WO2006022295A1 - Anti-ephrin b2 antibody - Google Patents

Abstract

It is intended to provide an antibody or an antibody fragment capable of binding to ephrin B2 and inhibiting the angiogenetic activity of ephrin B2 and the proliferation activity of a mammary gland tissue. Namely, an antibody or an antibody fragment binding to ephrin B2 which is expressed in cells of a tissue with angiogenesis such as a tumor; a method of immunologically detecting ephrin B2 or a cell expressing ephrin B2 and a detection reagent therefor with the use of the antibody or the antibody fragment as described above; a diagnostic or a remedy for a disease, in which ephrin B2 participates, by using the antibody or the antibody fragment as described above; and a hybridoma producing the above-described antibody.

Description

 Specification

 Anti-Ephrin B2 antibody

 Technical field

 [0001] The present invention relates to an antibody or an antibody fragment that binds to Ephrin (Eph-receptor-interacting protein) B2 and inhibits the angiogenic activity of Ephrin B2 and the proliferation activity of mammary tissue, the antibody or the antibody Methods for immunological detection of ephrin B2 or ephrin B2-expressing cells using antibody fragments and reagents for detection or quantification, diagnostic or therapeutic agents for diseases involving angiogenesis using the antibodies or antibody fragments, and the antibodies Provide hybridomas to produce. The antibody is useful for treating various diseases involving ephrin B2. In addition, the antibody can specifically detect ephrin B2 or ephrin B2 expressing cells by immunological techniques, and is useful for diagnosis of various diseases involving ephrin B2. Background art

 [0002] Ephrin B2 and EphB4 (Erythopoietin producing hepatocellular carcinoma B4) are membrane proteins expressed on vascular endothelium. Since ephrin B2 is selectively expressed in arterial endothelial cells and vascular smooth muscle cells around the artery, and EphB4 is selectively expressed in venous endothelial cells (Patent Document 1, Non-Patent Documents 1 to 3), ephrin B2 and EphB4 are It can be a marker that can distinguish arteries and veins, which has been difficult until now. In ephrin-B2 gene-deficient mice, angiogenesis is impaired both in arteries and veins, and is fatal in the early stages of development (Non-patent Document 1). In addition, ephrin B2 and EphB4 play an important role in angiogenesis, as ephrin B2 and Z or EphB4 gene-deficient mice are impaired in angiogenesis and lethal in the early embryonic period. (Non-Patent Documents 1 and 4).

 [0003] Angiogenesis that occurs in various pathological conditions is mainly formed to assist the infiltration of oxygen, nutrients, inflammatory cells, etc. into the affected tissue, and these environmental factors in the bloodstream are mainly used in the arteries. Be replenished. Therefore, in order to improve the pathology by inhibiting angiogenesis related to various pathological conditions, it is an appropriate means to inhibit arterial neoplasia.

[0004] It is considered effective to suppress the formation of arteries by suppressing the function of ephrin B2 as a means for specifically suppressing the formation of arteries. However, the function of ephrin B2 The function of ephrin B2 in the developmental stage of angiogenesis or angiogenesis in the matured body is unknown.

[0005] So far, polyclonal antibodies that react with ephrin B2 have been obtained by immunizing rabbits against the extracellular domain of human ephrin B2, which has been predicted to have the amino acid sequence of mouse ephrin B2, Epherin-B2 (P -20), the amino acid sequences of the polyclonal antibodies Epherin-B2 (H-83) and mouse ephrin B2 obtained by immunizing a rabbit with a polypeptide corresponding to amino acids 168-235 of the amino acid sequence of human ephrin B2 Polyclonal antibodies against human ephrin B2 of the polyclonal antibody Epherin-B2 (C-20) obtained by immunizing goats with the extracellular domain of human B2 estimated from E. coli (Non-patent Document 5) ). These are polyclonal antibodies that react with human, mouse and rat ephrin B2 and have been confirmed to be usable in Western blotting and immunohistochemical staining. Polyclonal antibody Epherin-B2 (H-83) can be used for immunoprecipitation

[0006] Monoclonal antibodies that react specifically with ephrin B2 are monoclonal antibodies whose subclass is IgM prepared for use in the cytofluorescent antibody method (Non-patent Document 6), and hamsters that react with mouse ephrin B2. A monoclonal antibody that specifically binds to ephrin B2 and inhibits the binding of mouse ephrin B2 and mouse Ep hB2, which is one of the receptors for ephrin B2, to 60% in an in xiim binding test. Known (Patent Document 2).

 [0007] Ephrin B2 has been suggested to be involved in angiogenesis An antibody that binds to ephrin B2 and inhibits the angiogenic activity of ephrin B2 is not known. In addition, ephrin B2 is expressed in mammalian glandular epithelial cells as a function of ephrin B2 other than vascular tissues, and is involved in hormone-dependent morphogenesis in adult mammals and ephrin B2 expression. It has been pointed out that the decrease may be involved in canceration of the gland (Non-patent Document 7). However, ephrin B2 is not known to be involved in breast tissue growth. Patent Document 1: US Pat. No. 6,579,683 specification

 Patent Document 2: US Patent Publication No. 2002-0136726

Non-Patent Document 1: Cell, 93, 741-753 (1998) Non-Patent Document 2: Dev. Biol, 230, 151 (2001)

 Non-Patent Document 3: Blood, 98, 1028 (2001)

 Non-Patent Document 4: Mol. Cell, 4, 403 (1999)

 Non-Patent Document 5: SANTA CRUZ BIOTECHNOLOGY website, [on line], [Heisei

Search on August 21, 2016], Internet http://www.scbt.com/catalog/action.lasso?— r esponse = product— search— list. Html & —Error = no— results. Html & — token. Order — Ia = & search—f ield = ephnnB2 & -nothing.x = 8 & -nothing.y = 17

 Non-Patent Document 6: Arterioscler Thromb Vase Biol, 23, 190 (2003)

 Non-Patent Document 7 Journal of Cell Science, 111, 2741 (1998)

 Disclosure of the invention

 Problems to be solved by the invention

[0008] An object of the present invention is to provide an antibody or an antibody fragment that binds to ephrin B2 and inhibits the angiogenic activity and the proliferation activity of mammary tissue of ephrin B2, and ephrin B2 using the antibody or antibody fragment. It is an object of the present invention to provide an immunological detection method and a reagent for detection or quantification, a diagnostic or therapeutic agent for a disease involving ephrin B2 using the antibody or the antibody fragment, and a hyperidoma that produces the antibody. .

 Means for solving the problem

The present invention relates to the following (1) to (23).

 [0010] (1) An antibody or antibody fragment that binds to ephrin B2 and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue,

 (2) The antibody or antibody fragment of (1), wherein ephrin B2 is a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2,

 (3) The antibody or antibody fragment according to (1) or (2), wherein the antibody is a monoclonal antibody,

(4) The antibody or antibody fragment of any one of (1) to (3), wherein the class of the monoclonal antibody is IgG,

(5) The monoclonal antibody is a monoclonal antibody produced from Hypridoma VERB2 (FERM BP-10100) (1) to (4) !, any monoclonal antibody or antibody fragment, (6) Any one of the monoclonal antibodies or antibodies according to (1) to (4), wherein the monoclonal antibody is a monoclonal antibody that binds to an epitope to which a monoclonal antibody produced from Hypridoma VERB2 (FERM BP-10100) binds. fragment,

 (7) A method of immunologically detecting ephrin B2 using any one of the antibodies or antibody fragments of (1) to (6),

 (8) The method of (7), wherein the detection method is an immune yarn and woven dyeing method,

 (9) The method of (7), wherein the detection method is an immune cell staining method,

 (10) A reagent for detection of ephrin B2 using any one of the antibodies or antibody fragments according to (1) to (6),

 (11) A reagent for quantifying ephrin B2 using any one of the antibodies or antibody fragments according to (1) to (6),

 (12) A diagnostic agent for a disease involving ephrin B2 using the antibody or antibody fragment of any one of (1) to (6),

 (13) The diagnostic agent of (12), wherein the disease involving ephrin B2 is a disease involving angiogenesis,

 (14) The diagnostic agent according to (13), wherein the disease involving angiogenesis is a disease selected from the group consisting of solid tumors, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness,

(15) The diagnostic agent of (12), wherein the disease involving ephrin B2 is a disease that causes proliferation of mammary tissue.

 (16) The diagnostic agent of (15), wherein the disease causing mammary gland tissue growth is a mammary gland type or breast cancer power is selected.

 (17) A therapeutic agent for a disease involving ephrin B2 comprising any one of the antibodies or antibody fragments as an active ingredient according to (1) to (6),

 (18) The therapeutic agent for (17), wherein the disease involving ephrin B2 is a disease involving angiogenesis,

 (19) The therapeutic agent according to (18), wherein the disease involving angiogenesis is a disease selected from the group consisting of solid tumors, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness,

(20) The disease involving ephrin B2 is a disease that causes proliferation of mammary gland tissue (17) Medicine,

 (21) The disease that causes the growth of mammary gland tissue is selected as a group power that can also be a mammary gland type or breast cancer power.

 (22) From (3) to (6) !, a hybridoma that produces any monoclonal antibody, and

 (23) The hybridoma of (22), in which the high-pridoma is high-pridoma VERB2 (FERM BP-10100).

 The invention's effect

 [0011] According to the present invention, an antibody or an antibody fragment that binds to ephrin B2 and inhibits angiogenic activity and mammary tissue proliferation activity of ephrin B2, ephrin B2 or ephrin B2 using the antibody or the antibody fragment Methods for immunological detection of expressed cells and reagents for detection or quantification, diagnostic or therapeutic agents for diseases involving ephrin B2 using the antibody or antibody fragment, and hybridomas producing the antibody .

 [0012] This specification includes the contents described in the specification and Z or drawings of Japanese Patent Application No. 2004-244433, which is the basis of the priority of the present application.

 Brief Description of Drawings

 [0013] FIG. 1 shows the results of sub-clustering of the produced VERB2 antibody.

 FIG. 2 shows the results of staining rat and mouse ephrin B2 by flow cytometry using the prepared VERB2 antibody.

 FIG. 3 shows the results of flow cytometry using the prepared VERB2 antibody. The left column expresses ephrin B2! /, N! / And BaF / 3 cells, and the right column shows the reactivity of each antibody to BaF / 3 / ephrin B2 cells expressing ephrin B2. Indicates. From the top, the figure shows the results of the reaction without the primary antibody, when the control antibody was used as the primary antibody, and the VERB2 antibody was used as the primary antibody.

 FIG. 4 shows the results of staining human ephrin B2 by flow cytometry using the prepared VERB2 antibody.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The present invention relates to ephrin B2 binding and angiogenic activity of ephrin B2 and mammary tissue. The present invention relates to an antibody or an antibody fragment that inhibits the growth activity of.

 [0015] The ephrin B2 angiogenic activity includes, for example, angiogenesis activity related to the growth or metastasis formation of solid tumor, angiogenesis activity related to the pathogenesis or promotion of diabetic retinopathy or rheumatoid arthritis.

 [0016] The proliferative activity of mammary gland tissue of ephrin B2 includes, for example, the proliferative activity of mammary cells, fibrocytes, or fibroepithelial cells.

 [0017] The ephrin B2 may be derived from any biological species. Specifically, the human ephrin B2, DN having a DNA sequence represented by SEQ ID NO: 1 and an amino acid sequence represented by SEQ ID NO: 2, respectively. Examples include mouse ephrin B2 whose A sequence is represented by SEQ ID NO: 3 and amino acid sequence is represented by SEQ ID NO: 4, rat ephrin B2 whose DNA sequence is represented by SEQ ID NO: 5 and amino acid sequence is represented by SEQ ID NO: 6, and the like.

 [0018] Ephrin B2 is a polypeptide having the amino acid sequence shown by SEQ ID NO: 2, 4 or 6, and one or more amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2, 4 or 6 And a polypeptide having an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 2, preferably having an amino acid sequence having 80% or more homology. A polypeptide, more preferably a polypeptide having an amino acid sequence having 90% or more homology, most preferably a polypeptide having an amino acid sequence having 95% or more homology, which is substantially identical to ephrin B2. And polypeptides having the same activity.

[0019] A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or attached in the amino acid sequence represented by SEQ ID NO: 2, 4, or 6, is described in [Molecular Cloning, AL Manual, second Edition (and old bpnng Harbor Laboratory Press, 1989), rrent Protocols in Molecular Biology (John Wiley & Sons 1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci "USA, 79 , 6409 (1982), Gene, 3 4, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985)] Using a mutagenesis method, for example, it can be obtained by introducing a site-specific mutation into DNA encoding a polypeptide having the amino acid sequence shown by SEQ ID NO: 2, 4 or 6. Deletion, substitution or addition Amino acids The number of is not particularly limited, but is preferably 1 to several tens, for example 1 to 20, more preferably 1 to several, for example 1 to 5.

[0020] The numerical value of homology described in the present invention is a numerical value calculated using a homology search program known to those skilled in the art, unless otherwise specified. For amino acid sequences, such as numerical values calculated using default parameters in BLAST [J. Mol. Biol, 215, 403 (1990)], BLAST2 [Nucleic Acids Res., 25, 33 89 (1997); Genome Res., 7, 649 (1997); http://www.ncbi.nlm.nih.gOv/Education/B LASTinfo / information3.html]! /, Numerical values calculated using default parameters, etc. Can be given.

[0021] The default parameter 1 is 5 when G (Cost to open gap) is a base sequence, 11 when amino acid sequence, 2 when -E (Cost to extend gap) is base sequence, amino acid 1 for sequence, 1 q (Penalty for nucleotide mismatch; force 3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, 11 if -W (wordsize) is a base sequence 3 residues for amino acid sequences, 20 if -y (Dropoff (X) for blast extensions in bits) is blastn, 7 for programs other than blastn,-X (X dropoff value for gapped alignment in bit s) force Si 5 and — Z (final X dropoff value for gapped alignment in bits) ¾ ^s 50 for blastn, 25 for programs other than blastn (http: 〃 www.ncbi.nlm.nih.gov/ blast / html /blastcgihelp.html).

 [0022] A polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 can be produced by methods known to those skilled in the art, for example, the amino acid represented by SEQ ID NO: 2, 4 or 6 It can be produced by culturing a transformant in which a part of the DNA encoding the sequence is deleted and an expression vector containing this is introduced. In addition, based on the polypeptide or DNA thus prepared, one or more amino acids are deleted, substituted or added in the partial sequence of the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 by the same method as described above. A polypeptide having the determined amino acid sequence can be obtained.

[0023] As an antibody that binds to ephrin B2 of the present invention and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue, the ability to include polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies are used. . [0024] Examples of the monoclonal antibody include antibodies produced by wild and hybridomas, and recombinant antibodies produced by transformants transformed with an expression vector containing the antibody gene.

 [0025] In the case of ibridoma, for example, cells that express the above-mentioned ephrin B2 are prepared as antigens, and antibody-producing cells having antigen specificity are induced from animals immunized with the antigens. And can be prepared. The monoclonal antibody of the present invention can be obtained by culturing the hybridoma or by administering the hybridoma cell to an animal to cause ascites cancer, and separating and purifying the culture medium or ascites.

 [0026] As the animal to be immunized with the antigen, any animal can be used as long as it can produce wild and hybridomas, but mouse, rat, wild, muster, rabbit heron and the like are preferably used. In addition, monoclonal antibodies, etc. produced by obtaining cells having antibody-producing ability from such animals, immunizing the cells with idim, and then fusing with myeloma cells and producing the ability of hybridomas are also included in the present invention. Included in antibodies.

 [0027] Specific examples of the monoclonal antibody of the present invention include rat antibody VERB2 produced by Hypridoma VERB2. Hypridoma VERB2 was entrusted to the Patent Biological Depositary Center of the National Institute of Advanced Industrial Science and Technology (AIST, Tsukuba 1-chome, 1-chome, 1-chome, 1-chuo, 6th), based on the Budapest Treaty as of August 19, 2004. Deposited as BP-10100.

 [0028] The monoclonal antibody of the present invention also includes a monoclonal antibody that binds to an epitope to which the monoclonal antibody produced by the above-mentioned hyperidoma VERB2 (FERM BP-10100) binds.

 [0029] A gene recombinant antibody is obtained by modifying the monoclonal antibody of the present invention using a gene recombination technique. The recombinant antibody includes an antibody produced by genetic recombination, such as a humanized antibody, human antibody or antibody fragment. Among the recombinant antibodies, those having the characteristics of a monoclonal antibody and having a low antigenicity and an extended blood half-life are preferable as therapeutic agents.

[0030] The humanized antibody in the present invention refers to a human chimeric antibody and a human CDR (Complementarit y Determining Region; complementarity determining region; hereinafter referred to as CDR).

[0031] A human chimeric antibody is composed of a heavy chain variable region (hereinafter referred to as VH) and a light chain variable region (hereinafter referred to as VL) of an antibody of a non-human animal, and a heavy chain constant region (hereinafter referred to as VL) of a human antibody. Hereinafter, it refers to an antibody consisting of CH) and a light chain constant region (hereinafter referred to as CL).

[0032] The human chimeric antibody of the present invention contains VH and VL from a hybridoma that produces a monoclonal antibody that binds to ephrin B2 and inhibits ephrin B2 vascularization and breast tissue growth activity. Obtain the encoded cDNA, insert it into an expression vector for animal cells having the genes encoding CH and CL of the human antibody, construct a human chimeric antibody expression vector, introduce it into animal cells, express it, and manufacture can do.

 [0033] The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as "hlg"), but is preferably of the hlgG class, and more preferably hIgGl, hIgG2 belonging to the hlgG class. Any of the subclasses such as hIgG3 and hIgG4 can be used. As the CL of the human chimeric antibody, any of those belonging to hlg can be used, and those of κ class or λ class can be used.

 [0034] The human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibody VH and VL.

 [0035] The human CDR-grafted antibody of the present invention is a non-human antibody produced from a hybridoma that produces a monoclonal antibody that binds to ephrin-2 and inhibits the vascularization activity of ephrin-2 and the proliferation activity of mammary tissue. VH and VL CDR amino acid sequences of human animal antibodies were grafted to VH and VL FRs of any human antibody to construct a cDNA encoding the V region and have genes encoding the human antibody CH and CL Each can be inserted into an animal cell expression vector to construct a human CDR-grafted antibody expression vector and introduced into an animal cell for expression and production.

[0036] As the CH of the human CDR-grafted antibody, if it belongs to hlg! /, It may be anything! /, But the hlgG class is preferred, and hIgGl, hIgG2, Any of the subclasses hIgG3 and hIgG4 can be used. In addition, as the CL of the human CDR-grafted antibody, any of those belonging to hlg can be used. Togashi.

 [0037] A human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from antibody-producing transgenic animal power.

 [0038] Naturally occurring antibodies in the human body can be cultured by isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibody. The antibody can be purified from the culture supernatant.

 [0039] A human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared for human B cell force into the phage gene. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be recovered using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule having two complete heavy chains and two complete light chains by genetic engineering techniques.

 [0040] A human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells. Specifically, for example, a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into a mouse ES cell, and transplanting the ES cell into an early mouse embryo, followed by generation. Human antibody-producing transgenic animal-powered human antibodies can be produced in the culture supernatant by obtaining and culturing human antibody-producing hybridomas by a conventional hyperidoma production method for non-human animals. Can produce and accumulate human antibodies.

 [0041] Examples of the antibody fragment of the present invention include peptides containing Fab, F (ab ') 2, Fab', scFv, diabody, dsFv and CDR.

 [0042] Fab is a fragment obtained by treating IgG with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain. It is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity bound by disulfide bond.

[0043] The Fab of the present invention can be obtained by treating a monoclonal antibody that binds to ephrin B2 and inhibits the angiogenic activity and mammary tissue growth activity of ephrin B2 with the proteolytic enzyme papain. . Alternatively, the DNA encoding the Fab of the antibody is expressed in a prokaryotic expression vector. Fab can be produced by inserting a vector into a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism.

 [0044] F (ab ') 2 was constructed by decomposing the lower part of two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and was composed of two Fab regions joined at the hinge region. This fragment has an antigen binding activity with a molecular weight of about 100,000.

 [0045] F (ab ') 2 of the present invention is obtained by treating a monoclonal antibody that binds to ephrin B2 and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue with the protease pepsin. be able to. Alternatively, the following Fab ′ can be produced by thioether bond or disulfide bond.

 [0046] Fab 'is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab') 2.

 [0047] Fab 'of the present invention binds to ephrin B2 of the present invention and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue. F (ab') 2 is a reducing agent dithiothreitol. It can be obtained by processing. Alternatively, there is one prokaryotic expression vector that encodes the Fab ′ fragment of the antibody! / ヽ is expressed in a eukaryotic expression vector and introduced into the prokaryotic or eukaryotic vector. And Fab ′ can be produced.

 [0048] scFv is 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). An antibody fragment having antigen-binding activity.

 [0049] The scFv of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that binds to ephrin B2 of the present invention and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue, construct a DNA encoding scFv, insert the DNA into a prokaryotic expression vector or eukaryotic expression vector, introduce the expression vector into a eukaryotic cell, scFv can be manufactured.

 [0050] A diabody is an antibody fragment in which scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity. The bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.

[0051] The diabody of the present invention binds to ephrin B2 of the present invention, and angiogenesis of ephrin B2 Obtains cDNA that encodes VH and VL of monoclonal antibodies that inhibit the activity and proliferation activity of mammary tissue, and constructs the DNA encoding scFv so that the length of the amino acid sequence of P is 8 residues or less A diabody can be produced by inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.

[0052] dsFv is obtained by binding a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue 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 three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Protein Enginee ring, 7, 697-704, 1994).

 [0053] The dsFv of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that binds to ephrin B2 of the present invention and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue, dsFv-encoding DNA is constructed, the DNA is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector is introduced into a prokaryotic or eukaryotic expression, and expressed. dsFv can be manufactured.

 [0054] The peptide containing CDR is constituted by including at least one region of CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via an appropriate peptide linker.

 [0055] A peptide comprising the CDR of the present invention encodes the CDRs of monoclonal antibodies VH and VL that bind to ephrin B2 of the present invention and inhibit the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue. Is a prokaryotic expression vector! / ヽ is inserted into a eukaryotic expression vector and expressed by introducing the expression vector into prokaryotic or eukaryotic cells, including CDR Peptides can be produced.

 [0056] The peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorylmethyloxycarbonyl method) or the t Boc method (t_butyloxycarbon method). it can.

[0057] The antibody of the present invention binds to the ephrin B2 of the present invention, and the antibody or antibody fragment that inhibits the angiogenic activity of the ephrin B2 and the proliferation activity of the mammary gland tissue contains a radioisotope, It includes derivatives of antibodies in which molecular drugs, high molecular drugs, proteins, etc., are conjugated chemically or genetically.

 [0058] The derivative of the antibody of the present invention binds to ephrin B2 of the present invention and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of breast tissue, or the H chain or L chain of an antibody fragment. N-terminal side or C-terminal side of the antibody, appropriate substituents or side chains in the antibody or antibody fragment thereof, and sugar chains in the antibody or antibody fragment. It can be produced by combining drugs, proteins, etc. by chemical methods (Introduction to Antibody Engineering, Osamu Kanmitsu, Jinjinshokan, 1994).

 [0059] In addition, it binds to ephrin B2 of the present invention and encodes an antibody or an antibody fragment that inhibits the angiogenic activity and mammary tissue growth activity of ephrin B2, and binds ヽ protein. The DNA can be ligated, inserted into an expression vector, the expression vector is introduced into an appropriate host cell and expressed, and then produced.

[0060] Examples of the radioisotope, ¹³¹ 1, ¹²⁵ 1 and the like, for example, can be conjugated to an antibody due chloramine T method.

 [0061] As low molecular weight drugs, there are alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexate, daunomycin, bleomycin, mitomycin C, daunorubicin, doxorubicin and the like. Anticancer agents such as antibiotics, hormonal agents such as plant anolekaloids such as vincristine, vinblastine, vindesine, tamoxifen, dexamethasone (Clinical Oncology, edited by Japan Clinical Oncology Society, Cancer and Chemotherapy, 1996), or Hyde mouth cortisone , Steroids such as prednisone, non-steroidal drugs such as aspirin and indomethacin, immunomodulators such as gold thiomalate and p-silamine, immunosuppressants such as cyclophosphamide and azathioprine, and chlorphenol maleate And anti-inflammatory agents such as antihistamines such as min and clemacitin (Inflammation and anti-inflammatory therapy, Ishiyaku Shuppan Co., Ltd., 1982). For example, daunomycin and antibody can be bound by binding between daunomycin and the amino group of the antibody via dartal aldehyde, or by binding the amino group of daunomycin to the carboxyl group of the antibody via water-soluble carpositimide. Can be given.

[0062] Polymeric drugs include polyethylene glycol (hereinafter referred to as PEG), albumi , Dextran, polyoxyethylene, styrene maleic acid copolymer, polybutylpyrrolidone, pyran copolymer, hydroxypropyl methacrylamide and the like. By binding these macromolecular compounds to antibodies or antibody fragments, (1) improved stability against various chemical, physical or biological factors, and (2) significant increase in blood half-life. (3) Expected effects such as loss of immunogenicity and suppression of antibody production (Bioconjugate Pharmaceutical, Yodogawa Shoten, 1993). For example, a method of conjugating PEG with an antibody includes a method of reacting with a PEGylation modifying reagent (Bioconjugate Pharmaceutical, Yodogawa Shoten, 1993). PEGylation modifying reagents include lysine ε-amino group modifiers (JP-A 61-17 8926), aspartic acid and glutamic acid carboxyl group modifiers (JP-A 56-23 587), arginine gua- And a dino group modifier (JP-A-2-117920).

 [0063] Examples of the protein include cyto force-in which activates immunocompetent cells, such as human interleukin 2, human granulocyte macrophage colony stimulating factor, human macrophage colony stimulating factor, human interleukin 12 and the like. . In addition, toxins such as ricin and diphtheria toxin having an activity of directly damaging cancer cells can be used. For example, for a fusion antibody with a protein, cDNA encoding the protein is linked to cDNA encoding the antibody or antibody fragment to construct a DNA encoding the fusion antibody, and the DNA is used for prokaryotic or eukaryotic organisms. A fusion antibody can be produced by inserting into an expression vector and expressing the expression vector by introducing it into a prokaryotic or eukaryotic organism.

 [0064] Examples of the drug when the fusion antibody is used as a detection method, a quantification method, a detection reagent, a quantification reagent or a diagnostic agent include labels used in usual immunoassay methods. Examples of the label include enzymes such as alkaline phosphatase, peroxidase, and luciferase, luminescent substances such as attalidum esters and mouth fins, and fluorescent substances such as FITC and RITC.

 [0065] The biological activity of ephrin B2 may be any biological activity as long as ephrin B2 has, for example, an activity involved in angiogenesis. The activity involved in angiogenesis includes, for example, arteriogenesis and invasion / migration of vascular endothelial cells.

[0066] The method for producing the antibody of the present invention will be specifically described below. [0067] (1) Preparation of antigen

 Ephrin B2 used in the production of the antibody of the present invention is described in [Molecular Cloning, A Laboratory Manual, Second Edition (and old bpnng Harbor Laboratory Press, 1989), Cur rent Protocols in Molecular Biology (John Wiley & Sons, 1987—1997). )] Can be produced by expressing DNA encoding ephrin B2 in a host cell, for example, by the following method. The ephrin B2 to be expressed may be expressed in a soluble portion excluding the membrane-binding domain, which may be expressed in a membrane-bound state in the same manner as the wild-type ephrin B2. The ephrin B2 to be expressed may be a full-length polypeptide of ephrin B2 or a part thereof.

 [0068] First, a recombinant vector is prepared by inserting a full-length cDNA including the cDNA of the portion encoding the polypeptide downstream of the promoter of an appropriate expression vector. If necessary, prepare a DNA fragment of an appropriate length containing the ephrin B2 coding portion based on the full-length cDNA, and use the DNA fragment in place of the full-length cDNA. Also good. Subsequently, a transformant producing a polypeptide can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.

 [0069] As the host cell, any strain can be used as long as it can express the target gene, such as Escherichia coli and animal cells.

 [0070] As the expression vector, one that can autonomously replicate or integrate into a chromosome in the host cell to be used and contains an appropriate promoter at a position where DNA encoding ephrin B2 can be transcribed is used. .

 [0071] When a prokaryotic organism such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryotic organism, and at the same time is used in a promoter, a ribosome binding sequence, and the present invention. A vector containing DNA and a transcription termination sequence is preferred. The recombinant vector may contain a gene that controls the promoter.

[0072] Examples of expression vectors include pBTrp2, pBTacl, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega) , PQE-8 (manufactured by QIAGEN), pKYPIO (JP 58-110600), pKYP200 [A gricultural Biological Chemistry, 48, 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 2 77 (1989)], pGELl [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescriptll SK (-Stratagene), pTrs30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], p Trs32 [prepared from E. coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [E. coli IGHA2 (FERM BP- 400), JP 60-221091], pGKA2 [E. coli IGKA2 (FERM BP-6 798), JP 60-221091], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, Examples include pC194, pEG400 [j. BacterioL, 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), pME18SFL3, and the like.

 [0073] The promoter may be any as long as it can function in the host cell to be used. For example, promoters derived from Escherichia coli or phage, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, T7 promoter, etc. can be mentioned. In addition, artificially designed and modified promoters such as tandem promoter, tac promoter, lacT7 promoter, letl promoter, etc. in which two Ptrps are connected in series can also be used.

 [0074] Further, as the above recombinant vector, Shine'Dalgarno, which is a ribosome binding sequence, is used.

 (Shine-Dalgarno) It is preferable to use a plasmid in which the distance between the sequence and the start codon is adjusted to an appropriate distance (eg 6 to 18 bases)! In the present invention! / In the base sequence of DNA encoding ephrin B2, the base can be substituted so that it becomes the optimal codon for expression in the host, thereby reducing the target ephrin B2 production rate. It can be improved. Furthermore, a transcription termination sequence is not necessarily required for gene expression in the above recombinant vector, but it is preferable to place a transcription termination sequence immediately below the structural gene.

[0075] As a prokaryote used as such a host cell, for example, a prokaryote belonging to the genus Escherichia can be used, and specifically, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Otsuki-recruited DH1 Otsuki-fungal MC1000, Otsuki-funded KY3276, Otsuki-funded W1485, Otsuki-funded JM109, E. coli HB101, E. coli No.49, E. coli W3110, E. coli NY49, etc. can be used. [0076] As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

 [0077] When ephrin B2 used in the production of the antibody of the present invention is produced in E. coli, ephrin B2 is soluble in the cytoplasm, insoluble granules in the cytoplasm, or periplasmic space depending on the type of vector. It can be expressed in an expression mode such as a soluble form.

 [0078] When animal cells are used as hosts, examples of expression vectors include pcDNAU pcD M8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (199 0)], pAS3 — 3 (JP-A-2-27075), pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 130 7 (1987)], pAGE210, pME18SFL3, and the like.

 [0079] Any promoter can be used as long as it can function in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, early sv40 Examples include promoters, retrovirus promoters, metallotine promoters, heat shock promoters, SRa promoters, and the like. In addition, an enhancer of the IE gene of human CMV may be used together with a promoter.

 [0080] As host cells, human cells, Namalwa cells, monkey cells, COS cells, Chinese's cells, CHO cells, Muster cells, and HBT5637 cells (Japanese Patent Laid-Open No. Sho 6 3-299). ) Etc.

 [0081] As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, the electopore position method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method ( JP-A-2-227075) and the ribofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

[0082] As a method of gene expression, in addition to direct expression, [Molecular Cloning, A Laboratory Manual, Second Edition (Cold bpnng Haroor Laboratory Press, 1989)] , Fusion protein expression, etc. When expressed in a cell derived from a product, ephrin B2 to which a sugar or a sugar chain has been added can be obtained.

 [0083] The transformant obtained as described above is cultured in a medium, and the ephrin B2 is produced and accumulated in the culture and collected from the culture to produce the ephrin B2 used in the present invention. can do. The method for culturing the transformant in a medium can be carried out according to a usual method used for culturing a host.

 [0084] When a microorganism transformed with a recombinant vector using an inducible promoter as a promoter is cultured, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalatatopyranoside, etc., or with a recombinant vector using the trp promoter. Indole acrylic acid or the like may be added to the medium.

[0085] As a medium for culturing a transformant obtained using animal cells as a host, a commonly used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM Medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (1950)] or these A medium obtained by adding fetal calf serum or the like to the medium can be used. Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and 5% CO. Also required during culture

 2

 If necessary, antibiotics such as kanamycin and penicillin may be added to the medium. As described above, a transformant derived from a microorganism or animal cell having a recombinant vector incorporating a DNA encoding ephrin B2 used in the production of the antibody of the present invention is cultured according to a normal culture method, and ephrin Ephrin B2 used in the production of the antibody of the present invention can be produced by producing and accumulating B2 and collecting it from the culture.

 [0086] As a gene expression method, in addition to direct expression, [Molecular Cloning, A Laboratory Manual, Second Edition (Cold bpnng Haroor Laboratory Press, 1989]] [secretion production according to the method described in this ti etc. Fusion protein expression can be performed.

Ephrin B2 can be produced in a host cell or distributed outside the host cell. Depending on the host cell used and the structure of ephrin B2 to be produced, an appropriate method can be selected.

 [0087] When ephrin B2 is produced in or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Lou et al. [Proc. Natl. Acad. Sci., U SA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990), JP 05-336963 or WO 94/23021], etc. The product can be actively secreted out of the host cell. Further, in accordance with the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.

 [0088] Ephrin B2 can be isolated and purified from the above culture or the like, for example, as follows.

 [0089] When ephrin B2 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a Mantongaurin homogenizer, A cell-free extract is obtained by disrupting the cells with dynomill or the like. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as ruaminoethyl (DEAE) -Sepharose and DIAION HPA-75 (Mitsubishi Chemical), cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia) Such as chromatography, hydrophobic chromatography using resins such as butyl sepharose and ferrule sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purified preparations can be obtained using methods such as electrophoresis alone or in combination.

[0090] When ephrin B2 is expressed by forming an insoluble granule in the cytoplasm, the cells are similarly collected and then crushed and centrifuged to obtain an insoluble granule of ephrin B2 as a precipitate fraction. Recover. The recovered insoluble granules of the protein are dissolved with a protein denaturant. By diluting or dialyzing the soluble solution, the protein is returned to a normal three-dimensional structure. After that, a purified preparation of ephrin B2 can be obtained by the same isolation and purification method as described above.

 [0091] When a derivative such as ephrin B2 or a sugar modification product thereof is secreted extracellularly, a derivative such as ephrin B2 or a sugar modification product thereof can be recovered in the culture supernatant. That is, the culture supernatant is removed from the culture supernatant by treating the culture with a technique such as centrifugation as described above, and the same isolation and purification method as described above is used from the culture supernatant. As a result, a purified preparation can be obtained.

 [0092] When ephrin B2 is produced on the outer membrane, the cells are collected and disrupted, and then the cell membrane fraction is solubilized with an appropriate surfactant or the like and isolated in the same manner as described above. A purified sample can be obtained by using a purification method. In addition, when ephrin B2 is produced on the outer membrane, the expressed cells can be recovered and used as an immunogen.

 [0093] Ephrin B2 can also be produced by chemical synthesis methods such as the Fmoc method (fluorylmethyloxycarbonyl method) and the tBoc method (t-ptyloxycarbonyl method). Alternatively, chemical synthesis can be performed using peptide synthesizers such as Advanced ChemTech, Norkin Enomer, Pharmacia, Protein Technology Instrument, Synthece U-Vega, PerSeptive, Shimadzu Corporation.

 [0094] Ephrin B2 obtained by the above method, a peptide having a partial sequence of ephrin B2 or an ephrin B2-expressing cell can be used as an antigen.

 [0095] (2) Immunization of animals and preparation of antibody-producing cells

 3 to 20-week-old mice, rats or hamsters are immunized with the antigen prepared as described above, and antibody-producing cells in the spleen, lymph nodes, and peripheral blood of the animals are collected.

 [0096] Immunization is carried out in the subcutaneous or intravenous or intraperitoneal region of an animal together with an appropriate adjuvant (for example, Freund's complete adjuvant, aluminum hydroxide gel and pertussis vaccine). Is administered. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (cushion serum albumin) or KLH (Keyhole Limpet hemocyanin) is prepared and used as an immunogen.

[0097] The antigen is administered 5 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the serum reacts with the antigen using an enzyme immunoassay (Antibodies-A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)). The Mice, rats or hamsters whose serum showed a sufficient antibody titer against the antigen used for immunization are used as a source of antibody-producing cells.

 [0098] Polyclonal antibodies can be prepared by separating and purifying the serum. The polyclonal antibody strength binds to ephrin B2 and has the activity of inhibiting the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue by the method described in (6) below. Can do.

 [0099] When the antibody-producing cells are fused with myeloma cells, 3 to 7 days after the final administration of the antigen, tissue containing antibody-producing cells such as spleen is removed from the immunized mouse, rat or hamster, and the antibody Producing cells are collected. When using spleen cells, spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged (1200 rpm, 5 minutes), the supernatant discarded, and Tris monosalt Treat with ammonia buffer (PH7.65) for 1-2 minutes to remove erythrocytes and wash with MEM medium 3 times to provide antibody producing cells for fusion.

 [0100] (3) Preparation of myeloma cells

As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (derived from BALB / c mice) myeloma cell line P3- X63Ag8- Ul (P3-U1) (Current Topics in Microbiology and Immunology, 18, 1, 1978), P3— NSl / 1 — Ag41 (NS— 1) (European J. Immunology, 6, 511, 1976), SP2 / 0-Agl4 (SP-2) (Nature, 276, 269, 197 8), P3- X63- Ag8653 (653) ( J. Immunology, 123, 1548, 1979), P3-X63-Ag8 (X63) (Nature, 256, 495, 1975) and the like are used. These cell lines include 8-Azaguanin medium [RP MI- 1640 medium click 'Retamin (1.5 mM), 2-mercaptoethanol ⁽⁵ X 10- 5 M), Jentama leucine (10 μ g / ml) and fetal calf A culture medium supplemented with serum (FCS) (hereinafter referred to as normal medium) and further subcultured with 8-azaguanine (15 g / ml) Normal] Normal 3-4 days before cell fusion Pass to culture medium and secure a cell count of 2 x 10 ⁷ or more on the day of fusion.

 [0101] (4) Cell fusion

The above antibody-producing cells and myeloma cells are thoroughly washed with MEM medium or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter distilled water, pH 7.2). Antibody-producing cells: Myeloma cells = 5-10: 1 After mixing, centrifuge (1200 rpm, 5 minutes), discard the supernatant, loosen the precipitated cells, and mix well. At ° C , 2 g of polyethylene glycol 1000 (PEG-1000), 0.2 ml of a mixture of 2 ml of MEM and 0.7 ml of dimethylsulfoxide are added to 1 X 10 ⁸ antibody-producing cells, and ME M every 1-2 minutes. Add 1-2 ml of medium several times, and then add MEM medium to a total volume of 50 ml. After centrifuge separation (900 rpm, 5 minutes), discard the supernatant, loosen the cells gently, and then gently suck the cells with a pipette and blow out the cells into the HAT medium [normal medium with hypoxanthine (104M), thymidine ( 1.5 X 10- ⁵ M) and suspended aminopterin (4 X 10- ⁷ M) in Ca卩example media] 100 ml. Dispense this suspension into a 96-well culture plate at 100 1 / well at 37 ° C in a 5% CO incubator. Incubate for 14 days.

 2

 [0102] After culturing, a portion of the culture supernatant is taken to produce an antibody that binds to ephrin B2 and inhibits the angiogenic activity of ephrin B2 and the proliferation activity of mammary tissue by the hybridoma selection method described below. No, select a wel that includes an bridridoma. Next, the cloning was repeated twice by the limiting dilution method (1st time using HT medium (medium excluding HAT medium power aminopterin) and 2nd time using normal medium). Are selected as monoclonal antibody-producing hyperpridoma strains.

 [0103] (5) Preparation of monoclonal antibody

It is obtained in (4) above to 8-10 week old mice or nude mice treated with pristane (0.5 ml of 2,6, 10, 14-tetramethylpentadecane (Pristane) administered intraperitoneally and reared for 2 weeks). Anti-ephrin B2 monoclonal antibody-producing hybridoma cells 2 × 10 ^{6 to} 5 × 10 ⁷ cells / mouse are injected intraperitoneally. Hypridoma becomes ascites tumor in 10-21 days. Also collect the ascites of this mouse force, centrifuge (3000 rpm, 5 minutes) to remove the solids, pour salt with 40-50% ammonium sulfate, force prillic acid precipitation, DEAE-Sepharose column, protein Purify using an A-column or gel filtration column and cleave the IgG or IgM fraction to obtain a purified monoclonal antibody.

 [0104] The subclass of the antibody is determined by enzyme immunoassay using a sub-clustering kit. The amount of protein is quantified using the Raleigh method and absorbance at 280 ° C.

 [0105] (6) How to select High Pridorma

Binds to ephrin B2 and inhibits angiogenesis activity and mammary tissue growth activity of ephrin B2 having angiogenesis inhibition activity and breast tissue growth inhibition activity of the present invention Examples of a method for selecting a hyperidoma producing a monoclonal antibody include the following methods.

 [0106] First, a hybridoma producing a monoclonal antibody capable of specifically binding to ephrin B2 is selected using the following method. The antigen protein or cells expressing the antigen protein are coated on a 96-well plate, and the reaction is carried out using the high-pridoma culture supernatant or the purified antibody obtained by the above method as the first antibody. Examples of the antigenic protein include ephrin B2 prepared in (1) above, and specifically, soluble mouse ephrin B2-Fc (Blood, 98, 1028, 2001). Examples of the cells expressing the antigen protein include cells expressing ephrin B2 prepared in (1) above. Specifically, the cells expressing mouse ephrin B2 described in (1) of Example 1 are expressed. Rati cells.

 [0107] After the first antibody reaction, the plate is washed and the second antibody is added.

 [0108] The second antibody is an antibody obtained by labeling an antibody capable of recognizing the immunoglobulin of the first antibody with piotin, an enzyme, a chemiluminescent substance or a radiation compound. Specifically, if a mouse is used in the preparation of the hyperidoma, an antibody capable of recognizing mouse immunoglobulin is used as the second antibody.

 [0109] After the reaction, a reaction according to the substance labeled with the second antibody is performed, and it is selected as a hyperidoma that produces a monoclonal antibody that reacts specifically with the antigen. At this time, confirm that it does not react with other ephrin families, ephrin B1 and ephrin B3, by the same method.

 [0110] It is confirmed, for example, by the method described in Example 2, (4) or (5) that the obtained monoclonal antibody has angiogenesis inhibitory activity and mammary gland tissue growth inhibitory activity. It is out.

 [7] (7) Disease diagnosis method using the antibody of the present invention

 By measuring ephrin B2 in a biological sample using the antibody or antibody fragment of the present invention, a disease associated with ephrin B2 can be diagnosed.

[0112] The biological sample to be measured for ephrin B2 in the present invention includes ephrin B2 to be measured, such as tissue cells, blood, plasma, serum, knee fluid, urine, stool, tissue fluid, and culture fluid. Is not particularly limited as long as it may contain ephrin B2-expressing cells. [0113] Examples of diseases associated with ephrin B2 include diseases involving angiogenesis. Specific examples of diseases involving angiogenesis include growth or metastasis formation of solid tumors such as breast cancer, prostate cancer, colon cancer, stomach cancer, and lung cancer, arthritis in rheumatoid arthritis, retinopathy of diabetes, premature infant retina Diseases in which the pathology progresses due to angiogenesis abnormalities such as psoriasis and psoriasis.

 [0114] In addition to diseases involving angiogenesis, ephrin B2 is specifically expressed in the mammary epithelium, and therefore diseases involving ephrin B2 include diseases that cause breast tissue proliferation. The Specific examples of diseases that cause growth of mammary tissue include breast adenoma and breast cancer.

 [0115] The diagnosis of a disease associated with angiogenesis can be performed, for example, as follows.

 [0116] Biological strength of a plurality of healthy subjects Using the antibody or antibody fragment of the present invention, or a derivative thereof, a biological sample collected from several healthy individuals, ephrin B2 is detected or measured using the following immunological detection method. Investigate the abundance of ephrin B2 or ephrin B2 expressing cells in a biological sample of a healthy person. Similarly, the abundance of ephrin B2 or ephrin B2 expressing cells is also examined in the biological sample of the subject, and the abundance is compared with the abundance of healthy subjects. When the abundance of ephrin B2 or ephrin B2-expressing cells in the subject is increased compared to healthy subjects, it can be diagnosed that the cancer is positive.

 [0117] The diagnostic agent containing the antibody or antibody fragment of the present invention or a derivative thereof may contain a reagent for performing an antigen-antibody reaction and a reagent for detecting the reaction, depending on the target diagnostic method. Examples of the reagent for performing the antigen-antibody reaction include a buffer and a salt. Detection reagents include antibodies or antibody fragments, or derivatives thereof, or conventional immunological methods such as labeled secondary antibodies that recognize antibodies or antibody fragments, or derivatives thereof, and substrates corresponding to the labels. Examples include reagents used in detection methods.

 [0118] As a method for quantifying ephrin B2 or ephrin B2-expressing cells in the present invention, any known method may be mentioned. For example, immunological measurement methods can be mentioned.

[0119] An immunoassay is a method of measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. The immunoassay includes radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), and luminescence immunoassay. Standard methods (luminescent immunoassay), Western blotting, physicochemical detection methods (TI A, LAPIA, PCIA) and the like can be mentioned. As long as it is a method for detecting or measuring an antigen, any method may be used, but an immunoprecipitation method or a fluorescent cell staining method is preferable.

[0120] Radioactive-labeled immunoantibody method (RIA) includes, for example, reacting an antibody of the present invention with an antigen or a cell expressing the antigen, and further reacting a radiolabeled anti-immunoglobin antibody or binding fragment. Then, measure with a scintillation counter.

 [0121] As an enzyme immunoassay (EIA or ELISA), for example, an antigen of the present invention is reacted with an antigen or a cell expressing the antigen, and a labeled anti-immunoglobulin antibody or binding fragment is used. After the reaction, the coloring dye is measured with an absorptiometer. For example, a sandwich ELISA method is used. As the label used in the enzyme immunoassay, any known enzyme label (edited by Koji Ishikawa et al., Enzyme immunoassay, Medical School) can be used as described above. For example, alkaline phosphatase label, peroxidase label, luciferase label, piotin label and the like can be used.

 [0122] Sandwich ELISA is a method in which an antibody is bound to a solid phase and then measured! /, The antigen is trapped, and the second antibody reacts with the trapped antigen. In the ELISA method, two types of antibodies or antibody fragments that recognize the measured antigen and have different antigen recognition sites are prepared, and one of the antibodies or antibody fragments is preliminarily placed on a plate (for example, 96 wells). Plate) and label the second antibody or antibody fragment with a fluorescent substance such as FITC, an enzyme such as peroxidase, or piotin. After the above-mentioned antibody-adsorbed plate is reacted with cells or its lysate, tissue or its lysate, cell culture supernatant, serum, pleural effusion, ascites, ophthalmic fluid, etc., separated from the living body, then labeled. The detected monoclonal antibody or antibody fragment is reacted, and a detection reaction is performed according to the labeling substance. The concentration of the test sample can be calculated from a calibration curve prepared by diluting antigens with known concentrations stepwise. As antibodies used in the sandwich ELISA method, antibody fragments such as Fab, Fab ′, F (ab), etc., which may be either polyclonal antibodies or monoclonal antibodies, are used.

 2

May be. The combination of the two types of antibodies used in the sandwich ELISA method may be a combination of monoclonal antibodies or antibody fragments that recognize different epitopes, A combination of a polyclonal antibody and a monoclonal antibody or antibody fragment may be used.

 [0123] The fluorescence immunoassay (FIA) is described in the literature [Monoclonal Antibodies: Principles and practice, Third edition (Academic Press, 1996); Monoclonal antibody experiment manual (Kodansha Scientific, 1987)]. Method. As the label used in the immunofluorescence assay, as described above, any known fluorescent label (Akira Kawao, fluorescent antibody method, Soft Science) can be used. For example, FITC tag, RITC tag, etc. can be used.

 [0124] As described above, the label used in the luminescent immunoassay as described above is any known [Imai Kazuhiro, Bioluminescence and Chemiluminescence, Yodogawa Shoten; Clinical Examination 42 (1998) ] Of the luminescent material label. For example, an atelidum ester label, a mouth fin label, or the like can be used.

 [0125] In the Western plot method, antigens or cells expressing the antigens were fractionated by SDS-polyacrylamito electrophoresis (Antibodies- A Laboratory Manual (Cold bpnng Haroor Laboratory, 1988)), and then the gel was PVDF membrane. Or an anti-mouse IgG antibody that has been blotted on a trocellulose membrane, reacted with an antibody or antibody fragment that recognizes the antigen on the membrane, and further provided with a fluorescent substance such as FITC, an enzyme label such as peroxidase, and a piotin label This is a method of confirming by visualizing the label after reacting the binding fragment. An example of Western blotting is shown below.

 [0126] Cells and tissues expressing ephrin B2 are lysed, and 0.1 to 30 / zg of protein mass per lane is run by SDS-PAGE under reducing conditions. The migrated protein is transferred to a P VDF membrane, and reacted with PBS containing 1% BSA (hereinafter referred to as BSA-PBS) for 30 minutes at room temperature to perform blocking. Here, the monoclonal antibody of the present invention is reacted, washed with Tween-PBS, and peroxidase-labeled goat anti-mouse IgG is incubated for 2 hours at room temperature. Tephrin B2 can be detected by washing with Tween—PBS and detecting the band to which the monoclonal antibody is bound using ECL Western blotting detection reagents (Amersham).

[0127] Specifically, the physico-chemical detection method is to bind ephrin B2 as an antigen to an antibody or antiserum using an antibody or antiserum that specifically binds to ephrinB2. In More aggregates are formed and this aggregate is detected. Other physicochemical detection methods include the capillary method, the one-dimensional immunodiffusion method, the immunoturbidimetric method, and the latex immunoturbidimetric method. [Proposal of clinical test methods, Kanbara Publishing, 499 (1998 )] ₀

[0128] For example, in latex immunoturbidimetry, an antibody or antigen is sensitized and a carrier such as polystyrene latex having a particle size of about 0.1 to 1 μm is used to cause an antigen-antibody reaction with the corresponding antigen or antibody. Then, the scattered light in the reaction solution increases and the transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, ephrin B2 can be measured.

 [0129] Since the antibody of the present invention can bind to ephrin B2, it is preferably used for detection of ephrin B2-expressing cells.

 [0130] For detection of ephrin B2-expressing cells, known immunological measurement methods can be used, but immunoprecipitation, fluorescent cell staining, immunohistochemical staining, and immunohistological staining are preferably used. It is done. In addition, fluorescent antibody staining using the FMAT8100HTS system (Applied Biosystems) can be used.

 [0131] In immunoprecipitation, ephrin B2-expressing cells and the like are reacted with the monoclonal antibody or antibody fragment of the present invention, and then a carrier having specific binding ability to immunoglobulin such as protein G-sepharose is added. In this method, the antigen-antibody complex is precipitated. Yes! You can also do it by the following method.

 [0132] The above-described antibody of the present invention is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS. If the antibody is purified and is in a purified state such as a culture supernatant of a hyperidoma strain, anti-mouse immunoglobulin or rat immunoglobulin or protein A or G is added. Preliminarily immobilize on ELISA 96-well plate and block with BSA-PBS, then dispense and bind the Hypridoma strain culture supernatant. Discard the BSA-PBS, wash well with PBS, and then react with the lysate of cell tissue expressing ephrin B2. Immunoprecipitates are extracted from the well-washed plate with SDS-P AGE sample buffer and detected by Western blotting as described above.

[0133] Immune cell staining and immunohistochemical staining methods refer to cells or tissues that express an antigen. In some cases, after treatment with a surfactant or methanol to improve the passage of the antibody, the antibody of the present invention is reacted, and further, a fluorescent label such as fluorescin'isothiocyanate (FITC), peroxidase, etc. After reacting with an anti-immunoglobulin antibody or binding fragment that has been labeled with an enzyme or piotin, the label is visualized and viewed under a microscope, or a fluorescent-labeled antibody and a cell are reacted to produce a flow cytometer. This is a fluorescent antibody staining method (flow cytometry) analyzed by For example, the method described in the literature [Monoclonal Anti bodies: Principles ana practice, Third edition (Academic Press, 1996), Monochrome ~~ n 饥. Body experiment manual (Kodansha Scientific, 1987)] etc. is used. be able to. In particular, since the antibody of the present invention specifically recognizes ephrin B2 and can bind to the extracellular region, it is preferably used for detection of cells expressing ephrin B2 by flow cytometry.

 [0134] In addition, the fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystems) consists of the formed antibody-antigen complex and a free antibody or antigen not involved in the formation of the antibody-antigen complex. This is a homogenous assay method that can measure the amount of antigen or antibody without separating the antibody.

 [0135] (8) A method for treating cancer using the antibody of the present invention

 The antibody or antibody fragment of the present invention binds to ephrin B2 and can treat diseases involving ephrin B2. Diseases involving ephrin B2 include diseases associated with angiogenesis. Diseases involving angiogenesis include growth or metastasis of solid tumors such as breast cancer, prostate cancer, colon cancer, stomach cancer, and lung cancer, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and psoriasis. Examples include diseases whose pathology progresses due to abnormal angiogenesis. The therapeutic agent of the present invention includes a therapeutic agent for cancer involving ephrin B2, and cancer treatment by antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or apoptosis-inducing action. Agent is included.

[0136] Antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) possessed by an antibody can be measured, for example, by the method described in JP-A-6-205694. An antibody having such activity can be used as a therapeutic agent for a disease because it can damage cells in which a specific antigen is expressed in χίχ2. Especially for the HgG class Human chimeric antibodies having human antibody constant regions, human CDR-grafted antibodies, and human antibodies are effectively used as therapeutic agents (Cancer Res., 56, 1118, 1996).

 [0137] Since the antibody of the present invention can recognize ephrin B2, it can recognize ephrin B2-expressing cells present in vivo. Therefore, human-type chimeric antibodies, human-type CDR-grafted antibodies and human antibodies having human IgG class antibody constant regions containing the variable region CDRs of the antibodies damage ephrin B2-expressing cells in vivo or in idim. Proliferation or metastasis of solid tumors such as breast cancer, prostate cancer, colon cancer, gastric cancer, lung cancer, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity and psoriasis It is useful for the treatment of diseases whose pathology progresses due to abnormalities.

 [0138] In addition to diseases involving angiogenesis, ephrin B2 is specifically expressed in the mammary epithelium. Therefore, it binds to ephrin B2 of the present invention and exhibits angiogenic activity and activity of ephrin B2. Antibodies or antibody fragments that inhibit the growth activity of mammary gland tissue are useful in the treatment of diseases that cause mammary tissue growth. Specific examples of diseases that cause growth of mammary tissue include, for example, breast adenoma and breast cancer.

 [0139] The therapeutic agent containing the antibody or antibody fragment of the present invention or a derivative thereof may contain only the antibody or antibody fragment or the derivative thereof as an active ingredient, but is usually a drug. It is desirable to provide a pharmaceutical formulation prepared by any method well known in the pharmaceutical arts, mixed with one or more physically acceptable carriers.

 [0140] The route of administration is preferably oral, for which it is desirable to use the most effective treatment, or parenteral, such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous. In the case of an antibody or peptide preparation, intravenous administration is desirable. Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

[0141] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, darikols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p- Hydroxybenzoic acid ester It can be manufactured using preservatives such as strawberry, flavors such as strawberry flavor and peppermint as additives. For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, It can be produced by using a binder such as bull alcohol, hydroxypropyl cellulose, gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.

 [0142] Suitable formulations for parenteral administration include injections, suppositories, sprays and the like. An injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The propellant is prepared using a carrier that does not irritate the antibody or antibody fragment itself or the recipient's oral cavity and respiratory mucosa and disperses the compound as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Depending on the nature of the antibody and the carrier used, preparations such as aerosols and dry powders are possible. In addition, these parenteral preparations can be supplemented with the ingredients exemplified as additives for oral preparations.

 [0143] The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc. The usual adult dose is 10 g / kg to 8 mg / kg per day.

 [0144] Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

 Example

[0145] [Example 1]

 Preparation of anti-ephrin B2 monoclonal antibody

 (1) Preparation of Rati cells expressing mouse ephrin B2 as antigen

A mouse full-length ephrin B2 gene expression plasmid was prepared according to a known method (Blood, 98, 1028, 2001). Rati cells (Cancer Research, 46, 4787, 1986) were used as cells to immunize rats. The gene transfer was carried out using a ribofunction method (lipofectamine 2000; manufactured by Invitrogen). Transfected cells were supplemented with pesilin (100 units / ml) and streptomycin (100 μg / ml) (both from GIBCO) in DMEM medium containing 10% serum. G418 (manufactured by GIBCO) was added at a final concentration of 500 μg / ml for surviving cells to serve as transgenic cells. These cells are seeded one by one in one well of a 96-well culture plate, the expanded cells are transferred to a 10 cm culture dish, and the culture dish force that occupies 80% of the cells in the culture dish is also EDTA. The cells were collected, and some of the cells were continuously cultured, and RNA and cell extracts were prepared from the remaining cells.

 [0146] The extract was electrophoresed on an SDS gel, transferred to a nitrocellulose filter, and then subjected to Western blotting using a commercially available ephrin B2 polyclonal antibody (manufactured by SANTA CRUZ). Since Rati expresses rat ephrin B2, cells that have had a strong band detected with ephrin B2 antibody in cells that have not been transfected into non-transfected cells are candidates for cells into which mouse ephrin B2 has been introduced. chosen. Whether the mouse ephrin B2 gene is actually introduced is determined by the primer for mouse ephrin B2 RT-PCR (5, -ATC TGTCTGCTTGGTCTTTATCAAC-3 '(SEQ ID NO: 7)) and the plasmid CAG promoter used for gene transfer. The PCR product was analyzed with the primer (5, -CTCTAGAGCCTCTGCTA ACCATGTTC-3 ′ (SEQ ID NO: 8)), which is the base sequence of the above. The resulting gene product is 387 bp.

 [0147] (2) Purification of soluble mouse extracellular ephrin-B2 human Fc fusion protein (ephrin-B2-Fc) used in the screening of hyperpridoma

 The expression plasmid of ephrin B2-Fc gene and its soluble protein were purified according to the method described in Blood, 98, 1028, 2001.

 [3] (3) Preparation of BaF / 3 cells (BaF / 3 / Ephrin B2) expressing mouse full-length ephrin B2 used in flow cytometry

 A mouse full-length ephrin B2 gene expression plasmid was prepared according to a known method (Blood, 98, 1028, 2001). BaF / 3 cells not expressing mouse ephrin B2 (Cell, 41, 727, 1985) were used as cells for gene transfer.

[0149] A ribofunction (lipofectamine 2000; manufactured by invitrogen) was used for gene introduction. Transfected cells were supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml) (V, both from GIBCO) in RPMI medium containing 10% serum, and G418 (GIBCO Manufactured at a final concentration of 500 μg / ml, and viable cells were used as transgenic cells. 96 holes of these cells Seed each cell in a well of the culture plate, transfer the proliferated cells to a 10 cm culture dish, collect the culture dish force cells that occupied 80% of the culture dish, and partly fine cells. As the cell culture continued, RNA and cell extracts were made from the remaining cells.

 [0150] The extract was electrophoresed on an SDS gel, transferred to a nitrocellulose filter, Western blotted with a commercially available ephrin B2 polyclonal antibody (manufactured by SANTA CRUZ), and ephrin B2 bands were detected. Was established as a transgenic cell. Furthermore, whether or not the mouse ephrin B2 gene has been introduced is determined by the nucleotide sequence of the mouse ephrin B2 RT-PCR primer (5, -ATCTGTCTGCTTGGTCTTTATCAAC-3, (SEQ ID NO: 9)) and the CAG promoter of the plasmid used for gene transfer. Analysis was performed with a certain primer (5, -CTCTAGAG CCTCTGCTAACCATGTTC-3, (SEQ ID NO: 10)) that can confirm the PCR product. The resulting gene product is 387 bp.

 [0151] (4) Animal immunity and preparation of antibody-producing cells

Rati cells (2 × 10 ⁸ cells) expressing mouse ephrin B2 obtained in (1) above were suspended in 500 1 phosphate buffer (PBS), and 8 weeks old female Fisher rats (manufactured by SLC, Japan) Was administered intraperitoneally. This operation was performed every 2 weeks for a total of 4 doses. Blood was collected from the tail vein, the serum antibody titer was examined by the enzyme immunoassay shown below, and the spleen was removed 3 days after the final immunization from the rat that showed a sufficient antibody titer. The spleen is shredded in RPMI medium (manufactured by SIGMA), loosened with forceps while rubbing the spleen cut into a stainless steel mesh, centrifuged (1200 rpm, 5 minutes), and then a cell strainer with a 40 μm pore. Cell mass was removed using BD Falcon. These were centrifuged (1200 rpm, 5 minutes), and the supernatant was discarded and used for cell fusion.

 [0152] (5) Enzyme immunoassay (ELISA)

Regarding the measurement of the culture supernatant of antiserum and hyperidoma derived from rats administered with Rati cells expressing mouse ephrin B2 obtained in (4), the soluble soluble phase obtained in (2) was used as an antigen. Mouse ephrin B2-Fc was used. Dispense 1-10 μg / ml soluble mouse ephrin-2 Fc into a 96-well EIA plate (BD Falcon) at 50 μ1 / well each, and leave at 4 ° C overnight. It was adsorbed. After washing, PBS containing 1% bovine serum albumin (BSA) (hereinafter referred to as 1% BSA-PBS) was reacted at 100 1 / ulker for 1 hour at room temperature to block the remaining active groups. Discard 1% BSA-PBS and immunize rat antiserum and hive Ridoma culture supernatant was dispensed at 50 μl / well and allowed to react for 2 hours. After washing with 0.05% Tween-PBS, peroxidase-labeled goat anti-rat immunoglobulin (manufactured by Biosource) was added with 50 μl / well and allowed to react at room temperature for 1 hour, washed with 0.05% Tween-PBS. Color was developed using [3,3 ′, 5,5′-tetramethylbenzidine] (manufactured by Wako Chemical), and the absorbance at OD450 nm was measured.

[6] (6) Preparation of mouse myeloma cells

8-azaguanine-resistant mouse myeloma cell line X63-AG8 was cultured in RPMI medium containing 10% bovine serum, and at least 5 X 10 ⁷ cells were secured at the time of cell fusion and used as the parent strain for cell fusion

[0154] (7) Production of High Pridoma

The rat splenocytes obtained in (4) above and the myeloma cells obtained in (6) above were mixed at a ratio of 2: 1 (1 × 10 ⁸ : 5 × 10 ⁷ ) and centrifuged (1200 rpm 5 minutes), the supernatant was discarded, and the precipitated cells were thoroughly loosened. Then, 1 ml of a polyethylene glycol solution (manufactured by SIGMA) was added at 37 ° C. with stirring, and the mixture was allowed to stand for 2 minutes. Then, 20 ml of RPMI medium warmed to 37 ° C was added for 20 minutes while shaking the 50 ml tube in which the cells were suspended. After centrifugation (900 rpm, 5 minutes, room temperature), the supernatant was discarded. After loosening the cells gently, the cells were sucked and sucked with a pipette, and the cells were gently suspended in 50 ml of RPMI medium supplemented with 10% bovine serum containing 1% of HAT (GIBCO).

 [0155] Dispense this suspension into a 96-well culture plate at a rate of 100 μ1 / well, and add 5% CO ink.

 Incubator was cultured at 37 ° C for 10-14 days under 5% CO 2 in the incubator. This culture supernatant was used in Example (5).

 2

 Select a well that reacts strongly with soluble mouse ephrin B2 obtained in (2) and does not react with other soluble ephrin B1 or B3 obtained in (2), and further HT medium (GIBCO) The hybridoma strain (hereinafter referred to as VERB2) that produces anti-mouse ephrin B2 monoclonal antibody was established by repeating cloning twice and changing to normal medium. The monoclonal antibody produced by Hypridoma strain VERB2 is called VERB2 antibody.

 [0156] The antibody class of the VERB2 antibody was determined by an enzyme immunoassay using a sub-clustering kit [manufactured by Zymed]. The results are shown in Fig. 1.

[0157] The monoclonal antibodies established in the present invention were IgGl and kappa classes. [8158] (8) Purification of VERB2 antibody

8 weeks old KSN nude female mice (manufactured by SLC) treated with pristane were injected intraperitoneally with each of 5-20 × 10 ⁶ cells Z of the hybridoma strain VERB2 obtained in (7). After 10 to 21 days, nobridoma became ascites cancer. Ascites was collected from ascitic fluid (l ~ 8ml / animal), centrifuged (3000rpm, 5min) to remove solids, and then ammonium sulfate precipitation (Antibodies 'A'Laboratory' Mayuual) And further purified using a Protein A column (manufactured by amersham) as necessary. In the reaction experiment of the antibody of the present invention using the flow cytometry shown below, a purified VERB2 antibody was used that was made into a biotin using a biotin binding kit (manufactured by SIGMA).

 [Example 2]

 Confirmation of the reactivity of the antibody of the present invention

 (1) Analysis of monoclonal antibodies in rat and mouse ephrin B2 expressing cells using flow cytometry

The specificity of the VERB2 antibody was confirmed using immune cell staining according to the following procedure. Cells transfected gene mouse ephrin B2 cell lines Rati and Rati cells expressing the rat ephrin B2 voluntarily (Rati / Ephrin B2) l X 10 ⁶ cells capacity 1.5ml enters tube (manufactured by Eppendor ί¾) The suspension was dispensed in 100 1 in an immune cell staining buffer (PBS containing 5% bovine serum). After centrifugation at 4 ° C and 5000rpm for 1 minute using a micro high-speed cooling centrifuge (manufactured by TOMY Kogyo), remove the supernatant and add 50 μl of purified biotinylated antibody (final concentration 0.1 to 5 μg / ml) And reacted at 4 ° C for 30 minutes. After the reaction, 500 1 immune cell staining buffer was added to each well, centrifuged at 4 ° C and 5000 rpm for 1 minute, the supernatant was removed, and the cells were washed. This washing operation was further performed twice, and then an immune cell staining buffer 50 1 containing avidin-FITC (Pharmingen) at a concentration of 1/50 was added and reacted at 4 ° C for 30 minutes. After the reaction, the same washing operation as described above was performed three times, and then analysis was performed using a flow cytometer (manufactured by Becton).

[0160] The results are shown in FIG. When the antibody was not added to the cells and only avidin-FITC was added, when the VERB2 antibody was added, a slight reaction was observed in the Rati cells. This means that it also crosses ephrin B2 of VERB2 antibody S rat. However, a stronger reaction was observed in cells overexpressing mouse ephrin B2. [0161] Next, using BaF / 3 cells and BaF / 3 / ephrin B2 cells described in Example 1, (3), whether or not the VERB2 antibody reacts with mouse ephrin B2 was examined. Cells were stained by the above method and observed using a flow cytometer.

 [0162] The results are shown in FIG. When BaF / 3 cells not expressing ephrin B2 were stained with VERB2 antibody, no reaction was observed as in the negative control (FIG. 3, left panel). On the other hand, no reaction was observed in BaF / 3 / Ephrin B2 cells when no primary antibody was used or when a control antibody was used as the primary antibody, whereas the reaction was achieved by using VERB2 antibody as the primary antibody. Recognized (right side of Fig. 3). Based on the above, it was found that VERB2 is an antibody that recognizes ephrin B2 in rats and mice.

 [0163] In addition, in order to confirm that the present VERB2 antibody reacts with human ephrin B2, using human prostate tumor cell line PC3 cells (ATCC CRL-1435) in which human ephrin B2 is expressed, staining is performed as described above. I did it.

 [0164] Figure 4 shows the experimental results. No reaction was observed when no primary antibody was used or when a control antibody was used as the primary antibody, whereas a fluorescent reaction was confirmed by using VERB2 antibody as the primary antibody. Therefore, it was found that the VERB2 antibody recognizes human ephrin B2.

 [0165] At the same time, a similar investigation was performed using a commercially available polyclonal antibody (manufactured by SANTA CRUZ), but the commercially available polyclonal antibody had the same results as the above-mentioned control antibody. Therefore, it was found that the commercially available polyclonal antibody cannot be used for flow cytometry.

 [0166] (2) Immunohistochemical staining with monoclonal antibody

 It has been reported that ephrin B2 is expressed in arteries but not in veins during fetal life. However, the strength that has been proved by detecting RNA. It has not been proved that ephrin B2 is expressed as a protein. Thus, expression analysis was performed using tissue sections of mouse embryos using the VERB2 antibody.

[0167] The fetus was removed from the uterus of the 13th day of pregnancy under a stereomicroscope (Leica) and permeated into PBS containing paraformaldehyde (Wako) at a concentration of 4% for 2 hours at 4 ° C. The tissue was fixed. Remove PBS containing paraformaldehyde and remove fetal tissue in PBS for 2 hours at 4 ° C. And soaked in PBS. The fetuses were dehydrated with PBS containing 40% methanol, 70% methanol and 100% methanol at 4 ° C for 20 minutes, respectively. The fetus was shredded with a knife and permeated with a methanol solution containing 50 o / o polyester wax (Daiichi Kagaku Co., Ltd.) and 100% polyester powder at 42 ° C for 30 to 60 minutes. Further, this tissue was embedded in wax in a cassette, sliced into 8 m using a microtome (manufactured by Yamato Kagaku Co., Ltd.), fixed on a slide glass and dried. The tissue sections thus prepared were dewaxed with 100% ethanol, soaked in methanol containing 0.6% hydrogen peroxide and 0.2% sodium azide for 30 minutes at room temperature, and endogenous peroxidase-free I went live. Subsequently, the solution was left to stand for 5 minutes in the order of PBS containing 70% methanol and then PBS to make it hydrophilic. PBS (blocking solution) containing 5% goat serum and 1% BSA was placed on the tissue slice and allowed to permeate at room temperature for 30 minutes to block the secondary antibody. The blocking solution was discarded, and the VERB2 antibody was diluted with a blocking solution to a final concentration of 0.1-5 / zg / ml and reacted at room temperature or 4 ° C for 6-12 hours. The antibody was discarded, and it was allowed to permeate in PBS (washing solution) containing 0.05% Tween20 at 4 ° C for 10 minutes, and the washing solution was discarded. This operation was repeated three times. Peroxidase-labeled goat anti-rat immunoglobulin (Biosource) was diluted with blocking solution to a concentration of 1/100 to 1/500, placed on a tissue section, and allowed to react at room temperature for 1 hour. The secondary antibody was discarded, and the sections were washed 3 times with the washing solution as described above. To a tissue section, diaminobenzidine (DAB; manufactured by Doujin Chemical Co., Ltd.) dissolved in PBS at a concentration of 250 g / ml in a DAB solution lml, a solution containing 51% of 1.5% hydrogen peroxide was added as a coloring solution. We used the VERB2 antibody to analyze the expression of ephrin B2.

 [0168] The VERB2 antibody showed a dark blue reaction in the dorsal artery, but no reaction in the vena cava. This indicates that the VERB2 antibody is an antibody that can be stained specifically in arteries in mouse fetuses.

 [0169] (3) Expression analysis of vascular ephrin B2 in tumor tissue using VERB2

Although expression of ephrin B2 has been promoted in the fetal period, detailed expression has not yet been clarified in adult lesions. Therefore, we examined the expression of ephrin B2 in tumor tissues where adult neovascularization occurs using the VERB2 antibody. Lewis lung carcinoma Lung cancer cells implanted in C5 7B1 / 6 mice (manufactured by SLC) The tumor force was also taken out of the tumor tissue, treated by the same method as in (1) of this example, tissue sections were prepared, and stained with VERB2 antibody by the same method as in (1) of this example.

 [0170] In the mesenchymal tissue surrounding the tumor, staining was confirmed in arterial vascular endothelial cells covered with smooth muscle cells and vascular endothelial cells without smooth muscle coating, and these cells expressed ephrin B2 I was able to confirm. It was also confirmed that ephrin B2 was expressed in almost all blood vessels inside the tumor. Based on the above, VERB2 antibody recognizes almost all endothelial cells in tumors, indicating that VERB2 antibody is effective in the diagnosis of tumors and the suppression of angiogenesis.

 [0171] (4) Inhibition of angiogenesis by VERB2 antibody

 Angiogenesis in adult tissues is observed only when the pathology occurs, but during fetal life, organ formation is vigorous and mice become lethal if angiogenesis does not progress. Therefore, VERB2 antibody was administered to pregnant mice and examined whether it could affect fetal angiogenesis.

 [0172] An 8 week-old C57B1 / 6 pregnant mouse (purchased from SLC) was injected with VERB2 antibody or control antibody (B220) from 9th day of pregnancy, when IgG from the mother penetrated into the fetus transplacentally. Dilute 1-5 mg with PBS, administer intraperitoneally on the 9th, 10th, 11th, and 12th gestation. On the 13th day, remove the fetus from the uterus of the pregnant mouse under a stereomicroscope (manufactured by Leica). I watched it.

 [0173] Compared to the fetus of the womb that received the control antibody, the fetus of the womb that received the VERB2 antibody had an undeveloped vasculature with narrowed yolk sac blood vessels. In the head, the blood vessels were significantly thinner and the number of blood vessels decreased compared to the fetus of the mother fetus administered with the control antibody. Also, as the number of blood vessels decreased, red blood cells were not transported, and the fetus was anemic.

 [0174] From the above, it has been found that VERB2 remarkably suppresses angiogenesis, and the antibody of the present invention is used as a therapeutic agent for suppressing angiogenesis that leads to worsening of the disease state in tumors and chronic inflammatory diseases. It was shown that it can be used.

 [0175] (5) Induction of failure of mammary tissue formation by VERB2

Ephrin Β2 has been reported to be specifically expressed in the mammary epithelium connecting only blood vessels (Journal of Cell Science, 111, 2741 1998) Because knockout mice are lethal in the early stages of development, functional analysis of ephrin B2 in the mammary gland that can be analyzed in pregnant mice has not been performed. Therefore, we observed the function of ephrin B2 in relation to mammary gland development by administering VERB2 antibody to pregnant mice. Similar to the schedule described in (4) above, VERB2 antibody and control antibody were administered to pregnant mice.

[0176] When 2.4 mg of VERB2 antibody was administered, all fetuses died by the 14th day of pregnancy, but when lmg of VERB2 antibody was administered, newborn mice were confirmed. The newborn was born normally but found to be delayed. However, it was found that when the VERB2 antibody was administered at the same time as the birth and the newborn was transferred to the cage of a pregnant mouse, the growth of the mouse whose growth was delayed was normal. It was found that the administration of VERB2 antibody delayed the development of mammary glands in pregnant mice, resulting in a decrease in the amount of breast milk given to the newborn, resulting in delayed growth of the newborn. Therefore, it was shown that the VERB 2 antibody has a therapeutic effect on diseases (breast adenoma, breast cancer, etc.) that cause proliferation of breast tissue.

 [0177] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

 [I] An antibody or antibody fragment that binds to ephrin B2 and inhibits ephrin B2's angiogenic activity and mammary tissue growth activity.

 [2] The antibody or antibody fragment according to claim 1, wherein ephrin B2 is a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 2.

[3] The antibody or antibody fragment according to claim 1 or 2, wherein the antibody is a monoclonal antibody.

[4] The antibody or antibody fragment according to any one of claims 1 to 3, wherein the class of the monoclonal antibody is IgG.

 5. The monoclonal antibody or antibody fragment according to any one of claims 1 to 4, wherein the monoclonal antibody is a monoclonal antibody produced from Hypridoma VERB2 (FERM BP-10100).

 [6] The monoclonal antibody is a monoclonal antibody that binds to an epitope to which a monoclonal antibody produced from Hypridoma VERB2 (FERM BP-10100) binds.

The monoclonal antibody or antibody fragment of any one of -4.

[7] A method for immunologically detecting ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.

8. The method according to claim 7, wherein the detection method is an immunohistochemical staining method.

9. The method according to claim 7, wherein the detection method is an immune cell staining method.

[10] A reagent for detecting ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.

 [II] A quantitative drug for ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.

 [12] A diagnostic agent for a disease associated with ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.

 13. The diagnostic agent according to claim 12, wherein the disease involving ephrin B2 is a disease involving angiogenesis.

[14] The diagnosis according to claim 13, wherein the disease in which angiogenesis is involved is a disease selected from solid tumors, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and fresh water. medicine.

 [15] The diagnostic agent according to claim 12, wherein the disease involving ephrin B2 is a disease causing proliferation of mammary tissue.

 [16] The diagnostic agent according to claim 15, wherein the disease causing proliferation of the mammary gland tissue is a disease that is selected as a mammary gland type or a group power of breast cancer.

[17] A therapeutic agent for a disease involving ephrin B2, comprising the antibody or antibody fragment according to any one of claims 1 to 6 as an active ingredient.

[18] The therapeutic agent according to claim 17, wherein the disease involving ephrin B2 is a disease involving angiogenesis.

 [19] The therapeutic agent according to claim 18, wherein the disease involving angiogenesis is a disease selected from the group consisting of solid tumors, rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and dryness.

 [20] The therapeutic agent according to claim 17, wherein the disease involving ephrin B2 is a disease causing proliferation of mammary gland tissue.

 [21] The therapeutic agent according to claim 20, wherein the disease that causes growth of mammary tissue is a disease that is selected from the group of mammary gland or breast cancer.

[22] A hybridoma producing the monoclonal antibody according to any one of claims 3 to 6.

23. The hybridoma according to claim 22, wherein the high-pridoma is high-pridoma VERB2 (FERM BP-10100).