JP2007320850A - Anti-ephrin b2 antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody or an antibody fragment binding to ephrin (eph-receptor-interacting protein) B2 and inhibiting binding of ephrin B2 to eph B4 and inhibiting biological activity of ephrin B2 thereby. <P>SOLUTION: The present invention provide 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. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention inhibits the biological activity of ephrin B2 by binding to ephrin (Eph-receptor-interacting protein) B2 and inhibiting the binding of ephrin B2 to EphB4 (Erythropoietin producing hepatocellular carcinoma B4). Antibody or antibody fragment, immunological detection method of ephrin B2 or ephrin B2-expressing cell using the antibody or antibody fragment and detection reagent or quantification reagent, disease involving angiogenesis using the antibody or antibody fragment Diagnostic or therapeutic agents and hybridomas that produce the antibodies are provided. The antibody is useful for the treatment of various diseases involving ephrin B2. In addition, the antibody can specifically detect ephrin B2 or ephrin B2 expressing cells by an immunological technique, and is useful for diagnosis of various diseases in which ephrin B2 is involved.

  Ephrin B2 and EphB4 are ligands and their receptors that are expressed on vascular endothelial cells, both of which are membrane proteins. 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 between arteries and veins that have been difficult. In ephrin B2 gene-deficient mice, angiogenesis is impaired both in arteries and veins, and it is lethal in the early stages of development (Non-patent Document 1). In addition, in ephrin B2 and / or EphB4 gene-deficient mice, vascularization is impaired and lethal in the early embryonic period, so ephrin B2 and EphB4 play an important role in angiogenesis (non-patented) References 1, 4).

  And, in the knockout mouse of either the ephrin B2 gene or the EphB4 gene, blood vessels induced by angiogenesis are not entirely formed regardless of arteries or veins (Non-patent Document 1). This indicates that ephrin B2 and EphB4 interact with each other, and suppression of the expression of either ephrin B2 or EphB4 suppresses the entire angiogenesis regardless of the artery or vein.

  Under conditions where ephrin B2 is excessively present, the generation of venous endothelial cells is suppressed, and conversely, under conditions where EphB4 is excessively present, the generation of arterial endothelial cells is suppressed (Non-patent Document 3). That is, when EphB4 and ephrin B2 are bound, a signal via tyrosine phosphorylation is transmitted into each cell expressing EphB4 or ephrin B2, and cell proliferation is suppressed. This is because, for example, when arterial tissue and venous tissue are close to each other or adhered at the end of the capillary, ephrin B2 and EphB4 of each endothelial cell bind to each other, thereby suppressing cell proliferation as described above, It is thought that angiogenesis is stopped. It is considered that the function of suppressing proliferation when the arterial tissue and the venous tissue are close to each other or adhered is a function for preventing the arteries and veins from being easily fused in vivo (Non-patent Document 5).

  In particular, angiogenesis that occurs in various pathological conditions is mainly formed to assist infiltration of oxygen, nutrients, inflammatory cells, etc. into the affected tissue, and these environmental factors in the bloodstream are mainly transmitted through the arteries. To be replenished. Therefore, when angiogenesis related to various pathological conditions is inhibited to improve the pathological conditions, it is an appropriate means to inhibit arterial neovascularization.

  As a means for specifically suppressing the formation of arteries, it is considered effective to suppress the function of ephrin B2 to suppress the formation of arteries. However, the functions of ephrin B2 and EphB4 have many unclear points, and the functions of ephrin B2 and EphB4 for angiogenesis at the developmental stage or angiogenesis in the matured body are unknown.

  So far, for polyclonal antibodies that react with ephrin B2, polyclonal antibodies Epherin-B2 (P-20) obtained by immunizing rabbits with the extracellular domain of human ephrin B2 deduced from the amino acid sequence of mouse ephrin B2; Human ephrin B2 deduced from the amino acid sequence of polyclonal antibody Epherin-B2 (H-83) and mouse ephrin B2 obtained by immunizing rabbits with a polypeptide corresponding to amino acid sequence 168 to 235 of the amino acid sequence of human ephrin B2 Polyclonal antibodies against three types of human ephrin B2, known as polyclonal antibody Epherin-B2 (C-20) obtained by immunizing a goat with the extracellular domain of are known (Non-patent Document 6). These are polyclonal antibodies that react with ephrin B2 of human, mouse and rat and have been confirmed to be usable for Western blotting and immunohistochemical staining. The polyclonal antibody Epherin-B2 (H-83) can be used for immunoprecipitation.

Monoclonal antibodies that specifically react with ephrin B2 include monoclonal antibodies whose subclass is IgM (Non-patent Document 7) and hamster monoclonal antibodies that react with mouse ephrin B2. Thus, a monoclonal antibody that specifically binds to ephrin B2 and inhibits the binding of mouse ephrin B2 and mouse EphB2, which is one of the receptors for ephrin B2, to 60% in an in vitro binding test is known ( Patent Document 2).

US Pat. No. 6,657,683 US Patent Publication No. 2002-0136726 Cell, 93, 741-753 (1998) Dev. Biol., 230, 151 (2001) Blood, 98, 1028 (2001) Mol. Cell, 4, 403 (1999) J. Cell Sci., 116, 2461 (2003) SANTA CRUZ BIOTECHNOLOGY company homepage, [on line], [searched August 21, 2004], Internet <http://www.scbt.com/catalog/action.lasso?-response=product_search_list.html&-Error=no_results .html & -token.order_id = & search_field = ephrinB2 & -nothing.x = 8 & -nothing.y = 17> Arterioscler Thromb Vasc Biol., 23, 190 (2003)

  An object of the present invention is to provide an antibody or antibody fragment that inhibits the biological activity of ephrin B2 by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4 receptor, and ephrin B2 using the antibody or antibody fragment 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 hybridoma producing the antibody.

The present invention relates to the following (1) to (23).
(1) An antibody that binds to ephrin B2 and inhibits the biological activity of ephrin B2 by inhibiting the binding of ephrin B2 to erythropoietin producing hepatocyte cellular carcinoma B4 (EphB4; Erythropoietin producing hepatocellular carcinoma B4) Antibody fragment.

(2) The antibody or antibody fragment according to (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 according to any one of (1) to (3), wherein the class of the monoclonal antibody is IgG.
(5) The monoclonal antibody or antibody fragment according to any one of (1) to (4), wherein the monoclonal antibody is a monoclonal antibody produced from hybridoma VERB2 (FERM ABP-10100).

(6) The monoclonal antibody or antibody fragment according to any one of (1) to (4), wherein the monoclonal antibody is a monoclonal antibody that binds to an epitope to which a monoclonal antibody produced from hybridoma VERB2 (FERM ABP-10100) binds. .
(7) A method for immunologically detecting ephrin B2 or ephrin B2 expressing cells using the antibody or antibody fragment according to any one of (1) to (6).

(8) The method according to (7), wherein the detection method is an immunohistochemical staining method.
(9) The method according to (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 (1) to (6).

(11) A reagent for quantifying ephrin B2 using the antibody or antibody fragment according to any one of (1) to (6).
(12) A diagnostic agent for a disease involving ephrin B2 using the antibody or antibody fragment according to any one of (1) to (6).

(13) The diagnostic agent according to (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 psoriasis.
(15) The diagnostic agent according to (12), wherein the disease involving ephrin B2 is a disease causing proliferation of mammary tissue.

(16) The diagnostic agent according to (15), wherein the disease causing proliferation of mammary gland tissue is a disease selected from the group consisting of mammary gland species or breast cancer.
(17) A therapeutic agent for a disease involving ephrin B2 comprising the antibody or antibody fragment according to any one of (1) to (6) as an active ingredient.
(18) The therapeutic agent according to (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 psoriasis.
(20) The therapeutic agent according to (17), wherein the disease involving ephrin B2 is a disease causing proliferation of mammary gland tissue.
(21) The therapeutic agent according to (20), wherein the disease causing proliferation of mammary gland tissue is a disease selected from the group consisting of mammary gland species or breast cancer.

(22) A hybridoma that produces the monoclonal antibody according to any one of (3) to (6).
(23) The hybridoma according to (22), wherein the hybridoma is a hybridoma VERB2 (FERM ABP-10100).

  According to the present invention, an antibody or an antibody fragment that inhibits the biological activity of ephrin B2 by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4, 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 antibodies or antibody fragments, and hybridomas producing the antibodies are provided.

  The present invention relates to an antibody or antibody fragment that inhibits the biological activity of ephrin B2 by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4.

  Ephrin B2 may be derived from any biological species. Specifically, human ephrin B2 whose DNA sequence is represented by SEQ ID NO: 1 and amino acid sequence is represented by SEQ ID NO: 2, and DNA sequence is SEQ ID NO: 3. Mouse ephrin B2 whose 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.

  Ephrin B2 includes a polypeptide having the amino acid sequence shown by SEQ ID NO: 2, 4 or 6, and an amino acid sequence in which 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 represented by SEQ ID NO: 2, preferably a polypeptide having an amino acid sequence having 80% or more homology, more preferably A polypeptide having an amino acid sequence having a homology of 90% or more, most preferably a polypeptide having an amino acid sequence having a homology of 95% or more, which has substantially the same activity as ephrin B2, etc. can give.

  A polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 is [Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (John Wiley & Sons 1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene , 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985)] and the like. 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. The number of amino acids to be deleted, substituted or added 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. .

  Unless otherwise specified, the homology values described in the present invention may be those calculated using a homology search program known to those skilled in the art, but for the base sequence, BLAST [J. Mol. Biol., 215, 403 (1990)], for amino acid sequences such as numerical values calculated using default parameters, BLAST2 [Nucleic Acids Res., 25, 3389 (1997); Genome Res., 7, 649 (1997); http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] and numerical values calculated using default parameters.

  Default parameters are 5 when G (Cost to open gap) is a base sequence, 11 when an amino acid sequence, 2 when -E (Cost to extend gap) is a base sequence, 1 when an amino acid sequence, -q (Penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, and -W (wordsize) is a base sequence, 11 residues. 3 residues, 20 if -y (Dropoff (X) for blast extensions in bits) is blastn, 7 for programs other than blastn, 15 for -X (X dropoff value for gapped alignment in bits) and -Z It is 50 when (final X dropoff value for gapped alignment in bits) is blastn, and 25 for programs other than blastn (http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

  A polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 can be produced by a method known to those skilled in the art, for example, encoding the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 It is possible to prepare by culturing a transformant into which a part of the DNA to be deleted is deleted and an expression vector containing the DNA is introduced. Further, based on the polypeptide or DNA thus prepared, one or more amino acids were 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 an amino acid sequence can be obtained.

  Antibodies that inhibit the biological activity of ephrin B2 by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4 include polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies. Used.

  Examples of the monoclonal antibody include an antibody produced by a hybridoma and a gene recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.

  A hybridoma is prepared by, for example, preparing a cell expressing the above ephrin B2 as an antigen, inducing an antibody-producing cell having antigen specificity from an animal immunized with the antigen, and further fusing it with a myeloma cell. can do. The monoclonal antibody of the present invention can be obtained by culturing the hybridoma or administering the hybridoma cell to an animal to cause ascites cancer, and separating and purifying the culture medium or ascites.

Any animal can be used as the animal to be immunized with the antigen as long as it can produce a hybridoma, but mice, rats, hamsters, rabbits, and the like are preferably used. In addition, monoclonal antibodies produced from hybridomas obtained by obtaining cells having antibody-producing ability from such animals, immunizing the cells in vitro , and then fusing with myeloma cells are also antibodies of the present invention. Is included.

  Specific examples of the monoclonal antibody of the present invention include rat antibody VERB2 produced by hybridoma VERB2. Hybridoma VERB2 was received by the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (August 19, 2004, 1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan) as of August 19, 2004. Receipt number FERM ABP-10100 Has been deposited.

  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 hybridoma VERB2 (FERM ABP-10100) binds.

  A gene recombinant antibody is obtained by modifying the above-described 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, a human antibody or an antibody fragment. A recombinant antibody having characteristics of a monoclonal antibody, low antigenicity, and extended blood half-life is preferable as a therapeutic agent.

  The humanized antibody in the present invention includes a human chimeric antibody and a human CDR (Complementarity Determining Region; hereinafter referred to as CDR) grafted antibody.

  The human chimeric antibody comprises 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 CH) of a human antibody. ) And a light chain constant region (hereinafter referred to as CL).

  The human chimeric antibody of the present invention encodes VH and VL from a hybridoma that produces a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4. CDNA is obtained and inserted into an animal cell expression vector having genes encoding human antibody CH and CL to construct a human chimeric antibody expression vector, which is introduced into animal cells for expression and production be able to.

  The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as hIg), but is preferably of the hIgG class, and more preferably hIgG1, hIgG2, hIgG3 belonging to the hIgG class, Any of the subclasses such as hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

  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.

  The human CDR-grafted antibody of the present invention is a non-human antibody produced from a hybridoma that produces a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4. An animal having a gene encoding the human antibody CH and CL by constructing a cDNA encoding the V region in which the VH and VL CDR amino acid sequences of the antibody of the animal are grafted on the VH and VL FR of any human antibody A human CDR-grafted antibody expression vector can be constructed by inserting it into an expression vector for cells, and introduced into animal cells for expression and production.

  The CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but the hIgG class is preferable, and any of the subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class may be used. it can. The CL of the human CDR-grafted antibody may be any CL as long as it belongs to hIg, and those of κ class or λ class can be used.

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

  The antibody naturally present in the human body can be cultured in lymphocytes that produce the antibody by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, and cloning. The antibody can be further purified.

  The 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 from human B cells into the phage gene. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected 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 comprising two complete heavy chains and two complete light chains by genetic engineering techniques.

  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, transplanting the ES cell into an early mouse embryo, and generating the mouse embryo. A human antibody production method from a human antibody-producing transgenic animal is obtained by obtaining and culturing a human antibody-producing hybridoma by a hybridoma production method performed in a normal non-human animal. Production can be accumulated.

  Examples of the antibody fragment of the present invention include a peptide containing Fab, F (ab ′) 2, Fab ′, scFv, diabody, dsFv and CDR.

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

  The Fab of the present invention can be obtained by treating a monoclonal antibody that inhibits the biological activity of ephrin B2 with proteolytic enzyme papain by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4. Alternatively, a Fab can be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. it can.

  F (ab ') 2 has a molecular weight of about 100,000, which is obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with the enzyme pepsin. It is a fragment having the antigen binding activity.

  F (ab ') 2 of the present invention binds to ephrin B2 and inhibits the binding of ephrin B2 and EphB4, thereby treating a monoclonal antibody that inhibits the biological activity of ephrin B2 with proteolytic enzyme pepsin. Obtainable. Alternatively, Fab ′ described below can be prepared by thioether bond or disulfide bond.

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

  Fab ′ of the present invention binds to ephrin B2 of the present invention, and inhibits the binding of ephrin B2 and EphB4, thereby inhibiting F (ab ′) 2 which inhibits the biological activity of ephrin B2 from the reducing agent dithiothreitol. It can be obtained by processing. Alternatively, Fab ′ is produced by inserting a DNA encoding the Fab ′ fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism. can do.

  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) and has antigen-binding activity. It is an antibody fragment having

  The scFv of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4. Constructs a DNA encoding scFv, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and introduces the expression vector into a prokaryotic or eukaryotic expression to produce scFv can do.

  A diabody is an antibody fragment obtained by dimerizing scFv 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.

  The diabody of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4. The DNA encoding scFv is constructed so that the length of the amino acid sequence of P is 8 residues or less, the DNA is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector is inserted into a prokaryotic expression vector. A diabody can be produced by expressing it by introduction into a living organism or a eukaryotic organism.

  dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are linked via a disulfide bond between the cysteine residues. The amino acid residue substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. (Protein Engineering, 7, 697-704, 1994).

  The dsFv of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4. Construct dsFv-encoding DNA, insert the DNA into a prokaryotic expression vector or eukaryotic expression vector, and introduce the expression vector into prokaryotic or eukaryotic cells to produce dsFv can do.

  The peptide containing CDR is composed of at least one region of CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.

  The peptide comprising the CDR of the present invention encodes CDRs of VH and VL of a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4. A peptide containing CDR is produced by inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism. be able to.

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

  The antibody of the present invention binds to ephrin B2 of the present invention and inhibits the binding of ephrin B2 and EphB4, thereby inhibiting the biological activity of ephrin B2 into an antibody or antibody fragment, a radioisotope, a small molecule drug In addition, a derivative of an antibody in which a high molecular drug, a protein, or the like is chemically or genetically bound is included.

  The derivative of the antibody of the present invention binds to ephrin B2 of the present invention and inhibits the binding of ephrin B2 and EphB4, thereby inhibiting the biological activity of ephrin B2 or the H chain or L chain of the antibody fragment. Radioisotope, low molecular weight drug, high molecular weight drug, protein on N-terminal side or C-terminal side, appropriate substituent or side chain in antibody or antibody fragment thereof, and sugar chain in antibody or antibody fragment Can be produced by combining them by chemical methods (Introduction to Antibody Engineering, Osamu Kanmitsu, Jinshokan, 1994).

  In addition, it binds to ephrin B2 of the present invention and inhibits the binding of ephrin B2 and EphB4, thereby encoding the protein to be bound with DNA encoding an antibody or antibody fragment that inhibits the biological activity of ephrin B2. The DNA can be ligated and inserted into an expression vector, and the expression vector can be introduced into an appropriate host cell and expressed.

Examples of the radioisotope include ¹³¹ I and ¹²⁵ I, and can be bound to an antibody by, for example, the chloramine T method.

  Low molecular weight drugs include: alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexate, antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin and doxorubicin, vincristine, Anticancer agents such as plant alkaloids such as vinblastine and vindesine, hormone agents such as tamoxifen and dexamethasone (clinical oncology, edited by Japan Clinical Oncology Society, Cancer and Chemotherapy, 1996), or steroids such as hydrocortisone and prednisone, Non-steroidal drugs such as aspirin and indomethacin, immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, chlorpheniramine maleate, Anti-inflammatory agents such as antihistamines such as macithin (Inflammation and anti-inflammatory therapy, Ishiyaku Shuppan Co., Ltd., 1982). For example, as a method of binding daunomycin and antibody, a method of binding between daunomycin and the amino group of the antibody via glutaraldehyde, a method of binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carbodiimide, etc. Is given.

  Examples of the polymer drug include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, and hydroxypropyl methacrylamide. By binding these macromolecular compounds to antibodies or antibody fragments, (1) improved stability against various chemical, physical or biological factors, (2) significant increase in blood half-life, ( 3) Expected effects such as loss of immunogenicity and suppression of antibody production (Bioconjugate Pharmaceuticals, Yodogawa Shoten, 1993). For example, as a method of binding PEG and an antibody, a method of reacting with a PEGylation modifying reagent and the like can be mentioned (Bioconjugate Pharmaceutical, Yodogawa Shoten, 1993). Examples of PEGylation modifying reagents include lysine ε-amino group modifiers (JP-A 61-178926), aspartic acid and glutamic acid carboxyl group modifiers (JP-A 56-23587), and arginine guanidino group modification. And the like (Japanese Patent Laid-Open No. 2-117920).

  Examples of proteins include cytokines that activate immunocompetent cells, such as human interleukin 2, human granulocyte macrophage colony stimulating factor, human macrophage colony stimulating factor, and human interleukin 12. 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, a cDNA encoding the antibody is constructed by linking a cDNA encoding the protein to a cDNA encoding the antibody or antibody fragment, and the DNA encoding the fusion antibody is constructed, and the DNA is used as a prokaryotic or eukaryotic expression vector. Into which the expression vector is introduced into prokaryotes or eukaryotes and expressed to produce a fusion antibody.

  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 immunological measurement methods. Examples of the label include enzymes such as alkaline phosphatase, peroxidase and luciferase, luminescent substances such as acridinium ester and lophine, and fluorescent substances such as FITC and RITC.

  The biological activity of ephrin B2 may be any biological activity possessed by ephrin B2, and examples thereof include an activity involved in angiogenesis. Examples of the activity involved in angiogenesis include arteriogenesis and induction of invasion / migration of vascular endothelial cells.

Below, the manufacturing method of the antibody of this invention is demonstrated concretely.
(1) Preparation of Antigen Ephrin B2 used in the production of the antibody of the present invention is [Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (John Wiley & Sons, 1987-1997)] and the like can be produced by expressing DNA encoding ephrinB2 in a host cell, for example, by the following method. The ephrin B2 to be expressed may be expressed in a state of being bound to the membrane similarly to the wild-type ephrin B2, or a soluble portion excluding the membrane-binding domain may be expressed. The ephrin B2 to be expressed may be a full-length polypeptide or a part of the ephrin B2.

  First, a recombinant vector is prepared by inserting a full-length cDNA containing the cDNA encoding the polypeptide into the downstream of the promoter of an appropriate expression vector. In this case, if necessary, a DNA fragment of an appropriate length containing a portion encoding ephrin B2 is prepared based on the full-length cDNA, and the DNA fragment can be used in place of the full-length cDNA. Good. Subsequently, a transformant producing a polypeptide can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.

  Any host cell can be used as long as it can express the target gene, such as Escherichia coli and animal cells.

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

  When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryote, and at the same time, comprises a promoter, a ribosome binding sequence, DNA used in the present invention and a transcription termination sequence. It is preferable that it is a vector containing. The recombinant vector may further contain a gene that controls the promoter.

  Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 ( QIAGEN), pKYP10 (JP 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescriptII SK (-) (Stratagene), pTrs30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrs32 [E. coli JM109 / pTrS32 ( Prepared from FERM BP-5408), pGHA2 [prepared from E. coli IGHA2 (FERM BP-400), JP 60-221091], pGKA2 [prepared from E. coli IGKA2 (FERM BP-6798), JP 60-221091] , PTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), pME18SFL3, etc. Give be able to.

  Any promoter can be used as long as it can function in the host cell to be used. For example, promoters derived from Escherichia coli, phages, etc., such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, T7 promoter and the like can be mentioned. An artificially designed and modified promoter such as a tandem promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a letI promoter can also be used.

  Moreover, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases) as the recombinant vector. In the base sequence of DNA encoding ephrin B2 used in the present invention, the base can be substituted so as to be an optimal codon for expression in the host, thereby reducing the production rate of the target ephrin B2. 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.

  As the prokaryote used as such a host cell, for example, a prokaryote belonging to the genus Escherichia can be used. Specifically, E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276 E. coli W1485, E. coli JM109, E. coli HB101, E. coli No.49, E. coli W3110, E. coli NY49, etc. can be used.

  As a method for introducing the 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)].

  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 as an insoluble granule or soluble in the periplasmic space depending on the type of vector. It can be expressed in an expression pattern such as as a mold.

  When animal cells are used as hosts, examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (1990)], pAS3-3 (special (Kaihei 2-227075), pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210 And pME18SFL3.

  Any promoter can be used as long as it can function in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter , Metallothionein promoter, heat shock promoter, SRα promoter and the like. In addition, an IE gene enhancer of human CMV may be used together with a promoter.

  Examples of host cells include human cells such as Namalwa cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, and HBT5637 cells (Japanese Patent Laid-Open No. 63-299). .

  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, an electroporation method [Cytotechnology, 3, 133 (1990)], a calcium phosphate method (Japanese Patent Laid-Open No. Hei 2). -227075) and lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

  In addition to direct expression, gene expression methods include secretory production, fusion protein expression, etc. according to the methods described in [Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989)]. It can be performed. When expressed in cells derived from eukaryotes, ephrin B2 to which sugars or sugar chains have been added can be obtained.

  The ephrin B2 used in the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the ephrin B2 in the culture, and collecting the ephrin B2 from the culture. . The method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.

  When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, 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-thiogalactopyranoside or the like with a recombinant vector using the trp promoter. Indoleacrylic acid or the like may be added to the medium.

As a medium for culturing a transformant obtained by using an animal cell 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 fetal calf serum in these media Etc. can be used. The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and the presence of 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

  As described above, ephrin B2 is obtained by culturing a transformant derived from a microorganism, animal cell, or the like carrying a recombinant vector incorporating DNA encoding ephrin B2 used in the production of the antibody of the present invention according to a normal culture method. Can be produced and accumulated and collected from the culture to produce ephrin B2 used in the production of the antibody of the present invention.

  In addition to direct expression, gene expression methods include secretory production, fusion protein expression, etc. according to the methods described in [Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989)]. It can be performed.

  There are two methods for producing ephrin B2: a method for production in the host cell, a method for secretion outside the host cell, and a method for production on the host cell outer membrane. By changing, an appropriate method can be selected.

  When ephrin B2 is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Roh et al. [Proc. Natl. Acad. Sci , USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990), Japanese Patent Laid-Open No. 05-336963 or WO94 / 23021], etc. Can also be actively secreted. Further, according to the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

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

  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 suspended by an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. To obtain a cell-free extract. 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, diethylamino Anion exchange chromatography using resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing etc. A purified sample can be obtained by using alone or in combination.

  When ephrin B2 is expressed by forming insoluble granules in the cytoplasm, the cells are similarly collected and then disrupted and centrifuged to collect the insoluble granules of ephrin B2 as a precipitate fraction. The recovered insoluble granules of the protein are solubilized with a protein denaturant. By diluting or dialyzing the solubilized solution, the protein is returned to a normal three-dimensional structure, and then a purified preparation of ephrin B2 can be obtained by the same isolation and purification method as described above.

  When a derivative such as ephrin B2 or a sugar modification product thereof is secreted outside the cell, 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 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. Thus, a purified sample can be obtained.

  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, etc., and the same isolation and purification method as above is used. A purified preparation can be obtained. In addition, when ephrin B2 is produced on the outer membrane, the expressed cells can be recovered and used as an immunogen.

  Ephrin B2 can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

  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.

(2) Animal immunity and preparation of antibody-producing cells
3 to 20-week-old mice, rats, hamsters or the like 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.

  Immunization is carried out by administering the antigen subcutaneously or intravenously or intraperitoneally to the animal together with an appropriate adjuvant (for example, Freund's Adjuvant, aluminum hydroxide gel and pertussis vaccine, etc.). When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limpet hemocyanin) is prepared and used as an immunogen.

  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 by using an enzyme immunoassay [Antibodies-A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)]. 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.

  Polyclonal antibodies can be prepared by separating and purifying the serum. The method described in (6) below, wherein the polyclonal antibody has an activity of inhibiting the biological activity of ephrin B2 by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4. Can be examined.

  When the antibody-producing cells and myeloma cells are fused, the tissue containing the antibody-producing cells such as the spleen is removed from the immunized mouse, rat or hamster 3-7 days after the final administration of the antigen. Collect. When using spleen cells, the spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged (1200 rpm, 5 minutes), the supernatant is discarded, and Tris-ammonium chloride buffer is used. The solution (pH 7.65) is treated for 1 to 2 minutes to remove red blood cells, washed 3 times with MEM medium, and provided as antibody producing cells for fusion.

(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-U1 (P3-U1) (Current Topics in Microbiology and Immunology, 18, 1, 1978), P3-NS1 / 1-Ag41 (NS-1) (European J. Immunology, 6, 511, 1976), SP2 / 0-Ag14 (SP-2) (Nature, 276, 269, 1978), P3-X63-Ag8653 (653) (J.Immunology , 123, 1548, 1979), P3-X63-Ag8 (X63) (Nature, 256, 495, 1975). These cell lines contain 8-azaguanine medium (RPMI-1640 medium containing glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 ⁻⁵ M), gentamicin (10 μg / ml) and fetal calf serum (FCS). Medium added with 8-azaguanine (15 μg / ml) to the added medium (hereinafter referred to as normal medium)], but subcultured to normal medium 3-4 days before cell fusion, Secure a cell count of 2 x 10 ⁷ or more.

(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). After mixing so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1 and centrifuging (1200 rpm, 5 minutes), the supernatant is discarded and the precipitated cells are thoroughly loosened. While stirring, at 37 ° C., add 0.2 to 1 ml of a mixture of 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM and 0.7 ml of dimethyl sulfoxide to 1 × 10 ⁸ antibody-producing cells, Add 1-2 ml of MEM medium several times every 2 minutes, then add MEM medium to a total volume of 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded, the cells are gently loosen, discharging from a graduated pipette, to gently cells HAT medium [normal medium in blowing hypoxanthine (10 ^-4 M), thymidine (Medium ^{supplemented with} 1.5 × 10 ⁻⁵ M) and aminopterin (4 × 10 ⁻⁷ M)] Suspend in 100 ml. This suspension is dispensed into a 96-well culture plate at 100 μl / well and cultured at 37 ° C. for 7 to 14 days in a 5% CO ₂ incubator.

  After culturing, a part of the culture supernatant is taken and the antibody that inhibits the biological activity of ephrin B2 is produced by binding to ephrin B2 and inhibiting the binding of ephrin B2 and EphB4 by the hybridoma selection method described later. Select the well containing the hybridoma to be used. Next, cloning was repeated twice by the limiting dilution method (first time using HT medium (medium obtained by removing aminopterin from HAT medium), and second time using normal medium), and a stable and strong antibody titer was observed. Are selected as monoclonal antibody-producing hybridoma strains.

(5) Preparation of monoclonal antibody To 8-10 week old mice or nude mice treated with pristane [0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane) was intraperitoneally administered and reared for 2 weeks] The anti-ephrin B2 monoclonal antibody-producing hybridoma cells obtained in (4) above are injected intraperitoneally 2 × 10 ^{6 to} 5 × 10 ⁷ cells / mouse. The hybridoma becomes ascites tumor in 10-21 days. Ascites was collected from this mouse, centrifuged (3000 rpm, 5 minutes) to remove solids, salted out with 40-50% ammonium sulfate, caprylic acid precipitation method, DEAE-Sepharose column, protein A column or Purify with a gel filtration column, collect IgG or IgM fractions, and use them as purified monoclonal antibodies.

The subclass of the antibody is determined by an enzyme immunoassay using a subcluster epiting kit. The amount of protein is calculated from the Raleigh method and absorbance at 280 nm.
(6) Hybridoma selection method As a method of selecting a hybridoma that produces a monoclonal antibody that inhibits the biological activity of ephrin B2 by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4. For example, the following methods can be mentioned.

  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 hybridoma culture supernatant or the purified antibody obtained by the above-described method is reacted as the first antibody. Examples of the antigen 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 the cells expressing ephrin B2 prepared in (1) above. Specifically, the cells expressing mouse ephrin B2 described in (1) of Example 1 are expressed. Rat1 cells.

After the first antibody reaction, the plate is washed and the second antibody is added.
The second antibody is an antibody obtained by labeling an antibody capable of recognizing the immunoglobulin of the first antibody with biotin, an enzyme, a chemiluminescent substance or a radiation compound. Specifically, if a mouse is used in the production of the hybridoma, an antibody capable of recognizing mouse immunoglobulin is used as the second antibody.

  After the reaction, a reaction according to the substance labeled with the second antibody is performed to select a hybridoma that produces a monoclonal antibody that reacts specifically with the antigen. At this time, it may be confirmed by the same method that it does not react with ephrin B1 and ephrin B3 which are other ephrin families.

  Then, using the culture supernatant of a hybridoma that produces a monoclonal antibody that can specifically bind to ephrin B2 selected as described above, a hybridoma that produces a monoclonal antibody that inhibits the binding of ephrin B2 and EphB4, for example, , And a method similar to the ephrin B2 and EphB2 binding inhibition experiment described in US Patent Publication No. 2002-0136726. Specifically, in the reaction system in which the binding between immobilized Glycophosphatidylinositol (hereinafter referred to as GPI) fusion ephrin B2 and Fc fusion EphB4 is detected using a labeled anti-Fc antibody, it specifically binds to the above ephrin B2. The hybridoma producing the monoclonal antibody that inhibits the binding of ephrin B2 and EphB4 is selected by adding the culture supernatant of the hybridoma that produces the monoclonal antibody capable of inhibiting the binding of GPI-fused ephrin B2 and Fc-fused EphB4. You can choose.

(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.

  The biological sample to be measured for ephrin B2 in the present invention includes ephrin B2 or ephrin B2 expressing cells to be measured, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, culture fluid, etc. If there is a possibility, it will not specifically limit.

  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, diabetic retinopathy, retinopathy of prematurity and psoriasis Diseases whose disease state progresses due to abnormal angiogenesis such as

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

Diagnosis of a disease associated with angiogenesis can be performed, for example, as follows.
About a biological sample collected from a plurality of healthy human bodies, the antibody or antibody fragment of the present invention, or a derivative thereof, is used to detect or measure ephrin B2 using the following immunological detection method. The abundance of ephrin B2 or ephrin B2 expressing cells in the biological sample is examined. 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.

  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. As a detection reagent, an ordinary immunological detection method such as an antibody or antibody fragment, or a derivative thereof, or a labeled secondary antibody that recognizes the antibody or antibody fragment, or a derivative thereof, or a substrate corresponding to the label And reagents used in the above.

  In the present invention, any known method can be used as a method for quantifying ephrin B2 or ephrin B2-expressing cells. For example, immunological measurement methods can be mentioned.

  An immunological measurement method is a method for measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Immunological measurement methods include radiolabeled immunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, western blotting and physical Examples include chemical detection methods (TIA, LAPIA, PCIA). Any method can be used as long as it detects or measures an antigen, and preferably an immunoprecipitation method or a fluorescent cell staining method.

  As a radioactive substance labeled immuno-antibody method (RIA), for example, an antibody of the present invention is reacted with an antigen or an antigen-expressing cell, and an anti-immunoglobulin antibody or binding fragment to which a radiolabel is applied is further reacted. And a method of measuring with a scintillation counter or the like.

  As an enzyme immunoassay (EIA or ELISA), for example, an antigen or a cell expressing the antigen is reacted with the antibody of the present invention, and further, a labeled anti-immunoglobulin antibody or a binding fragment is reacted, An example is a method of measuring a coloring dye 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, biotin label and the like can be used.

The sandwich ELISA method is a method in which after binding an antibody to a solid phase, an antigen to be measured is trapped and a second antibody is reacted with the trapped antigen. In the ELISA method, two kinds of antibodies or antibody fragments that recognize an antigen to be measured and having different antigen recognition sites are prepared, and one of the antibodies or antibody fragments is pre-plated (for example, a 96-well plate). The second antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, biotin and the like. Labeled after reacting cells or their lysates, tissues or their lysates, cell culture supernatant, serum, pleural effusion, ascites, ophthalmic fluid, etc., separated from the living body on the above-mentioned antibody-adsorbed plate A monoclonal antibody or antibody fragment is reacted, and a detection reaction according to the labeling substance is performed. The concentration of the test sample can be calculated from a calibration curve prepared by diluting antigens with known concentrations stepwise. As an antibody used in the sandwich ELISA method, either a polyclonal antibody or a monoclonal antibody may be used, and antibody fragments such as Fab, Fab ′, and F (ab) ₂ may be used. 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, or a combination of polyclonal antibodies and monoclonal antibodies or antibody fragments.

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

  As described above, the label used in the luminescent immunoassay as described above is luminescence of any known [Imai Kazuhiro, Bioluminescence and Chemiluminescence, Yodogawa Shoten; Clinical Laboratory 42 (1998)]. A body sign is given. For example, an acridinium ester label, a lophine label, or the like can be used.

  In Western blotting, an antigen or cells expressing the antigen are fractionated by SDS-polyacrylamide gel electrophoresis [Antibodies-A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)], and the gel is then separated into a PVDF membrane or nitrocellulose. Blotting was performed on the membrane, and the membrane was reacted with an antibody or antibody fragment that recognizes the antigen, and further reacted with a fluorescent substance such as FITC, an enzyme label such as peroxidase, or an anti-mouse IgG antibody or binding fragment with a biotin label. Then, it is a method of confirming by visualizing the label. An example of Western blotting is shown below.

Cells and tissues expressing ephrin B2 are lysed, and 0.1-30 μg of protein per lane is run by SDS-PAGE under reducing conditions. The migrated protein is transferred to a PVDF membrane and reacted with PBS containing 1% BSA (hereinafter referred to as BSA-PBS) at room temperature for 30 minutes to perform a blocking operation. Here, the monoclonal antibody of the present invention is reacted, washed with Tween-PBS, and peroxidase-labeled goat anti-mouse IgG is reacted at room temperature for 2 hours. Ephrin 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 (manufactured by Amersham).

  Specifically, the physicochemical detection method uses an antibody or antiserum that specifically binds to ephrin B2 to form an aggregate by binding the antigen ephrin B2 to the antibody or antiserum. And detecting this aggregate. Other physicochemical detection methods include the capillary method, the one-dimensional immunodiffusion method, the immunoturbidimetric method, and the latex turbidimetric method [Proposal for clinical testing methods, Kanbara Publishing, 499 (1998) ]].

  For example, in the latex immunoturbidimetric method, when an antibody or antigen-sensitized carrier such as polystyrene latex having a particle size of about 0.1 to 1 μm is used and an antigen-antibody reaction is caused by the corresponding antigen or antibody, Scattered light increases and transmitted light decreases. By detecting this change as absorbance or integrating sphere turbidity, ephrin B2 can be measured.

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

  For detection of ephrin B2-expressing cells, known immunological measurement methods can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, immunohistological staining, and the like are preferably used. Further, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystems) can also be used.

  The immunoprecipitation method is a method in which an ephrin B2-expressing cell is reacted with the monoclonal antibody or antibody fragment of the present invention, and then a carrier having a specific binding ability to immunoglobulin such as protein G-sepharose is added to the antigen-antibody complex. It is a method of settling. Alternatively, the following method can be used.

  The above-described antibody of the present invention is immobilized on a 96-well plate for ELISA, and then blocked with BSA-PBS. When the antibody is not purified, such as a hybridoma strain culture supernatant, the anti-mouse immunoglobulin, rat immunoglobulin, protein A or G is immobilized on an ELISA 96-well plate in advance. After phasing and blocking with BSA-PBS, the hybridoma strain culture supernatant is dispensed and bound. After discarding BSA-PBS and washing well with PBS, the lysate of cells and tissues expressing ephrin B2 is reacted. The immunoprecipitate is extracted from the well-washed plate with a sample buffer for SDS-PAGE and detected by the above Western blotting.

  In immunocytostaining and immunohistochemical staining, cells or tissues that have expressed an antigen are treated with a surfactant or methanol to improve the passage of the antibody in some cases, and then the antibody of the present invention is reacted. Furthermore, after reacting a fluorescent label such as fluorescein isothiocyanate (FITC), an enzyme label such as peroxidase, a biotin label or the like with an anti-immunoglobulin antibody or a binding fragment, the label is visualized and observed under a microscope. Or a fluorescent antibody staining method (flow cytometry) in which a fluorescent labeled antibody is reacted with cells and analyzed by a flow cytometer. For example, it can be performed using a method described in the literature [Monoclonal Antibodies: Principles and practice, Third edition (Academic Press, 1996), monoclonal antibody experiment manual (Kodansha Scientific, 1987)]. 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.

  The fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystems) separates the formed antibody-antigen complex from the free antibody or antigen that is not involved in the formation of the antibody-antigen complex. It is a homogeneous assay method in which the amount of antigen or antibody can be measured without any problem.

(8) Method of treating cancer using the antibody of the present invention The antibody or antibody fragment of the present invention binds to ephrin B2 and inhibits the binding of ephrin B2 and EphB4, thereby preventing diseases involving ephrin B2. Can be treated. Diseases involving ephrin B2 include diseases involving angiogenesis. Diseases involving angiogenesis include the growth or metastasis of solid tumors such as breast cancer, prostate cancer, colon cancer, gastric cancer, and lung cancer, arthritis in rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, and psoriasis Examples include diseases in which the disease state progresses due to abnormalities in neoplasia. The therapeutic agent of the present invention includes a therapeutic agent for cancer involving ephrin B2, and a therapeutic agent for cancer by antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or apoptosis-inducing action. included.

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. Since an antibody having such an activity can injure cells expressing a specific antigen in vivo , it can be used as a therapeutic agent for a disease. In particular, human chimeric antibodies, human CDR-grafted antibodies and human antibodies having human IgG class antibody constant regions are effectively used as therapeutic agents (Cancer Res., 56, 1118, 1996).

Since the antibody of the present invention can recognize ephrin B2, it can recognize ephrin B2-expressing cells present in the living body. Therefore, human chimeric antibodies, human CDR-grafted antibodies and human antibodies having human IgG class antibody constant regions including CDRs of the variable regions of the antibodies may damage ephrin B2-expressing cells in vivo or in vitro . It can be caused by abnormalities in angiogenesis such as growth or metastasis of solid tumors such as breast cancer, prostate cancer, colon cancer, gastric cancer, lung cancer, arthritis in chronic rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity and psoriasis. It is useful for the treatment of advanced diseases.

  In addition to diseases involving angiogenesis, since ephrin B2 is specifically expressed in mammary epithelium, by binding to ephrin B2 of the present invention and inhibiting the binding of ephrin B2 and EphB4, An antibody or antibody fragment that inhibits the biological activity of ephrin B2 is useful in the treatment of diseases that cause breast tissue proliferation. Specific examples of diseases that cause growth of mammary tissue include, for example, breast adenoma and breast cancer.

  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 derivative thereof as an active ingredient, but usually pharmacologically. It is desirable to provide a pharmaceutical formulation prepared by any method well known in the pharmaceutical arts, mixed with one or more acceptable carriers.

  It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous can be used. Alternatively, in the case of peptide preparations, intravenous administration can be desirably performed. Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

  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, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used 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, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The 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 cocoa butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the antibody or antibody fragment itself or the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the compound as fine particles. 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 these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

  The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 8 mg / kg per day for an adult.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to the following Example.

Preparation of Anti-Ephrin B2 Monoclonal Antibody (1) Preparation of Rat1 Cell Expressing Mouse Ephrin B2 as Antigen The mouse full-length ephrin B2 gene expression plasmid was prepared according to a known method (Blood, 98, 1028, 2001). Rat1 cells (Cancer Research, 46, 4787, 1986) were used as cells to immunize rats. For gene transfer, a lipofection method (lipofectamine 2000; manufactured by invitrogen) was used. Transfected cells are prepared by adding penicillin (100unit / ml) and streptomycin (100μg / ml) (both from GIBCO) to DMEM medium containing 10% serum, and finalizing G418 (GIBCO) at 500μg / ml for 7 days. The viable cells were used as transgenic cells by adding at a concentration. These cells are seeded one by one in a well of a 96-well culture plate, the proliferated cells are transferred to a 10 cm culture dish, and EDTA is used from the culture dish in which 80% of the cells in the culture dish occupy 80%. The cells were collected and a part of the cells was continuously cultured, and RNA and a cell extract were prepared from the remaining cells. The extract was electrophoresed on an SDS gel, transferred to a nitrocellulose filter, and then Western blotted using a commercially available ephrin B2 polyclonal antibody (SANTA CRUZ). Since Rat1 expresses rat ephrin B2, cells that have had a strong band detected with the ephrin B2 antibody in cells transfected with non-gene-transferred cells are candidates for cells into which mouse ephrin B2 has been introduced. chosen. Whether or not the mouse ephrin B2 gene is actually introduced is determined by the nucleotide sequence of the mouse ephrin B2 RT-PCR primer (5'-ATCTGTCTGCTTGGTCTTTATCAAC-3 '(SEQ ID NO: 7)) and the CAG promoter of the plasmid used for gene transfer. An analysis was performed as to whether a PCR product could be confirmed with a certain primer (5′-CTCTAGAGCCTCTGCTAACCATGTTC-3 ′ (SEQ ID NO: 8)). The resulting gene product is 387 bp.

(2) Purification of soluble mouse extracellular ephrin B2 human Fc fusion protein (ephrin B2-Fc) for use in hybridoma screening The expression plasmid of ephrin B2-Fc gene and its soluble protein can be purified using Blood, 98, 1028, This was performed according to the method described in 2001.

(3) Production of BaF / 3 cells (BaF / 3 / Ephrin B2) expressing mouse full-length ephrin B2 used in flow cytometry The known method (Blood, 98, 1028) for the expression plasmid of mouse full-length ephrin B2 gene , 2001). BaF / 3 cells not expressing mouse ephrin B2 (Cell, 41, 727, 1985) were used as cells for gene transfer.

  For the gene transfer, lipofection (lipofectamine 2000; manufactured by invitrogen) was used. For the transgenic cells, penicillin (100unit / ml) and streptomycin (100μg / ml) (both from GIBCO) are added to RPMI medium containing 10% serum, and G418 (GIBCO) is finally added to 500μg / ml for 7 days. The viable cells were used as transgenic cells by adding at a concentration. These cells are seeded one by one in a well of a 96-well culture plate, the proliferated cells are transferred to a 10 cm culture dish, and the cells are recovered from the culture dish in which 80% of the cells in the culture dish are occupied. Some of the cells were continuously cultured, and RNA and cell extracts were prepared from the remaining cells.

  The extract was electrophoresed on an SDS gel, transferred to a nitrocellulose filter, Western blotted with a commercially available ephrin B2 polyclonal antibody (SANTA CRUZ), and the cells in which the ephrin B2 band was detected were transfected into the cells. Established as. Whether or not the mouse ephrin B2 gene has been introduced is 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 introduction. It was analyzed whether the PCR product could be confirmed with the primer (5′-CTCTAGAGCCTCTGCTAACCATGTTC-3 ′ (SEQ ID NO: 10)). The resulting gene product is 387 bp.

(4) Immunization of animals and preparation of antibody-producing cells
Rat1 cells (2 × 10 ⁸ cells) expressing mouse ephrin B2 obtained in (1) above were suspended in 500 μl of phosphate buffer (PBS), and 8 week-old female Fisher rats (manufactured by SLC, Japan) It was administered intraperitoneally. This operation was performed every 2 weeks for a total of 4 doses. Blood was collected from the tail vein, and the serum antibody titer thereof was examined by the enzyme immunoassay shown below. The spleen was removed 3 days after the final immunization from a rat that showed a sufficient antibody titer. The spleen is shredded in RPMI medium (manufactured by SIGMA), the spleen cut into stainless meshes is rubbed with tweezers, centrifuged (1200 rpm, 5 minutes), and then a cell strainer (BD Falcon with a 40 μm pore) The cell mass was removed using These were centrifuged (1200 rpm, 5 minutes), and the supernatant was discarded and used for cell fusion.

(5) Enzyme immunoassay (ELISA)
Regarding the measurement of the antiserum and hybridoma culture supernatant derived from rats administered with Rat1 cells expressing mouse ephrin B2 obtained in (4), the soluble mouse ephrin obtained in (2) was used as an antigen. B2-Fc was used. To a 96-well EIA plate (BD Falcon), PBS diluted 1-10 μg / ml soluble mouse ephrin B2-Fc was dispensed at 50 μl / well respectively and allowed to stand overnight at 4 ° C. for adsorption. After washing, 100 μl / well of PBS containing 1% bovine serum albumin (BSA) (hereinafter referred to as 1% BSA-PBS) was added and reacted for 1 hour at room temperature to block remaining active groups. 1% BSA-PBS was discarded, and the culture supernatant of immunized rat antiserum and hybridoma 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 at 50 μl / well, allowed to react at room temperature for 1 hour, washed with 0.05% Tween-PBS, and then washed with TMB substrate solution [3, 3 Color was developed using “, 5,5′-tetramethylbenzidine” (manufactured by Wako Chemical Co.), and the absorbance at OD 450 nm was measured.

(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 5 × 10 ⁷ or more cells were secured at the time of cell fusion and used as a parent strain for cell fusion.

(7) Production of hybridoma
The rat splenocytes obtained in (4) above and the myeloma cells obtained in (6) above were mixed to 2: 1 (1 × 10 ⁸ : 5 × 10 ⁷ ) and centrifuged (1200 rpm 5 minutes), the supernatant was discarded, the precipitated cells were thoroughly loosened, 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, add 20 ml of RPMI medium warmed to 37 ° C over 20 minutes while shaking the 50 ml tube in which the cells are suspended. After centrifugation (900 rpm, 5 minutes, room temperature), discard the supernatant and gently After loosening the cells, the cells were gently suspended by suctioning and sucking with a pipette in 50 ml of RPMI medium supplemented with 10% bovine serum containing 1% HAT (GIBCO).

The suspension was dispensed by 100 [mu] l / well in a 96 well culture plate, 5% CO ₂ incubator, and cultured in CO ₂ 5% under 10 to 14 days at 37 ° C.. This culture supernatant was examined by the enzyme immunoassay described in Example (5), and wells that reacted strongly with the soluble mouse ephrin B2 obtained in (2) but did not react with other commercially available soluble ephrin B1 or B3. Then, the HT medium (GIBCO) and normal medium were selected and cloning was repeated twice to establish a hybridoma strain (hereinafter referred to as VERB2) that produced anti-mouse ephrin B2 monoclonal antibody. The monoclonal antibody produced by the hybridoma strain VERB2 is referred to as VERB2 antibody.

The antibody class of the VERB2 antibody was determined by enzyme immunoassay using a sub-clustering kit [manufactured by Zymed]. The result is shown in FIG.
The monoclonal antibodies established in the present invention were IgG1 and kappa classes.

(8) Purification of VERB2 antibody
The hybridoma strain VERB2 obtained in (7) was injected intraperitoneally into 8-week-old KSN nude female mice (manufactured by SLC) treated with pristane at 5 to 20 × 10 ⁶ cells / mouse. After 10 to 21 days, the hybridoma developed ascites tumor. Ascites was collected from mice with ascites (1-8 ml / mouse), centrifuged (3000 rpm, 5 minutes) to remove solids, and then purified by ammonium sulfate precipitation (Antibodies a Laboratory Manual). If necessary, further purification was performed using a Protein A column (manufactured by amersham). In the reaction experiment of the antibody of the present invention using the flow cytometry shown below, a purified VERB2 antibody biotinylated using a biotin binding kit (manufactured by SIGMA) was used.

Confirmation of reactivity of antibody of the present invention (1) Analysis of monoclonal antibody 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. Cell lines Rat1 and Rat1 that spontaneously express rat ephrin B2 Rat1 ephrin B2 gene-transfected cells (Rat1 / Ephrin B2) 1 × 10 ⁶ cells are immunized to a tube (Eppendorf) containing 1.5 ml capacity The cells were suspended in 100 μl of cell staining buffer (PBS containing 5% bovine serum) and dispensed. After centrifugation at 4 ° C and 5000rpm for 1 minute using a micro high-speed cooling centrifuge (manufactured by TOMY Industry), remove the supernatant, add 50 µl of biotinylated purified antibody (final concentration 0.1 to 5 µg / ml), and add 30 at 4 ° C. Reacted for 1 minute. After the reaction, 500 μl of 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. After this washing operation was further performed twice, 50 μl of an immune cell staining buffer 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).

  The results are shown in FIG. When VERB2 antibody was added compared to the case where only avidin-FITC was added without adding antibody to the cells, a slight reaction was observed in Rat1 cells. This means that the VERB2 antibody also crosses rat ephrin B2. However, a stronger response was seen in cells overexpressing mouse ephrin B2.

  Next, using BaF / 3 cells and BaF / 3 / Ephrin B2 cells described in Example 1 (3), it was examined whether the VERB2 antibody reacts with mouse ephrin B2. Cells were stained by the above method and observed using a flow cytometer.

  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 (left side of FIG. 3). On the other hand, in BaF / 3 / Ephrin B2 cells, no reaction was observed when no primary antibody was used or when a control antibody was used as the primary antibody, whereas a reaction was observed when VERB2 antibody was used as the primary antibody. (Right stage of FIG. 3). Based on the above, it was found that VERB2 is an antibody that recognizes rat and mouse ephrin B2.

  At the same time, the same examination was performed using a commercially available polyclonal antibody (manufactured by SANTA CRUZ). The results of the commercially available polyclonal antibody were the same as those of the control antibody. Therefore, it turned out that a commercially available polyclonal antibody cannot be used by flow cytometry.

(2) Immunohistochemical staining with monoclonal antibodies It has been reported that ephrin B2 is expressed in arteries but not in veins during fetal period. However, although proof by detecting RNA has been made, it has not been proved that ephrin B2 is expressed as a protein. Thus, expression analysis was performed using mouse fetal tissue sections using the VERB2 antibody.

  The fetus was removed from the uterus of the mouse on the 13th day of pregnancy under a stereomicroscope (Leica), and fixed in tissue by penetrating into PBS containing 4% paraformaldehyde (Wako) at 4 ° C for 2 hours. . PBS containing paraformaldehyde was removed, and fetal tissue was washed with PBS for 2 hours at 4 ° C. The fetuses were dehydrated with PBS containing 40% methanol, 70% methanol and 100% methanol for 20 minutes at 4 ° C, respectively. The fetus was shredded with a knife, and the solution was infiltrated with methanol solution containing 50% polyester wax (Daiichi Kagaku Co., Ltd.) and 100% polyester wax at 42 ° C. for 30 to 60 minutes, respectively. Further, this tissue was embedded in wax in a cassette, sliced into 8 μm 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 and infiltrated with methanol containing 0.6% hydrogen peroxide and 0.2% sodium azide at room temperature for 30 minutes to inactivate endogenous peroxidase. Went. Subsequently, the mixture 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 section and permeated at room temperature for 30 minutes to block the secondary antibody. The blocking solution was discarded, and the VERB2 antibody was diluted with the blocking solution to a final concentration of 0.1 to 5 μg / ml and reacted at room temperature or 4 ° C. for 6 to 12 hours. The antibody was discarded and permeated into 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 (manufactured by 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 three times with the washing solution as described above. Using 1 ml of DAB solution in which diaminobenzidine (DAB; manufactured by Doujin Chemical Co., Ltd.) was dissolved in PBS at a concentration of 250 μg / ml to a tissue section, a solution containing 5 μl of 1.5% hydrogen peroxide was used as a coloring solution. The expression of ephrin B2 by VERB2 antibody was analyzed.

  The VERB2 antibody showed a dark blue reaction in the dorsal artery, but no reaction in the vena cava. From this, it was shown that the VERB2 antibody is an antibody that can be stained specifically in an artery in a mouse fetus.

(3) Expression analysis of vascular ephrin B2 in tumor tissue using VERB2 Although expression analysis of ephrin B2 has been promoted in the fetal period, detailed expression has been revealed in adult lesions. Absent. Therefore, the expression of ephrin B2 in tumor tissues in which angiogenesis occurs in adults was examined using the VERB2 antibody. Lewis lung carcinoma planted in a C57Bl / 6 mouse (manufactured by SLC) Removed tumor tissue from a mouse having lung cancer cells as a tumor, processed in the same manner as (1) of this example, and prepared a tissue section The VERB2 antibody was stained by the same means as (1) of this example.

  In the mesenchymal tissue surrounding the tumor, staining can be confirmed in arterial vascular endothelial cells covered with smooth muscle cells and venous endothelial cells without smooth muscle coating, and these cells express ephrin B2 Was confirmed. In addition, it was confirmed that ephrin B2 was expressed in almost all blood vessels inside the tumor. Based on the above, the VERB2 antibody recognizes almost all endothelial cells in tumors, indicating that the VERB2 antibody is effective in the diagnosis of tumors and the suppression of angiogenesis.

(4) Inhibition of angiogenesis by VERB2 antibody Angiogenesis in adult tissues is observed only when pathological conditions occur, but in the fetal period, organ formation is vigorous and mice are lethal if angiogenesis does not progress. It becomes. Therefore, VERB2 antibody was administered to pregnant mice to examine whether it affects fetal angiogenesis.

  Eight weeks old C57Bl / 6 pregnant mice (purchased from SLC) receive VERB2 antibody or control antibody (B220) from 1 to 9 days after pregnancy, when IgG from the mother penetrates the fetus transplacentally. 5 mg was diluted with PBS and administered intraperitoneally on the 9th, 10th, 11th and 12th pregnancy. On the 13th day, the fetus was taken out from the uterus of the pregnant mouse under a stereomicroscope (manufactured by Leica) and observed for angiogenesis.

  Compared to the fetus of the mother who received the control antibody, the fetus of the mother that received the VERB2 antibody had a thin yolk sac blood vessel and an undeveloped blood vessel structure. Further, in the head, blood vessels were remarkably thinner and the number of blood vessels was reduced as compared with a fetus of a mother fetus administered with a control antibody. In addition, as the number of blood vessels decreased, red blood cells were not transported, and the fetus had an anemia color.

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

(5) Induction of disruption of mammary tissue formation by VERB2 Ephrin B2 has been reported to be specifically expressed not only in blood vessels but also in mammary epithelium (Journal of Cell Science, 111, 2741 1998). Since mortality 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. Thus, the function of ephrin B2 was observed with respect 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.

  When 2.4 mg of VERB2 antibody was administered, all fetuses died by the 14th day of pregnancy, but when 1 mg 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 newborn was transferred to the cage of a pregnant mouse not administered VERB2 antibody at the same time as the birth, the growth of the mouse whose growth was delayed was normal. It has been found that administration of VERB2 antibody delays the development of mammary glands in pregnant mice, so that the amount of breast milk given to the newborn is reduced, resulting in delayed growth of the newborn. Therefore, it was shown that the VERB2 antibody has a therapeutic effect on diseases (breast adenoma, breast cancer, etc.) that cause breast tissue proliferation.

The result of sub-clustering of the prepared VERB2 antibody is shown. The results of staining rat and mouse ephrin B2 by flow cytometry using the prepared VERB2 antibody are shown. The results of flow cytometry using the prepared VERB2 antibody are shown. The left column shows the reactivity of each antibody against BaF / 3 cells not expressing ephrin B2, and the right column shows the reactivity of each antibody against BaF / 3 / ephrin B2 cells expressing ephrin B2. 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.

Claims (23)

  An antibody or an antibody fragment that binds to ephrin B2 and inhibits the biological activity of ephrin B2 by inhibiting the binding of ephrin B2 to erythropoietin producing hepatocyte cellular carcinoma B4 (EphB4; Erythropoietin producing hepatocellular carcinoma B4) .   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.   The antibody or antibody fragment according to claim 1 or 2, wherein the antibody is a monoclonal antibody.   The antibody or antibody fragment according to any one of claims 1 to 3, wherein the class of the monoclonal antibody is IgG.   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 hybridoma VERB2 (FERM ABP-10100).   The monoclonal antibody or antibody fragment according to any one of claims 1 to 4, wherein the monoclonal antibody is a monoclonal antibody that binds to an epitope to which a monoclonal antibody produced from hybridoma VERB2 (FERM ABP-10100) binds.   A method for immunologically detecting ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.   The method according to claim 7, wherein the detection method is an immunohistochemical staining method.   The method according to claim 7, wherein the detection method is an immune cell staining method.   A reagent for detection of ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.   A reagent for quantifying ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.   A diagnostic agent for a disease involving ephrin B2 using the antibody or antibody fragment according to any one of claims 1 to 6.   The diagnostic agent according to claim 12, wherein the disease involving ephrin B2 is a disease involving angiogenesis.   The diagnostic agent according to claim 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.   The diagnostic agent according to claim 12, wherein the disease involving ephrin B2 is a disease causing proliferation of mammary gland tissue.   The diagnostic agent according to claim 15, wherein the disease causing proliferation of mammary gland tissue is a disease selected from the group consisting of mammary gland species or breast cancer.   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.   The therapeutic agent according to claim 17, wherein the disease involving ephrin B2 is a disease involving angiogenesis.   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.   The therapeutic agent according to claim 17, wherein the disease involving ephrin B2 is a disease causing proliferation of mammary gland tissue.   The therapeutic agent according to claim 20, wherein the disease causing proliferation of mammary gland tissue is a disease selected from the group consisting of mammary gland species or breast cancer.   The hybridoma which produces the monoclonal antibody of any one of Claims 3-6.   The hybridoma according to claim 22, wherein the hybridoma is a hybridoma VERB2 (FERM ABP-10100).

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