) and placed in a plastic culture dish having a diameter of 35πιιη. The total amount of the above mixed solution of plasmid DNA, linear baculovirus DNA and lipofectin was 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 fresh Sf900-II medium was added, and the mixture was further cultured at 27 ° C for 3 days to obtain another 1.5 ml of the culture supernatant containing the recombinant virus. Next, each of the recombinant viruses obtained for use in protein expression was propagated by the following procedure.

2 × 10 ⁷ S of Sf9 cells were suspended in 10 ml of Sf900-II medium, placed in a 175 cm ² flask (manufactured by Grainer Co., Ltd.) and left at room temperature for 1 hour to allow the cells to adhere to the flask. After standing, the supernatant was removed, and 15 ml of TMN-FH Insect Medium and 1 ml of the culture supernatant containing the recombinant virus were added and cultured at 27 ° C for 3 days. After the culture, the supernatant is centrifuged at 1,500Xg for 10 minutes to remove the cells, and the recombinant used for protein expression is removed. A virus solution was obtained.

 The virus titer of the obtained recombinant virus solution was calculated by the method described in PacuMouth Golds Evening-Evening-Kit 'Manual (Pharmingen).

^{Six 6} × 10 ⁶ Sf9 cells were suspended in 4 ml of Sf900-II medium, placed in a 60-cell diameter plastic culture dish for cell culture, and left at room temperature for 1 hour to allow the cells to adhere to the dish. After 1 hour at room temperature was added the recombinant Wirusu solution diluted 10 ³ fold with freshly Sf 900- II medium 400 l and Sf 900-11 media supernatant was removed to the next, the first 5 m 1 medium was removed氐Medium containing melting point [agai plaque agarose (Agai ^ plaque Agarose) manufactured by PfaMingen] [Sterile lagaplaque plus, lml agarose water solution and TMN- The mixture was mixed with 4 ml of FH Insect Medium and kept at 42 ° C.] into the Petri dish. After standing at room temperature for 15 minutes, a vinyl tape was spread on a Petri dish to prevent drying, the Petri dish was placed in a sealable plastic container, and cultured at 27 ° C for 6 days. After adding PBSlml containing 0.01% neutral red to the petri dish and further culturing for 1 day, the number of plaques 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 virus.

(3) Expression and purification of soluble human VEGF receptor KDR7N-FC in insect cells The soluble human VEGF receptor KDR-7N-FC 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 (manufactured by Grainer), allowed to stand at room temperature for 1 hour, and allowed to adhere to the flask. 10 The solution lml added including transfer one vector one pVL-KDR-7N-FC-derived recombinant virus obtained in (2) at a concentration of about 1~ 3X10 ⁸ PFl) / ml, were 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.

Fill the column with about 1 ml of Prosep A [Bioprocessing] and add 20 mM phosphoric acid. The column was washed with 10 ml of sodium buffer (PH7.2) at a flow rate of 1 ml / min. After washing, 760 ml of the culture solution containing the soluble human VEGF receptor KDR-7N-FC prepared above was passed through a Prosep A column at a flow rate of 100 ml / hour. After washing with 10 ml of 20 sodium phosphate buffer (PH7.2) 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 eluted. Was done. The protein contained in each fraction was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), and soluble human VEGF receptor, soluble human KDR-7N-Fc, was used as a solution in 7 ml (protein concentration 281 / zg / ml). ).

11. Inhibition of VEGF binding to VEGF receptor KDR and F11-1

 The activity of the anti-human VEGF monoclonal antibody described in 5 to inhibit the binding between human VEGF and human VEGF receptor KDR and Flt-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 / il / well to hydrophilize the PVDF membrane at the bottom of the plate. After washing with water, the soluble human VEGF receptor KDR-7N-Fc purified in step 10 is diluted with PBS to a concentration of 4 / _ig / ml, or the soluble human VEGF receptor Flt-17N

(W098 / 22616) was diluted with PBS to a concentration of 1.6 / g / ml, dispensed at 50 1 / well, and allowed to stand at 4 ° C overnight to adsorb. After washing the plate, 50% / well of 1% BSA-PBS was added, and the mixture was reacted at room temperature for 1 hour to block the binding residues remaining on the plate. After washing with PBS, purified monoclonal antibody KM1544 prepared in a 3-fold dilution series (0.005 to 42 g / ml) with the culture supernatant of hybridoma or 1% BSA-PBS solution containing 0.5M NaCl and KM1548 was dispensed in 50 il / Ueru was further 4 ng / ml of ¹²⁵ 1-labeled human Bok VEGF (manufactured by Amersham) 1 / Ueru added to 1.5 hours at room temperature. After washing with 0.05% tween-PBS, dry the wells at 50 ° C, add 10/1/1 / well of Microscint-0 (Packard), and use Topcount (Packard). The radioactivity of labeled human VEGF bound to each well was measured.

Fig. 5 shows the results. Both purified monoclonal antibody KM1544 and belly 1548 The binding of human VEGF to the human VEGF receptor KDR was inhibited in a degree-dependent manner. The concentrations (IC50) of the purified monoclonal antibodies KM1544 and KM1548 showing 50% inhibition of binding between human VEGF and human VEGF receptor KDR were 0.06 and 0.21 g / ml. Furthermore, both KM1544 and KM1548 inhibited the binding of human VEGF to the human VEGF receptor Fit-1 in a concentration-dependent manner.

 The concentrations (IC50) of purified monoclonal antibodies KM1544 and KM1548 showing 50% inhibition of binding between human VEGF and human VEGF receptor Fit-1 were 0.23 and 0. On the other hand, as a negative control antibody, a monoclonal antibody KM231 which belongs to the mouse IgGl class and does not react with human VEGF but binds to Cyaryl Lewis A [Anticancer Research, _10, 1579 (1990)] Was used. KM231 did not show any inhibitory activity. Furthermore, as a control antibody, a neutralizing monoclonal antibody (W098 / 22616) that belongs to the mouse igG2b class, does not react with human VEGF, and binds to the human VEGF receptor Flt-1 was used. # 50 selectively inhibited only the binding between human VEGF and the human VEGF receptor Fit-1 in a concentration-dependent manner. 12. Inhibition of autophosphorylation of VEGF receptor Fit-1 and KDR

 The autophosphorylation inhibitory activity of VEGF receptor FU-1 and KDR by the anti-human VEGF monoclonal antibody described in 5 was confirmed according to the following procedure.

Fit-1 autophosphorylation: Human VEGF receptor Fit-1 expression NIH3T3 cells (NIH3T3-FU-1) [Oncogene, 10, 135 (1995)] in 20% of 10% FCS-DMEM medium The cells were cultured in a 175 cm ² flask until they reached 5 to 10 × 10 ⁶ cels / flask. After that, the medium was replaced with 20 ml of 0.1% FCS-DMEM and cultured for 2 晚. After the culture, replace with 10 ml of 0.1% FCS-DMEM containing 0.1 mM sodium orthovanadate (V), add the anti-VEGF neutralizing antibody KM1544 to 10 g / ml, and place on ice. For 30 minutes. After the culture, human VEGF (manufactured by R & D) was added to the medium at a concentration of 20 ng / ml, and the cells were cultured on ice for 45 minutes. After incubation, the medium was removed from the flask, the cell Yabu碎buffer [20 mM Hepes (pH7. 4 ), 150 mM NaCl, 0. 2% Tr i tonX-100, 10% Glycero l, mM Na 3 V0 4, 10 mM Na ₄ P ₂ 0 ₇ , 5 ₂ ml of iM EDTA, 50 DI NaF, 1.5 mM MgCl ₂ , 1 mM PMSF, 10 / ml aprotinin, 5 / ml leupeptin] were added, and the cells were crushed to obtain a cell lysate. The cell lysate is centrifuged at 15,000 X g for 10 minutes, and 100 µl of goat anti-mouse IgG (H + L) Sepharose 4B (Zymed Laboratories) is added to the supernatant for 1 hour at 4 ° C. Mix gently. After centrifugation at 5,000 Xg for 1 minute, 30 l of goat anti-mouse 〖gG (H + L) Sepharose 4B and mouse anti-Flt-1 monoclonal antibodies KM1730 and ΚΜΠ37 (W098 / 22616) were added to the supernatant. 10 g each was added, and mixed gently at 4 ° C for 1C to perform immunoprecipitation. After centrifugation at 5,000 Xg for 1 minute, Sepharose was collected and washed with 1 ml of cell disruption buffer. The washing operation was repeated six times. After washing, the adsorbed protein was eluted from the collected Sepharose with SDS-PAGE sample buffer (2x concentration) containing 301 2-mercaptoethanol, and the entire amount was electrophoresed by SDS-PAGE, Western blotting. Was served. After blotting, 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 ig / ml) (Upstate Biotechnology Incoiotot) at 4 ° C. 1 晚 reaction. Washed with 0.05% Tween-PBS, Peruokishidaze labeled blanking evening anti Usagi IgG (10 ³ fold dilutions: Dako Corp.) was reacted at room temperature for 1 hour.

 After washing with 0.05% Tween-PBS, the band bound to the heron anti-phosphorylated tyrosine antibody was detected using ECL ™ Western blotting detection reagents (Amersham).

KDR Autophosphorylation human VEGF receptor KDR expressing NIH3T3 cells (NIH3T3-KDR) [oncogene (Oncogene), 10, 135 ( 1995)] to 5 in 175cm ² flasks with 10% FCS-DMEM medium 20ml The cells were cultured until they reached 10 × 10 ⁶ cells / flask. After cultivation, replace with 0.1 ml of 0.1% FCS-DMEM containing 0.1% ImM sodium orthovanadate (V), add anti-VEGF neutralizing antibody KM1544 to 10 ig / ml, and pre-treat on ice for 30 minutes Went. After 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 stimulation, remove the medium from the flask and add cell disruption buffer [20 mM Hepes (pH7.4), 150mM NaCl, 0.2 % TritonX-100, 10% Glycerol, 2 mM Na 3 V0 4, 10 mM Na 4 P 2 0 7, 5iM EDTA, 50 M NaF, 1.5mM MgCl 2, lmM PMSF, 2 g of [10 g / ml aprotinin, 5 / xg / ml leupeptin] was added to disrupt cells to obtain a cell lysate. Centrifuge the cell lysate at 15, OOOXg for 10 minutes, and add 100 1 of goat anti-mouse IgG (H + L) Sepharose 4B (manufactured by Zymed Laboratories) to the supernatant, and gentle at 4 ° C for 1 hour Mixed. 5.After centrifugation at OOOXg for 1 minute, add goat anti-mouse IgG (H + L) Sepharose 4B to the supernatant and add 30 xK mouse anti-KDR monoclonal antibody KM1668 (FERM BP-6216) to the supernatant at 4 ° C. 1 晚 The mixture was gently mixed and immunoprecipitated. 5. Centrifugation was performed for 1 minute at OOOXg to collect the Sepharose, and the collected Sepharose was washed with 1 ml of the cell disruption buffer. The washing operation by centrifugation was repeated six times. After washing, the adsorbed protein is eluted from the Sepharose recovered at 30 // 1 (2x concentration) SDS polyacrylamide gel electrophoresis sample buffer containing 2-mercaptoethanol, and the whole amount is SDS polyacrylamide. The samples were subjected to amid gel electrophoresis and western blotting.

After blotting, the PVDF membrane was reacted with 1% BSA-PBS at room temperature for 30 minutes to perform blocking, and 、 a heron anti-phosphorylated tyrosine antibody.

(Upstate

Biotechnology Incorpotated) at 4 ° C for 1 晚. Washed with 0.05% Tween-PBS, Peruokishidaze labeled blanking evening anti Usagi IgG (10 ³ fold dilutions: Dako Corp.) was reacted at room temperature for 1 hour.

 After washing with 0.05% Tween-PBS, the band bound to the heron anti-phosphorylated tyrosine antibody was detected using ECL ™ Western blotting detection reagents (Amersham).

 Fig. 6 shows the results. Although autophosphorylation was specifically observed in Fit-1 and KDR when VEGF was added, autophosphorylation of Flt-1 and KDR could be inhibited by the addition of anti-human VEGF neutralizing monoclonal antibody KM1544. Indicated.

13.Inhibition of vascular endothelial cell growth E-BM medium containing 48% microtiter plate (Iwaki Glass) supplemented with 5% (V / V) FCS, 10 ng / ml human recombinant epidermal growth factor, and 1 g / ml hydrocortisone The human skin-derived microvascular endothelial cells HMVEC (Kurabo Co., Ltd.) suspended in a basal medium (Kurabo Co., Ltd.) were added at 4000 cells / 800 1 / well. Next, purified monoclonal antibodies KM1544, KM1548, KM1543, and KM1540, each prepared in a 5-fold dilution series (final concentration: 0.02 to 12.5 / zg / ml) with the above basal medium, were added at 100 / ^ 1 / well. further, the basal medium human VEGF diluted with a (R & D) was added at 100 / xl / Ueru (final concentration 10 ng / ml), and cultured for 3-4 days in 37 ° C C0 ₂ incubator scratch. After culturing, remove the culture supernatant, add RPMI 1640 (manufactured by Gibco) containing 200% of 10% phenol red to each well, and further add Cell Reagent for Cell Coloring (Cell Culture). ing Kit (manufactured by Dojin Chemical Co., Ltd.) and cultured at 37 ° C. for 2 to 3 hours. After completion of the culture, was transferred to a 96-well microtiter plate for EIA, and the absorbance of 0D450M1 was measured. Relative cell proliferation activity was shown, where the proliferation of HMVEC when 10 ng / ml VEGF was added was 100%, and the proliferation of HMVEC without VEGF was 0%.

 Fig. 7 shows the results. KM1544, M1548 and KM1543 inhibited VEGF-dependent HV EC proliferation in a concentration-dependent manner. On the other hand, K1540 did not show any growth inhibitory activity. The antibody concentration required to completely suppress proliferation is 0.1 / g / ml (2.67) for KM1548 and 0.1 / il (13.33) for KM1544 (the molar ratio of antibody to VEGF in Kakko) Met. KM1543 inhibited VEGF-dependent proliferation of HMVEC in a concentration-dependent manner, but partially. 14. In vivo antitumor effect

Human fibrosarcoma HT-1080 (ATCC CCL-121) cut into approximately 8 thighs ³ was placed subcutaneously on the ventral side of Balb / c nu / nu nude mice (purchased from CLEA Japan; 6-8 week old male species) Transplanted

(Transplant date = Day 0). 1, 2, 3, 4, 8, 9, 10, 0, 1 1,

On days 14 and 15, 0.2 ml of the anti-human VEGF monoclonal antibody KM1544 or KM1548 (10, 50, mouse / day) dissolved in PBS was intravenously administered, and the tumor volume was increased over time. 2 X ab ² , a (j): major axis, b (mm): minor axis] were measured. With a negative control antibody A mouse IgGl control monoclonal antibody of the same antibody class, which reacts with Radicicol UCS1006 (Japanese Patent Laid-Open No. 6-298764) but does not react with human VEGF, and uses only PBS as an anti-human VEGF monoclonal antibody. The administration was performed in the same manner as the antibody. In the advanced cancer model, from day 8 after tumor implantation (when the tumor volume reaches 50 to 200 episodes ³ ), on days 9, 10, 11, 14, 15, 16, 17, 18, and 21, The antibody was administered intravenously. Five mice were used for each antibody administration group. Tumor growth inhibitory activity of the anti-VEGF monoclonal antibody was determined by T / C (volume of treatment group / volume of control group).

 Fig. 8 shows the results. In the early cancer model, the tumor volume on the 25th day after tumor implantation was significantly suppressed in the 10, 50, and 100 zg administration groups of KM1544 and the 10, 50, and 100 ig administration groups of KM1548, and T / C The values were 0.44, 0.14, 0.11 and 0.48, 0.17, 0.16, respectively.

 On the other hand, in the advanced cancer model, the tumor volume on the 33rd day after tumor implantation was significantly suppressed in the K1544 10, 50, lOOjLtg administration group and the KM1548 10, 50, 100 / g administration group, and T The / C values were 0.63, 0.44, 0.33 and 0.77, 0.44, 0.22, respectively. From the above results, it has been clarified that KM1544 and KM1548 show antitumor effects in early cancer and advanced cancer models.

 FIG. 9 shows the antitumor effect of KM1544 in the early cancer model and the advanced cancer model over time. It was revealed that KM1544 exhibited an antitumor effect in two models in a dose-dependent manner.

 The antitumor effect of the administration schedule of the antibody was examined in an advanced cancer model. The dosing schedule was tumor administration for 5 consecutive days from day 7 and continuous administration for 5 days from day 14 (50, 100 ig / mouse / day), and a total of 4 administrations at 2 day intervals from day 7 (125, 250 ig) / Mouse / day) and once weekly administration from day 7 X 2 weeks (25, 500 g / mouse / day).

The results are shown in FIG. KM1544 has T / C of 0.17 ~ The antitumor effect was almost equivalent to 0.24. These results indicate that KM1544 is extremely clinically useful because it shows the same antitumor effect even if administered once a week as continuous administration for 5 days.

Industrial applicability

 According to the present invention, there is provided a monoclonal antibody characterized by binding to human VEGF and inhibiting binding of human VEGF to human VEGF receptors KDR and F-1. The monoclonal antibody of the present invention can be used for the diagnosis or treatment of VEGF-related diseases, such as proliferation or metastasis of solid tumors, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and xerogeneity, in which the disease state is involved in the progression and malignancy of VEGF. Useful for

 "Sequence List Free Text"

 SEQ ID NO: 1 Description of Artificial Sequence: Synthetic DNA

 SEQ ID NO: 2—Description of Artificial Sequence: Synthetic DNA

 SEQ ID NO: 3—Description of Artificial Sequence: Synthetic DM

Claims

The scope of the claims

 1. A monoclonal antibody that binds to human VEGF and inhibits the binding of human VEGF to human VEGF receptor KDR and Fit-1.

 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is a monoclonal antibody belonging to the mouse IgGl subclass.

 3. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is KM1544 or KM1548.

 4. A hybridoma that produces the monoclonal antibody according to any one of claims 1 to 3.

 5. The hybridoma according to claim 4, wherein the hybridoma is a hybridoma KM1544 (FERM BP-6555) or a hybridoma KM 1548 (FERM BP-6556).

 6. A method for inhibiting binding of human VEGF to human VEGF receptor KDR and F11-1 using the monoclonal antibody according to any one of claims 1 to 3.

 7. A method for inhibiting the biological activity of human VEGF using the monoclonal antibody according to any one of claims 1 to 3.

 8. A method for immunologically detecting human VEGF using the monoclonal antibody according to any one of claims 1 to 3.

 9. A method for immunologically quantifying human VEGF using the monoclonal antibody according to any one of claims 1 to 3.

 10. A method for diagnosing a disease based on the growth or metastasis of a solid tumor or a disease progressing due to abnormal angiogenesis, using the monoclonal antibody according to any one of claims 1 to 3.

 1 1. A therapeutic agent for a disease based on the growth or metastasis of a solid tumor, or a disease whose disease state progresses due to abnormal angiogenesis, comprising the monoclonal antibody according to any one of claims 1 to 3 as an active ingredient.

12. The monoclonal antibody according to any one of claims 1 to 3 as an active ingredient. Diagnostic agent for diseases based on the growth or metastasis of solid tumors or whose disease progresses due to abnormal angiogenesis.