WO2012124334A1 - Antibody, pharmaceutical composition used for treatment of breast cancer, tumor test method, and reagent for testing tumor - Google Patents

Antibody, pharmaceutical composition used for treatment of breast cancer, tumor test method, and reagent for testing tumor Download PDF

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WO2012124334A1
WO2012124334A1 PCT/JP2012/001802 JP2012001802W WO2012124334A1 WO 2012124334 A1 WO2012124334 A1 WO 2012124334A1 JP 2012001802 W JP2012001802 W JP 2012001802W WO 2012124334 A1 WO2012124334 A1 WO 2012124334A1
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epha10
antibody
cancer
tumor
breast cancer
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PCT/JP2012/001802
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French (fr)
Japanese (ja)
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角田　慎一
一也 長野
堤　康央
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独立行政法人医薬基盤研究所
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Priority to JP2013504572A priority Critical patent/JP5956424B2/en
Publication of WO2012124334A1 publication Critical patent/WO2012124334A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to an antibody effective for treating breast cancer and a pharmaceutical composition containing the antibody, and a tumor test method using a tumor marker for diagnosing the onset of gastric cancer, prostate cancer and colon cancer, and the like. It relates to a reagent for tumor examination.
  • Cancer is a disease that divides the cause of adult death together with vascular diseases such as cerebral infarction and myocardial infarction.
  • Breast cancer which is a carcinoma that occurs in breast tissue, is commonly found around the world, and in western countries about 10% of women have the opportunity to get breast cancer in their lifetime. Therefore, development of an effective therapeutic agent for breast cancer is desired.
  • hormone-dependent which proliferates under the action of female hormones
  • hormone-independent which does not respond to female hormones.
  • Hormonal therapeutic agents for breast cancer include, for example, anti-estrogen agents and progesterone preparations.
  • hormone therapeutic agents can suppress the action of hormones such as estrogen that affect breast cancer development.
  • hormone therapy causes estrogen and the like to not work, and as a side effect thereof, symptoms similar to menopausal disorders occur, and osteoporosis and joint pain and the like in which bones become fragile and fractures easily occur.
  • Anticancer agents against breast cancer include, for example, doxorubicin, paclitaxel and the like, and it is possible to effectively carry out surgery by reducing cancerous lesions with the anti-cancer agent before surgery.
  • anticancer drugs include subjective side effects such as digestive symptoms such as nausea and vomiting, stomatitis, diarrhea, general fatigue, systemic symptoms such as allergic reaction, and bone marrow suppression such as leucopenia, thrombocytopenia, anemia, etc. And objective side effects such as organ damage may occur.
  • trastuzumab is an antibody drug targeting Her-2 and is effective for treating metastatic breast cancer having a high growth rate of breast cancer cells (Patent Document 1).
  • this antibody drug can be used only for Her-2 positive cases, and Her-2 positive cases are only about 20-30% (Non-patent Document 1).
  • trastuzumab resistance may occur due to factors such as changes in trastuzumab receptor binding while continuing the treatment (Non-patent Document 2).
  • TNBC triple negative case
  • a triple negative case which is a type of breast cancer that is all negative for estrogen receptor, progesterone receptor and Her-2, which account for about 15% of all breast cancer.
  • treatment with a bevacizumab preparation which is an angiogenesis inhibitor, is performed, thereby somewhat suppressing the possibility of preventing recurrence (Patent Document 2).
  • Patent Documents 3 and 4 no effective treatment has been established for this case, and the prognosis is extremely poor.
  • the recurrence rate within 5 years of the first diagnosis is about 15%, but for triple negative cases, the recurrence rate within 5 years of the first diagnosis is about 32%, and for triple negative cases Life expectancy is about 9 months if it recurs.
  • Patent Document 3 discloses in tissue collected from a living body Tumor test for gastric cancer / prostate cancer / colorectal cancer to determine the metastasis of cancer to tissue by measuring the absolute amount of cancer marker and comparing the measured absolute amount with a predetermined threshold value The method is described.
  • the present invention has been made in view of the above problems, and an object thereof is to provide an antibody and a pharmaceutical composition effective for treating breast cancer, particularly triple negative cases. Another object of the present invention is to provide a tumor inspection method and a reagent for tumor inspection which can accurately detect stomach cancer, prostate cancer and colon cancer.
  • the inventors of the present invention have found that antibodies that specifically bind to the extracellular domain of EphA10 are based on the new finding that EphA10 family molecules that are ligands of EphA10 act on EphA10-expressing breast cancer cells to promote cell proliferation.
  • the present invention has been accomplished by finding a therapeutic agent that inhibits the growth of breast cancer.
  • the present invention has the following constitution. 1. An antibody that specifically binds to the extracellular domain of EphA10. 2. The antibody according to 1 above, which binds to the fibronectin domain of EphA10. 3. The antibody according to 1 or 2, wherein the heavy chain variable domain comprises a complementarity determining region (CDR) defined by DYFIT (CDR1), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3). 4. The antibody according to any one of the above 1 to 3, wherein the light chain variable domain comprises complementarity determining regions (CDRs) defined by KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3). 5.
  • CDR complementarity determining region defined by KASQSVSKDVA
  • SASKRYT SASKRYT
  • QQDYSSPRT CDR3
  • the heavy chain variable domains are: QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4)
  • the antibody according to any one of the above 1 to 4, which comprises a framework region (FR) defined by 6.
  • FR framework region defined by SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDFIGSGYGTDFTFTISTVQAEDLAVYFC (FR3) and FGGGTKLEIKRA (FR4).
  • a pharmaceutical composition for treating breast cancer comprising the antibody according to any one of 1 to 6 as an active ingredient.
  • a tumor examination method characterized by detecting EphA10 as a tumor marker of stomach cancer, prostate cancer, and colon cancer about a living body sample separated from a living body.
  • a reagent for tumor examination which is used for a method of examining gastric cancer, prostate cancer, and colon cancer, which comprises an anti-EphA10 antibody or a primer capable of amplifying EphA10 mRNA for detecting EphA10 in a biological sample.
  • EphA10 is expressed in breast cancer cases at a higher rate than Her-2 and is expressed in about half of Her-2 negative cases, so that it is more effective than anti-Her-2 antibodies.
  • An antibody suitable for treatment and a pharmaceutical composition containing the antibody are obtained.
  • QOL quality of life
  • highly effective antibodies and pharmaceutical compositions can be obtained for triple negative cases where neither hormonal therapy nor trastuzumab can be expected to be effective and there are few options for postoperative drug therapy.
  • a tumor inspection method and a reagent for tumor inspection which can accurately detect gastric cancer, prostate cancer and colon cancer can be obtained.
  • FIG. 6 is a Western Blot showing that this monoclonal antibody recognizes a fibronectin region.
  • FIG. 6 shows that this monoclonal antibody is useful as a therapeutic agent for triple negative cases when MDA-MB-468 cells are used.
  • FIG. 6 shows that this monoclonal antibody is useful as a therapeutic agent for triple negative cases when MDA-MB-435 EphA10 cells are used.
  • FIG. 6 is a tissue microarray showing high positive rate of EphA10 in triple negative cases.
  • FIG. 6 is a tissue microarray showing that EphA10 is also expressed in gastric cancer, prostate cancer and colon cancer.
  • FIG. 6 is an mRNA analysis diagram showing that EphA10 is expressed not only in breast cancer cells but also in prostate cancer cells.
  • the antibody according to this embodiment targets the extracellular domain of EphA10 and specifically binds to it, thereby suppressing the growth of breast cancer cells.
  • EphA10 is a type of ephrin receptor, to which, for example, EphrinA3, EphrinA4 and EphrinA5 bind as ligands.
  • EphA10 has three isoforms, isoform1 is a full-length protein (GenBank Accession No. AJ872185), isoform 2 is extracellular domain only (GenBank Accession No. AJ872185), isoform 3 is intracellular C in isoform 1 The terminal SAM domain was deleted (GenBank Accession No. AJ872185).
  • Ephrin and Eph refer to ligand and receptor, respectively.
  • EphA10 is found to express protein in about half (about 49%) of breast cancer patients and about half (about 44%) in Her-2 negative patients It recognized. Furthermore, protein expression is observed in about 67% of triple negative cases.
  • the antibody may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody (for example, a bispecific antibody) formed from at least two complete antibodies, a human antibody, a humanized antibody, a camelised antibody, Chimeric antibodies, single chain Fv (scFv), single chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F (ab ') 2 fragments include, but are not limited to.
  • Antibodies include immunoglobulin molecules and immunologically active fragments thereof, ie, molecules that contain a binding site for a portion of EphA10.
  • the antibody according to the present embodiment binds to at least one extracellular domain of EphA10.
  • the extracellular domain of EphA10 includes, for example, a ligand binding domain and both the first and second fibronectin domains.
  • the monoclonal antibody can be of any isotype.
  • the monoclonal antibody may be, for example, mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, IgD or IgE.
  • the monoclonal antibody may also be, for example, human IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or IgE.
  • Any heavy chain may be paired with a kappa or lambda light chain.
  • a complete antibody molecule has two heavy (H) chain variable regions (VH) and two light (L) chain variable regions (VL).
  • the VH and VL regions can be subdivided into complementary determining regions (CDRs), interspersed with framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • the present inventors determined the VH and VL regions of heavy chain IgG2b, light chain ⁇ type, and hybridoma mRNA by RACE method (rapid amplification of cDNA ends), and the CDRs of VH were DYFIT (CDR1). ), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3). Moreover, CDRs of VL were KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3).
  • the FR of VH was QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4). Further, the FR of VL was a framework region (FR) defined by SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDRIGSGYGTDFTFTISTVQAEDLAVYFC (FR3), and FGGGTKLEIKRA (FR4).
  • FR1 framework region defined by SFVMTQTPKFLLVSAGDRITITC
  • FR2 WYQQKPGQSPKLLIY
  • GVPDRIGSGYGTDFTFTISTVQAEDLAVYFC FR3
  • FGGGTKLEIKRA FGGGTKLEIKRA
  • Antibodies useful for treating breast cancer may be derived from humanized monoclonal antibodies, which are one or more of the heavy chain variable region and the light chain variable region of a mouse (or other species) immunoglobulin. CDRs are introduced into human variable domains and are produced by substituting human residues into the corresponding framework regions of the mouse. The use of antibody components derived from humanized monoclonal antibodies eliminates the possibility of problems associated with the immunogenicity of mouse constant regions.
  • the antibody according to this embodiment includes an antibody derivative which is modified or conjugated by covalent bond of any kind of molecule and the antibody.
  • antibody derivatives include, for example, acetylation, glycosylation, amidation, PEGylation, phosphorylation, derivatization with known protecting / blocking groups, proteolytic cleavage, or intracellular ligands or other proteins Included are antibodies that have been modified by conjugation to.
  • a hybridoma that produces the antibody to be obtained can be cultured, and the antibody can be obtained by purification from the obtained culture supernatant by a conventional method.
  • the method for collecting the antibody from the obtained hybridoma is not particularly limited, and for example, it is possible to use a usual ascites fluid formation method, cell culture method and the like.
  • a mineral oil such as pristane is administered into the abdominal cavity of an animal of the same species as a mammal derived from myeloma cells, and then 1 ⁇ 10 6 to 1 ⁇ 10 9 hybridomas, preferably 1 ⁇ 10 7 to 1 ⁇ 10 8 is administered intraperitoneally and hybridomas are grown in large quantities. Then, ascites fluid or serum is collected after 1 to 4 weeks, preferably 2 to 3 weeks.
  • normal culture conditions eg, 37 ° C., 5
  • an animal cell culture medium such as 10 to 20% calf serum-containing IMDM, RPMI-1640, MEM, E-RDF or serum-free medium.
  • the cells can be cultured for 3 to 14 days at% CO 2 concentration, and antibodies can be obtained from the culture supernatant. Purification of the antibody may be carried out by known methods such as salting out with ammonium sulfate, ion exchange chromatography using anion exchangers such as DEAE cellulose, affinity chromatography using protein A sepharose etc., molecular sieve chromatography according to molecular weight and structure, etc. The method can be selected appropriately and purified.
  • a gene encoding an antibody is obtained from a hybridoma producing an antibody to be obtained, more specifically, a gene encoding an immunoglobulin heavy chain and a light chain is obtained, and the gene is expressed.
  • a vector can be prepared and introduced into host cells (mammalian cells, insect cells, microorganisms etc.) to produce the antibody.
  • gene encoding immunoglobulin heavy and light chains is subjected to genetic modification to introduce desired traits, and antibody chimeric proteins and small molecules using immunoglobulin heavy and light chain variable regions
  • the production of antibodies and scaffold antibodies can be carried out by those skilled in the art using known techniques.
  • the pharmaceutical composition according to the present embodiment contains the antibody according to the present embodiment as an active ingredient.
  • the pharmaceutical composition according to this embodiment can be formulated and administered according to a known method.
  • it can be orally or parenterally administered to humans or mammals as a solution as it is or as a pharmaceutical composition of a suitable dosage form.
  • the dose of the antibody to humans is not particularly limited, and can be, for example, 0.01 mg / kg to 50 mg / kg.
  • the pharmaceutical composition may also include a preservative that inhibits the growth of microorganisms or a buffer that helps maintain the pH within an acceptable range.
  • Preservatives include sodium azide, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, alkyl paraben such as butyl or benzyl alcohol, methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol and the like.
  • Buffering agents are phosphoric acid, citric acid and other organic acids.
  • the pharmaceutical composition may also contain, for example, an excipient, a stabilizer, a chelating agent such as EDTA, a salt, or an antibacterial agent.
  • an excipient such as ascorbic acid and methionine, polypeptides, proteins such as serum albumin, gelatin, or nonspecific immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, glycine, glutamine, asparagine, histidine, arginine,
  • amino acids such as lysine, monosaccharides such as glucose, mannose or dextrin, disaccharides, and other carbohydrates and saccharides such as sucrose, mannitol, trehalose or sorbitol.
  • the reagent for tumor examination is used for examination of stomach cancer, prostate cancer or colon cancer, and detects EphA10 as a tumor marker.
  • the determination method of the reagent for tumor examination may be any one that can confirm EphA10 in a biological sample, and may be qualitative or quantitative. For example, it may simply detect the presence or absence of EphA10, and may relatively or absolutely determine the expression level of EphA10.
  • the biological sample is derived from the blood or breast cancer tissue of a breast cancer patient or a patient having a possibility of developing breast cancer, and may preferably be a sample containing breast cancer cells.
  • the biological sample is a sample collected from a patient and may be a sample acquired for the examination method of the present invention, but is a sample acquired for use in other examinations or a sample collected by surgery May be For example, when a sample is subjected to an immunohistological staining test, paraffin sections prepared from a sample obtained from a patient can be used as a sample to be subjected to the test. For example, when subjecting a sample to Western Blot or RT-PCR, a protein extract or mRNA extract prepared from a sample obtained from a patient can be used as a sample to be subjected to the test.
  • EphA10 When EphA10 is examined by immunological techniques, an anti-EphA10 antibody is included in the test reagent.
  • an anti-EphA10 antibody When detecting expression of EphA10 at mRNA level, for example, when using RT-PCR, a primer capable of amplifying EphA10 mRNA is included in the test reagent. For example, if EphA10 is affected by the mutation of a gene that controls the expression of each gene, the expression of each protein is affected. The mutant form may be confirmed.
  • Detection of EphA10 expression at the protein level is preferably by immunological techniques.
  • an immunostaining method including a fluorescent antibody method, an enzyme antibody method, a heavy metal labeling antibody method, a radioactive isotope labeling antibody method
  • a separation by electrophoresis and a detection method by fluorescence an enzyme, a radioactive isotope, etc.
  • Western blot, fluorescent two-dimensional electrophoresis, and enzyme-linked immunosorbent assay (ELISA), dot blotting, and the like can be used.
  • the cells are judged to be gastric cancer cells, prostate cancer cells, and colon cancer cells.
  • the fusion protein (EphA10 EX -Fc chimera) of the extracellular domain (EphA10 EX ) of human EphA10 and the human IgG1 Fc domain is used as an antigen to immunize mice as an antigen, and hybridomas are prepared from spleen cells of mice with elevated antibody titer by a conventional method did. As shown in FIG. 1, it was screened by ELISA, from among the obtained hybridomas based on the binding to EphA10 EX, not bind to DR5-Fc as a negative control, 4 having affinity for EphA10 EX I picked a clone.
  • the CDRs of VH were DYFIT (CDR1), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3).
  • CDRs of VL were KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3).
  • the FR of VH was QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4).
  • FRs of VL were SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDRIGSGYGTDFTFTISTVQAEDLAVYFC (FR3) and FGGGTKLEIKRA (FR4). It was confirmed that three clones (clone 1, 2 and 4) produce an antibody whose variable region sequence is identical.
  • EphA10-pIRES2-DsRedVector was transfected into 293T cells and sorted by flow cytometry. The results are shown in FIG. As a result of flow cytometry analysis, it was confirmed that the prepared monoclonal antibody can recognize EphA10 expressed in cells.
  • Ephrin receptor type A family EphA1 to A8, A10
  • the specificity for recombinant proteins EphA1, EphA2, EphA5, EphA6, EphA10
  • ELISA ELISA
  • the binding activity of the anti-EphA10 EX monoclonal antibody was examined.
  • the binding activity was measured by surface plasmon resonance (SPR method) using Biacore 3000 (manufactured by GE Healthcare Biosciences).
  • SPR method surface plasmon resonance
  • Biacore 3000 manufactured by GE Healthcare Biosciences
  • 20 ⁇ g / mL of EphA10 EX -Fc was immobilized on a CM5 sensor chip.
  • the binding rate constant and dissociation rate constant were calculated using analysis software attached to the device.
  • the dissociation constant KD value of the monoclonal antibody is 3.9 ⁇ 10 ⁇ 9 M, and in general, it has a binding affinity that can be used for antibody drugs.
  • Human EphA10 EX is based on the gene homology with other Eph family molecules, and from the N-terminus, the Ligandbinding domain (hereinafter LBD) and two Fibronectin domains (the N-terminal side is denoted as FD1 and the C-terminal side as FD2) Configured First, a gene expression vector of EphA10 (the following (a) to (b)) in which the full length and each domain were deleted was prepared.
  • EphA10-Full (AA 34 to 1008, 109 kDa); full-length EphA10
  • B EphA10- ⁇ LBD (AA 212 to 1008, 86 kDa); LBD deleted (c) EphA10- ⁇ LBD-FD1 (AA 454 to 1008, 61 kDa); LBD, FD1 deleted (d) EphA 10- ⁇ LBD-FD 1 LBD, FD1, FD2-deleted pIRES2-DsRedExpress (Takara Bio) insert the EphA10 signal sequence (AA1-33), followed by the full-length and partial deletion EphA10 cDNA A mammalian expression vector was constructed.
  • Human fetal kidney cell line 293T cells were seeded at 2 ⁇ 10 5 cells / 1 mL / well in 12-well plates and cultured at 37 ° C. After 12 hours, the gene expression vectors of (a) to (d) and 1 mg of the Empty vector as a control were allowed to act on the cells together with the transfection reagent FuGENE HD (Roche). After 24 hours, the introduction of each expression vector was confirmed by the expression of the fluorescent protein DsRed, and then the cells were solubilized, and the lysate was subjected to SDS-PAGE, and further Western blot was performed using the anti-EphA10 EX monoclonal antibody as the primary antibody. Then, it was analyzed whether the expressed partially deleted EphA10 protein could be recognized by the anti-EphA10 EX monoclonal antibody.
  • the anti-EphA10 EX monoclonal antibody of the present invention recognizes the human EphA10 extracellular portion, and furthermore, the Fibronectin III domain (FD2) portion near the transmembrane domain.
  • the breast cancer cell growth inhibitory effect was evaluated by anti-EphA10 EX monoclonal antibody.
  • the tripro negative breast cancer cells MDA-MB-468 also expressing EphA10
  • the anti-EphA10 EX monoclonal antibody prepared at 80 mg / mL was added (final concentration; 20 mg / mL) at 25 mL / well, and after 15 minutes, the Ephrin-A5-Fc chimeric protein (prepared at 16 mg / mL) R & D was added at 25 mL / well (final concentration; 4 mg / mL).
  • the number of cells was evaluated using intracellular ATP content as an index (CellTiter-GloTM Luminescent CellViability Assay, Promega).
  • EphA10 expressing human triple negative cell line MDA-MB-468, EphA10 ligand candidate Ephrin-A5 Fc chimera and anti-EphA10 EX The monoclonal antibodies were allowed to react with each other to evaluate their antiproliferative activity. As a result, as shown in FIG. 5, it was demonstrated that the growth of breast cancer cells promoted by the addition of Ephrin-A5-Fc was completely suppressed by the addition of the anti-EphA10 EX monoclonal antibody.
  • MDA-MB-435 EphA10 cells stably expressing human EphA10 were used as EphA10 expressing triple negative cell model, and 5 ⁇ 10 6 cells were transplanted into Fat pad (milk fat tissue) of BALB / c-nu / nu mice. By doing this, a biliary cancer mouse was produced.
  • the first day (Day 0) is when the tumor volume reaches approximately 100 mm 3 and anti-EphA10 EX monoclonal antibody (200 ⁇ g / mouse) or saline as a control 4 times (Day 0, Day 3, Day 7, Day 10) intravenously Administered. Tumor volume was calculated by the following equation.
  • FIG. 6 shows that this monoclonal antibody is useful as a therapeutic agent for triple negative cases when using MDA-MB-435 EphA10 cells.
  • the vertical axis shows the percentage (%) of tumor volume on each day to the tumor volume on the first day.
  • anti-EphA10 EX monoclonal antibody or its humanized antibody may be an effective therapeutic agent for EphA10 positive triple negative breast cancer.
  • the monoclonal antibody against EphA10 EX is useful as a novel therapeutic agent for breast cancer including triple negative cases.
  • the tumor is aggressive and there is a high possibility of metastasis, and trastuzumab prescribed for HER-2 positive breast cancer or tamoxifen prescribed for hormone receptor positive breast cancer as targeted treatment
  • trastuzumab prescribed for HER-2 positive breast cancer or tamoxifen prescribed for hormone receptor positive breast cancer as targeted treatment
  • trastuzumab prescribed for HER-2 positive breast cancer or tamoxifen prescribed for hormone receptor positive breast cancer
  • Antibodies allow targeted treatment of most triple-negative cases and make a significant contribution to breast cancer treatment.
  • anti-EphA10 According to the EX monoclonal antibody, it can be cured in a short period of time, and complete recovery and reintegration can avoid social loss of human resources.
  • tissue microarray analysis Next, breast cancer tissue as a removed sample tissue was fixed using formaldehyde. The fixed breast cancer tissues were thoroughly washed with water, and then gradually increased in concentration from low concentration alcohol and sequentially transferred to absolute alcohol and sufficiently dehydrated. The alcohol was then removed using xylol and the fixed breast cancer tissue was placed in the melted liquid paraffin and thoroughly soaked, and then gradually cooled and embedded in a paraffin block. A cylindrical block of 0.6 to 2 mm in diameter is cut out of a paraffin block, and a large number of cylindrical test structures are collected, embedded in a new paraffin block, and then sliced and a sheet of tissue sections is arranged. The sectioned slice samples were placed on a slide glass to prepare a tissue microarray of breast cancer tissue.
  • nonspecific protein binding was blocked by incubating the cells in 10% BSA for 30 minutes. Thereafter, anti-EphA10 EX monoclonal antibody (5 ⁇ g / mL) was dropped as a primary antibody solution, and reacted at room temperature for 30 minutes. After washing with PBS, excess reagent was removed and a polymer reagent (Envision +, manufactured by DAKO) was dropped and reacted as a secondary antibody. After washing with PBS, the DAB solution was added dropwise to develop color. After washing with running water, the cells were stained with hematoxylin solution.
  • FIG. 7 is a tissue microarray of breast cancer tissue. As shown in FIG. 7, EphA10 positive in the triple negative case was 10/15, and the positive rate was about 67%.
  • a tissue microarray of stomach cancer tissue, prostate cancer tissue, and colon cancer tissue is prepared using specimens removed from a biopsy tissue sample, and an anti-EphA10 EX monoclonal antibody is used. Immunostaining. The results are shown in FIG. As shown in FIG. 8, it was found that EphA10 is also expressed in gastric cancer, prostate cancer, and colon cancer.
  • EphA10 gene expression was examined in breast cancer cells and prostate cancer cells.
  • breast cancer cells MDA-MB-468, MDA-MB-231, MDA-MB-435s, MCF-7, SKBR3, and MDA-MB-435 were used.
  • Androgen-dependent LNCaP, androgen-independent PC-3 and DU-145 were used as prostate cancer cells.
  • RT-PCR was performed using the following primers. Forward: CCAAGTGTGCCCTGACTACCTGTC Reverse: GTTC AGCCAAAGAGATGCCTAGGCTCAC The annealing is set at 57 ° C. for 1 minute, the extension reaction at 68 ° C. for 1 minute, the polymerase is 35 cycles of PCR using AmpliTaq Gold (Applied biosystems), and the PCR product is 1.5% TAE agarose gel Electrophoresis was performed using
  • EphA10 was found to be expressed not only in breast cancer cells but also in prostate cancer cells.
  • EphA10 has specificity to cancer tissues.
  • Her-2 negative cases and triple negative cases can be used, it can be applied to cases where treatment has been difficult or impossible until now.
  • gastric cancer, prostate cancer, or colon cancer it can be used to determine the resecting range of tissue, and also the selection of treatment methods for chemotherapy, radiation therapy, immunotherapy, etc. before and after surgery. Available to

Abstract

Provided are an antibody and a pharmaceutical composition, both of which are effective for the treatment of breast cancer, particularly a triple-negative case of breast cancer. An antibody capable of binding specifically to a extracellular domain of EphA10 is allowed to act. An example of the extracellular domain is a fibronectin domain. The EphA10 positive ratio is about 67% in a triple-negative case. In a biological sample separated from a living body, EphA10 can be detected as a tumor marker for stomach cancer, prostate cancer or colorectal cancer.

Description

抗体、乳がんの治療に用いられる医薬組成物、腫瘍検査方法、及び、腫瘍検査用試薬Antibody, pharmaceutical composition used for treatment of breast cancer, tumor test method, and reagent for tumor test
　本発明は、乳がんの治療に有効な抗体及びその抗体を含む医薬組成物、並びに、胃がん、前立腺がん及び大腸がんの発症等を診断するための腫瘍マーカーを用いた腫瘍検査方法、及び、腫瘍検査用試薬に関する。 The present invention relates to an antibody effective for treating breast cancer and a pharmaceutical composition containing the antibody, and a tumor test method using a tumor marker for diagnosing the onset of gastric cancer, prostate cancer and colon cancer, and the like. It relates to a reagent for tumor examination.
　がんは、脳梗塞や心筋梗塞に代表される血管系疾患とともに成人の死亡原因を二分する疾患である。乳房組織に発生するがん腫である乳がんは、世界中でよく見られるがんで、西側諸国では女性の約１０％が一生涯の間に乳がんに罹患する機会を有する。それゆえ乳がんに対する有効な治療薬の開発が切望されている。 Cancer is a disease that divides the cause of adult death together with vascular diseases such as cerebral infarction and myocardial infarction. Breast cancer, which is a carcinoma that occurs in breast tissue, is commonly found around the world, and in western countries about 10% of women have the opportunity to get breast cancer in their lifetime. Therefore, development of an effective therapeutic agent for breast cancer is desired.
　乳がんの発症は、女性ホルモンの働きで増殖するホルモン依存性のものと女性ホルモンに反応しないホルモン非依存性のものとがある。乳がんに対するホルモン療法剤は、例えば、抗エストロゲン剤やプロゲステロン製剤等がある。ホルモン療法剤を使用すると、乳がん発生に影響するエストロゲン等のホルモンの働きを抑制できる。しかしながらホルモン療法はエストロゲン等を働けなくさせるため、その副作用として更年期障害と同じような症状が発生し、骨がもろくなって骨折しやすくなる骨粗鬆症や関節痛等が発生する。 The onset of breast cancer is either hormone-dependent, which proliferates under the action of female hormones, and hormone-independent, which does not respond to female hormones. Hormonal therapeutic agents for breast cancer include, for example, anti-estrogen agents and progesterone preparations. The use of hormone therapeutic agents can suppress the action of hormones such as estrogen that affect breast cancer development. However, hormone therapy causes estrogen and the like to not work, and as a side effect thereof, symptoms similar to menopausal disorders occur, and osteoporosis and joint pain and the like in which bones become fragile and fractures easily occur.
　乳がんに対する抗がん剤として、例えばドキソルビシンやパクリタキセル等があり、手術前に抗がん剤でがん病変を小さくしておき、外科手術を有効に行うことができる。しかしながら、抗がん剤には、悪心・嘔吐、口内炎、下痢等の消化器症状、全身倦怠感、アレルギー反応等の全身症状等の自覚的副作用や、白血球減少、血小板減少、貧血等の骨髄抑制や臓器障害等の他覚的副作用が発生する場合がある。 Anticancer agents against breast cancer include, for example, doxorubicin, paclitaxel and the like, and it is possible to effectively carry out surgery by reducing cancerous lesions with the anti-cancer agent before surgery. However, anticancer drugs include subjective side effects such as digestive symptoms such as nausea and vomiting, stomatitis, diarrhea, general fatigue, systemic symptoms such as allergic reaction, and bone marrow suppression such as leucopenia, thrombocytopenia, anemia, etc. And objective side effects such as organ damage may occur.
　乳がんの細胞表面には、ヒト上皮増殖因子受容体２型（Her-2：humanepidermal growth factor receptor2）と呼ばれる突起の存在が発見されている。このHer-2を標的とした抗体医薬としてトラスツズマブがあり、乳がん細胞増殖速度の大きい転移性乳がんの治療に有効である（特許文献１）。しかしながら、この抗体医薬はHer-2陽性症例の場合にしか使用することができず、Her-2陽性症例は２０～３０％程度にすぎない（非特許文献１）。更にはHer-2陽性症例であっても治療を継続するうちに、トラスツズマブの受容体結合の変化等の要因によりトラスツズマブ抵抗性が生じうる場合がある（非特許文献２）。 On the cell surface of breast cancer, the presence of a process called human epidermal growth factor receptor type 2 (Her-2: human epidermal growth factor receptor 2) has been discovered. Trastuzumab is an antibody drug targeting Her-2 and is effective for treating metastatic breast cancer having a high growth rate of breast cancer cells (Patent Document 1). However, this antibody drug can be used only for Her-2 positive cases, and Her-2 positive cases are only about 20-30% (Non-patent Document 1). Furthermore, even in the case of Her-2 positive cases, trastuzumab resistance may occur due to factors such as changes in trastuzumab receptor binding while continuing the treatment (Non-patent Document 2).
　更には、エストロゲン受容体、プロゲステロン受容体、Her-2の全てが陰性のタイプの乳がん症例であるトリプルネガティブ症例（TNBC）があり、この症例は乳がん全体の約１５％を占めている。この症例に対しては、例えば血管新生阻害剤であるベバシズマブ製剤による治療が行われており、これにより再発防止可能性が幾分か抑制される（特許文献２）。しかしながら、この症例に対しては未だ有効な治療法が確立されておらず、予後は極めて不良である（非特許文献３、４）。他の乳がんのタイプでは初めの診断から５年以内の再発率は約１５％であるが、トリプルネガティブ症例では初めの診断から５年以内の再発率は約３２％であり、更にトリプルネガティブ症例では再発した場合の平均余命は約９ヶ月とされている。 Furthermore, there is a triple negative case (TNBC), which is a type of breast cancer that is all negative for estrogen receptor, progesterone receptor and Her-2, which account for about 15% of all breast cancer. In this case, for example, treatment with a bevacizumab preparation, which is an angiogenesis inhibitor, is performed, thereby somewhat suppressing the possibility of preventing recurrence (Patent Document 2). However, no effective treatment has been established for this case, and the prognosis is extremely poor (Non-patent Documents 3 and 4). For other breast cancer types, the recurrence rate within 5 years of the first diagnosis is about 15%, but for triple negative cases, the recurrence rate within 5 years of the first diagnosis is about 32%, and for triple negative cases Life expectancy is about 9 months if it recurs.
　次に、近年、腫瘍マーカーについての研究が盛んに行われており、既にがんのスクリーニングテストとして活用されている腫瘍マーカーや、現在機能を評価中の腫瘍マーカーが存在する。腫瘍マーカーとしては、がん発症や再発等の診断マーカー、予後予測マーカー、治療奏効性予測マーカー等としての活用が期待されており、例えば、特許文献３には、生体から採取された組織中のがんマーカーの絶対量を測定し、測定された絶対量と予め決められた閾値とを比較することにより組織へのがんの転移を判定する、胃がん・前立腺がん・大腸がんの腫瘍検査方法が記載されている。 Next, in recent years, studies on tumor markers have been actively conducted, and there are tumor markers that have already been used as screening tests for cancer and tumor markers whose function is currently being evaluated. As a tumor marker, it is expected to be used as a diagnostic marker for cancer development or recurrence, a prognostic prediction marker, a therapeutic response prediction marker, etc. For example, Patent Document 3 discloses in tissue collected from a living body Tumor test for gastric cancer / prostate cancer / colorectal cancer to determine the metastasis of cancer to tissue by measuring the absolute amount of cancer marker and comparing the measured absolute amount with a predetermined threshold value The method is described.
国際公開ＷＯ８９／６６９２号公報International Publication WO 89/6692 特表２０１０－５０４９４９号公報Japanese Patent Publication No. 2010-504949 特開２００８－０１７８３２号公報Japanese Patent Application Publication No. 2008-017832
　しかし、上述の検査方法では、ステージの進んだ進行がん、末期がんでの検出は可能であるものの、早期のがんを検出することは困難である。 However, although the above-mentioned inspection method can detect advanced stage cancer and advanced stage cancer, it is difficult to detect early stage cancer.
　本発明は上述の問題点に鑑みてなされたものであって、乳がん、特にトリプルネガティブ症例の治療に有効な抗体及び医薬組成物を提供することを目的とする。また、胃がん、前立腺がん、及び大腸がんを的確に検出することのできる腫瘍検査方法、及び、腫瘍検査用試薬を提供することを目的とする。 The present invention has been made in view of the above problems, and an object thereof is to provide an antibody and a pharmaceutical composition effective for treating breast cancer, particularly triple negative cases. Another object of the present invention is to provide a tumor inspection method and a reagent for tumor inspection which can accurately detect stomach cancer, prostate cancer and colon cancer.
　本発明者らは、EphA10のリガンドであるEphA10ファミリー分子をEphA10発現乳がん細胞に作用させることにより細胞増殖が促進されるという新知見に基づいて、EphA10の細胞外ドメインに特異的に結合する抗体が、乳がんの増殖を阻害する治療薬になることを見いだし、本発明を完成させた。 The inventors of the present invention have found that antibodies that specifically bind to the extracellular domain of EphA10 are based on the new finding that EphA10 family molecules that are ligands of EphA10 act on EphA10-expressing breast cancer cells to promote cell proliferation. The present invention has been accomplished by finding a therapeutic agent that inhibits the growth of breast cancer.
　即ち、本発明は、以下の構成からなる。
１．EphA10の細胞外ドメインに特異的に結合する抗体。
２．EphA10のフィブロネクチンドメインに結合する前記１に記載の抗体。
３．重鎖可変ドメインが、DYFIT（ＣＤＲ１）、EIYPGSGSIYYNENFKG（ＣＤＲ２）及びSWVPYAMDY（ＣＤＲ３）により規定される相補性決定領域（ＣＤＲ）を含む前記１又は２に記載の抗体。
４．軽鎖可変ドメインが、KASQSVSKDVA（ＣＤＲ１）、SASKRYT（ＣＤＲ２）及びQQDYSSPRT（ＣＤＲ３）により規定される相補性決定領域（ＣＤＲ）を含む前記１～３の何れか１つに記載の抗体。
５．重鎖可変ドメインが、QVLLQQSGPELVKPGASVKMSCKASGYTFT（ＦＲ１）、WVSQRTGQGLEWIG（ＦＲ２）、KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR（ＦＲ３）及びWGQGTSVTVSS（ＦＲ４）
により規定されるフレームワーク領域（ＦＲ）を含む前記１～４の何れか１つに記載の抗体。
６．軽鎖可変ドメインが、SFVMTQTPKFLLVSAGDRITITC（ＦＲ１）、WYQQKPGQSPKLLIY（ＦＲ２）、GVPDRFIGSGYGTDFTFTISTVQAEDLAVYFC（ＦＲ３）及びFGGGTKLEIKRA（ＦＲ４）により規定されるフレームワーク領域（ＦＲ）を含む前記１～５の何れか１つに記載の抗体。
７．前記１～６の何れか１つに記載の抗体を有効成分として含有することを特徴とする乳がんの治療に用いられる医薬組成物。
８．生体から分離された生体検体について、EphA10を、胃がん、前立腺がん、及び大腸がんの腫瘍マーカーとして検出することを特徴とする、腫瘍検査方法。
９．生体検体中のEphA10を検出するための、抗EphA10抗体又はEphA10のｍＲＮＡを増幅可能なプライマーを含む、胃がん、前立腺がん、及び大腸がんの検査方法に用いる、腫瘍検査用試薬。 That is, the present invention has the following constitution.
1. An antibody that specifically binds to the extracellular domain of EphA10.
2. The antibody according to 1 above, which binds to the fibronectin domain of EphA10.
3. The antibody according to 1 or 2, wherein the heavy chain variable domain comprises a complementarity determining region (CDR) defined by DYFIT (CDR1), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3).
4. The antibody according to any one of the above 1 to 3, wherein the light chain variable domain comprises complementarity determining regions (CDRs) defined by KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3).
5. The heavy chain variable domains are: QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4)
The antibody according to any one of the above 1 to 4, which comprises a framework region (FR) defined by
6. The antibody according to any one of the above 1 to 5, wherein the light chain variable domain comprises a framework region (FR) defined by SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDFIGSGYGTDFTFTISTVQAEDLAVYFC (FR3) and FGGGTKLEIKRA (FR4). .
7. A pharmaceutical composition for treating breast cancer comprising the antibody according to any one of 1 to 6 as an active ingredient.
8. A tumor examination method characterized by detecting EphA10 as a tumor marker of stomach cancer, prostate cancer, and colon cancer about a living body sample separated from a living body.
9. A reagent for tumor examination, which is used for a method of examining gastric cancer, prostate cancer, and colon cancer, which comprises an anti-EphA10 antibody or a primer capable of amplifying EphA10 mRNA for detecting EphA10 in a biological sample.
　本発明によれば、EphA10はHer-2よりも高い割合で乳がん症例に発現しており、しかもHer-2陰性症例の約半分に発現しているので、抗Her-2抗体よりも有効な乳がん治療に好適な抗体及びその抗体を含有する医薬組成物が得られる。また、女性ホルモンの働きを阻害するものでなく、一般的な抗がん剤に特有の副作用も極めて少ないので、乳がん治療におけるＱＯＬ（Quality of Life）を向上させることができる。特に、ホルモン療法もトラスツズマブも効果が期待できず、術後薬物療法の選択肢が少ないとされるトリプルネガティブ症例に対して、極めて有効な抗体及び医薬組成物が得られる。また、胃がん、前立腺がん、及び大腸がんを的確に検出することのできる腫瘍検査方法及び腫瘍検査用試薬が得られる。 According to the present invention, EphA10 is expressed in breast cancer cases at a higher rate than Her-2 and is expressed in about half of Her-2 negative cases, so that it is more effective than anti-Her-2 antibodies. An antibody suitable for treatment and a pharmaceutical composition containing the antibody are obtained. In addition, since it does not inhibit the action of female hormones and has very few side effects specific to general anticancer drugs, it is possible to improve the quality of life (QOL) in breast cancer treatment. In particular, highly effective antibodies and pharmaceutical compositions can be obtained for triple negative cases where neither hormonal therapy nor trastuzumab can be expected to be effective and there are few options for postoperative drug therapy. In addition, a tumor inspection method and a reagent for tumor inspection which can accurately detect gastric cancer, prostate cancer and colon cancer can be obtained.
ELISA法によるモノクローナル抗体のヒトEphA10の細胞外ドメインに対する結合性評価を示す図である。It is a figure which shows the binding property with respect to the extracellular domain of human EphA10 of the monoclonal antibody by ELISA method. 作製したモノクローナル抗体が、細胞に発現しているEphA10を認識することを確認するフローサイトメトリーによる図である。It is a figure by flow cytometry which confirms that the produced monoclonal antibody recognizes EphA10 expressed to a cell. 作製したモノクローナル抗体が、EphA10を特異的に認識することを確認するELISA法による図である。It is a figure by ELISA which confirms that the produced monoclonal antibody recognizes EphA10 specifically. 本モノクローナル抗体が、フィブロネクチン領域を認識することを示すWestern Blot図である。FIG. 6 is a Western Blot showing that this monoclonal antibody recognizes a fibronectin region. MDA-MB-468細胞を使用した場合において、本モノクローナル抗体が、トリプルネガティブ症例に対する治療薬として有用であることを示す図である。FIG. 6 shows that this monoclonal antibody is useful as a therapeutic agent for triple negative cases when MDA-MB-468 cells are used. MDA-MB-435EphA10細胞を使用した場合において、本モノクローナル抗体が、トリプルネガティブ症例に対する治療薬として有用であることを示す図である。FIG. 6 shows that this monoclonal antibody is useful as a therapeutic agent for triple negative cases when MDA-MB-435 EphA10 cells are used. トリプルネガティブ症例におけるEphA10の高い陽性率を示す組織マイクロアレイ図である。FIG. 6 is a tissue microarray showing high positive rate of EphA10 in triple negative cases. EphA10が、胃がん、前立腺がん、及び、大腸がんにおいても発現していることを示す組織マイクロアレイ図である。FIG. 6 is a tissue microarray showing that EphA10 is also expressed in gastric cancer, prostate cancer and colon cancer. EphA10が、乳がん細胞に発現するのみならず、前立腺がん細胞においても発現することを示すｍＲＮＡ解析図である。FIG. 6 is an mRNA analysis diagram showing that EphA10 is expressed not only in breast cancer cells but also in prostate cancer cells.
　以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, which are for the purpose of facilitating the understanding of the principle of the present invention, and the scope of the present invention is as follows: The present invention is not limited to the embodiment, and other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.
　（抗体）
　本実施形態に係る抗体は、EphA10の細胞外ドメインを標的とし、それに特異的に結合することにより、乳がん細胞の増殖を抑制する。 (antibody)
The antibody according to this embodiment targets the extracellular domain of EphA10 and specifically binds to it, thereby suppressing the growth of breast cancer cells.
　EphA10はエフリン受容体の一種であり、リガンドとして例えばEphrinA3、EphrinA4、及びEphrinA5が結合する。EphA10にはアイソフォームが３つあり、isoform1は全長からなるタンパク質であり（GenBank Accession No. AJ872185）、isoform 2は、細胞外ドメインのみ（GenBankAccession No. AJ872185）、isoform 3はisoform 1において細胞内Ｃ末端のSAMドメインが欠失したもの（GenBankAccession No. AJ872185）である。Ephrin及びEphは、各々リガンド及びレセプターを指す。 EphA10 is a type of ephrin receptor, to which, for example, EphrinA3, EphrinA4 and EphrinA5 bind as ligands. EphA10 has three isoforms, isoform1 is a full-length protein (GenBank Accession No. AJ872185), isoform 2 is extracellular domain only (GenBank Accession No. AJ872185), isoform 3 is intracellular C in isoform 1 The terminal SAM domain was deleted (GenBank Accession No. AJ872185). Ephrin and Eph refer to ligand and receptor, respectively.
　本発明者らがEphA10について解析を行った結果、EphA10は乳がん患者のほぼ半数（約４９％）でタンパク質の発現が認められ、Her-2陰性患者のほぼ半数（約４４％）でタンパク質発現が認められている。更には、トリプルネガティブ症例における約６７％においてタンパク質発現が認められている。 As a result of analysis by the present inventors on EphA10, EphA10 is found to express protein in about half (about 49%) of breast cancer patients and about half (about 44%) in Her-2 negative patients It recognized. Furthermore, protein expression is observed in about 67% of triple negative cases.
　抗体(免疫グロブリン)は、モノクローナル抗体、ポリクローナル抗体、少なくとも２つの完全な抗体から形成される複特異性抗体(例えば、二特異性抗体)、ヒト抗体、ヒト化抗体、ラクダ化(camelised)抗体、キメラ抗体、一本鎖Ｆｖ(ｓｃＦｖ)、一本鎖抗体、単一ドメイン抗体、ドメイン抗体、Ｆab断片、Ｆ(ab')２断片を包含するが、それらに限られない。抗体は、免疫グロブリン分子及びその免疫学的に活性な断片、即ちEphA10の一部分に対する結合部位を含む分子を含む。 The antibody (immunoglobulin) may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody (for example, a bispecific antibody) formed from at least two complete antibodies, a human antibody, a humanized antibody, a camelised antibody, Chimeric antibodies, single chain Fv (scFv), single chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F (ab ') 2 fragments include, but are not limited to. Antibodies include immunoglobulin molecules and immunologically active fragments thereof, ie, molecules that contain a binding site for a portion of EphA10.
　本実施形態に係る抗体は、EphA10の細胞外ドメインの少なくとも一つに結合する。EphA10の細胞外ドメインは、例えばリガンド結合ドメイン（Ligand binding domain）、第一及び第二のフィブロネクチンドメイン（Fibronectin domain）のいずれも包含する。 The antibody according to the present embodiment binds to at least one extracellular domain of EphA10. The extracellular domain of EphA10 includes, for example, a ligand binding domain and both the first and second fibronectin domains.
　モノクローナル抗体は、任意のアイソタイプであることができる。モノクローナル抗体は、例えば、マウスIgM、IgG1、IgG2a、IgG2b、IgG3、IgA、IgD、又はIgEであってもよい。また、モノクローナル抗体は、例えば、ヒトIgM、IgG1、IgG2、IgG3、IgG4、IgA1、IgA2、IgD、又はIgEであってもよい。任意の重鎖は、κ又はλ型の軽鎖と対形成してもよい。 The monoclonal antibody can be of any isotype. The monoclonal antibody may be, for example, mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, IgD or IgE. The monoclonal antibody may also be, for example, human IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or IgE. Any heavy chain may be paired with a kappa or lambda light chain.
　完全な抗体分子は、２つの重（Ｈ）鎖可変領域（ＶＨ）及び２つの軽（Ｌ）鎖可変領域（ＶＬ）を有する。ＶＨ及びＶＬ領域は、相補的決定領域（ＣＤＲ）に細分することができ、フレームワーク領域（ＦＲ）が散在している。 A complete antibody molecule has two heavy (H) chain variable regions (VH) and two light (L) chain variable regions (VL). The VH and VL regions can be subdivided into complementary determining regions (CDRs), interspersed with framework regions (FRs).
　本発明者らは、重鎖IgG2b、軽鎖κ型にて、ハイブリドーマｍＲＮＡからＲＡＣＥ法（rapidamplification of ｃＤＮＡ ends）にてＶＨ領域及びＶＬ領域の配列を決定したところ、ＶＨのＣＤＲが、DYFIT（ＣＤＲ１）、EIYPGSGSIYYNENFKG（ＣＤＲ２）及びSWVPYAMDY（ＣＤＲ３）であった。また、ＶＬのＣＤＲは、KASQSVSKDVA（ＣＤＲ１）、SASKRYT（ＣＤＲ２）及びQQDYSSPRT（ＣＤＲ３）であった。ＶＨのＦＲは、QVLLQQSGPELVKPGASVKMSCKASGYTFT（ＦＲ１）、WVSQRTGQGLEWIG（ＦＲ２）、KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR（ＦＲ３）及びWGQGTSVTVSS（ＦＲ４）であった。また、ＶＬのＦＲは、SFVMTQTPKFLLVSAGDRITITC（ＦＲ１）、WYQQKPGQSPKLLIY（ＦＲ２）、GVPDRFIGSGYGTDFTFTISTVQAEDLAVYFC（ＦＲ３）及びFGGGTKLEIKRA（ＦＲ４）により規定されるフレームワーク領域（ＦＲ）であった。 The present inventors determined the VH and VL regions of heavy chain IgG2b, light chain κ type, and hybridoma mRNA by RACE method (rapid amplification of cDNA ends), and the CDRs of VH were DYFIT (CDR1). ), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3). Moreover, CDRs of VL were KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3). The FR of VH was QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4). Further, the FR of VL was a framework region (FR) defined by SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDRIGSGYGTDFTFTISTVQAEDLAVYFC (FR3), and FGGGTKLEIKRA (FR4).
　乳がん治療に有用な抗体は、ヒト化モノクローナル抗体由来であってもよく、ヒト化モノクローナル抗体は、マウス（又は他の種の）免疫グロブリンの重鎖可変領域及び軽鎖可変領域から１つ以上のＣＤＲをヒト可変ドメイン内に導入し、ヒト残基をマウスの相当するフレームワーク領域に置換することにより産生される。ヒト化モノクローナル抗体由来の抗体成分の使用により、マウス定常部の免疫原性に付随する問題の可能性を排除する。 Antibodies useful for treating breast cancer may be derived from humanized monoclonal antibodies, which are one or more of the heavy chain variable region and the light chain variable region of a mouse (or other species) immunoglobulin. CDRs are introduced into human variable domains and are produced by substituting human residues into the corresponding framework regions of the mouse. The use of antibody components derived from humanized monoclonal antibodies eliminates the possibility of problems associated with the immunogenicity of mouse constant regions.
　本実施形態に係る抗体は、任意の種類の分子と抗体との共有結合により修飾又は複合化された、抗体誘導体を包含する。このような抗体誘導体として、例えば、アセチル化、グリコシル化、アミド化、ＰＥＧ化、リン酸化、既知の保護基／ブロック基による誘導体化、タンパク質分解的開裂、又は細胞内配位子又は他のタンパク質への結合により修飾されている抗体が挙げられる。 The antibody according to this embodiment includes an antibody derivative which is modified or conjugated by covalent bond of any kind of molecule and the antibody. Such antibody derivatives include, for example, acetylation, glycosylation, amidation, PEGylation, phosphorylation, derivatization with known protecting / blocking groups, proteolytic cleavage, or intracellular ligands or other proteins Included are antibodies that have been modified by conjugation to.
　本実施形態に係る抗体を取得する方法としては、取得したい抗体を産生するハイブリドーマを培養し、得られた培養上清から常法によって抗体を精製して取得することができる。取得したハイブリドーマから抗体を採取する方法は、特に限定されるものではないが、例えば通常の腹水形成法や細胞培養法等を用いることが可能である。腹水形成法においては、骨髄腫細胞由来の哺乳動物と同種の動物の腹腔内にプリスタン等の鉱物油を投与し、その後ハイブリドーマ１×１０６～１×１０９個、好ましくは１×１０７～１×１０８個を腹腔内に投与し、ハイブリドーマを大量に増殖させる。そして、１～４週間、好適には２～３週間後に腹水又は血清を採集する。細胞培養法においては、ハイブリドーマを１０～２０％仔ウシ血清含有IMDM、RPMI-1640、MEM、E-RDF又は無血清培地等の動物細胞培養培地中で、通常の培養条件（例えば37℃、５％ＣＯ２濃度）で３～１４日間培養し、その培養上清から抗体を取得することができる。抗体の精製は、硫安塩析法、DEAEセルロース等の陰イオン交換体を利用するイオン交換クロマトグラフィー、プロテインＡセファロース等を用いるアフィニティークロマトグラフィー、分子量や構造によってふるい分ける分子ふるいクロマトグラフィー等の公知の方法を適宜に選択して精製することが可能である。 As a method for obtaining the antibody according to the present embodiment, a hybridoma that produces the antibody to be obtained can be cultured, and the antibody can be obtained by purification from the obtained culture supernatant by a conventional method. The method for collecting the antibody from the obtained hybridoma is not particularly limited, and for example, it is possible to use a usual ascites fluid formation method, cell culture method and the like. In ascites fluid formation, a mineral oil such as pristane is administered into the abdominal cavity of an animal of the same species as a mammal derived from myeloma cells, and then 1 × 10 6 to 1 × 10 9 hybridomas, preferably 1 × 10 7 to 1 × 10 8 is administered intraperitoneally and hybridomas are grown in large quantities. Then, ascites fluid or serum is collected after 1 to 4 weeks, preferably 2 to 3 weeks. In the cell culture method, normal culture conditions (eg, 37 ° C., 5) are used in an animal cell culture medium such as 10 to 20% calf serum-containing IMDM, RPMI-1640, MEM, E-RDF or serum-free medium. The cells can be cultured for 3 to 14 days at% CO 2 concentration, and antibodies can be obtained from the culture supernatant. Purification of the antibody may be carried out by known methods such as salting out with ammonium sulfate, ion exchange chromatography using anion exchangers such as DEAE cellulose, affinity chromatography using protein A sepharose etc., molecular sieve chromatography according to molecular weight and structure, etc. The method can be selected appropriately and purified.
　また、別の方法としては、取得したい抗体を産生するハイブリドーマから抗体をコードする遺伝子、より詳細には免疫グロブリンの重鎖及び軽鎖をコードする遺伝子を取得して、該遺伝子を発現するためのベクターを作成し、宿主細胞（哺乳類細胞、昆虫細胞、微生物等）に導入して、該抗体を産生させることも可能である。このとき、免疫グロブリンの重鎖及び軽鎖をコードする遺伝子について、望む形質を導入するための遺伝子改変を行ったり、免疫グロブリンの重鎖及び軽鎖の可変領域を用いて抗体キメラタンパク質、低分子抗体やスキャフォールド抗体を作成することは、公知の技術を用いることで、当業者であれば実施することができる。 As another method, a gene encoding an antibody is obtained from a hybridoma producing an antibody to be obtained, more specifically, a gene encoding an immunoglobulin heavy chain and a light chain is obtained, and the gene is expressed. A vector can be prepared and introduced into host cells (mammalian cells, insect cells, microorganisms etc.) to produce the antibody. At this time, gene encoding immunoglobulin heavy and light chains is subjected to genetic modification to introduce desired traits, and antibody chimeric proteins and small molecules using immunoglobulin heavy and light chain variable regions The production of antibodies and scaffold antibodies can be carried out by those skilled in the art using known techniques.
　（医薬組成物）
　本実施形態に係る医薬組成物は、本実施形態に係る抗体を有効成分として含有する。 (Pharmaceutical composition)
The pharmaceutical composition according to the present embodiment contains the antibody according to the present embodiment as an active ingredient.
　本実施形態に係る医薬組成物は、公知の方法に従って製剤化し、投与する医薬組成物とすることができる。例えば、そのまま液剤として又は適当な剤型の医薬組成物として、ヒト又は哺乳類に対して経口的又は非経口的に投与することができる。抗体のヒトに対する投与量は、特に限定されるものではないが、例えば０．０１ｍｇ／ｋｇ～５０ｍｇ／ｋｇとすることが可能である。 The pharmaceutical composition according to this embodiment can be formulated and administered according to a known method. For example, it can be orally or parenterally administered to humans or mammals as a solution as it is or as a pharmaceutical composition of a suitable dosage form. The dose of the antibody to humans is not particularly limited, and can be, for example, 0.01 mg / kg to 50 mg / kg.
　医薬組成物は、微生物の増殖を抑制する防腐剤又はｐＨを許容範囲に保つのに役立つ緩衝剤を含んでもよい。防腐剤は、アジ化ナトリウム、オクタデシルジメチルベンジルアンモニウムクロライド、塩化ヘキサメトニウム、塩化ベンザルコニウム、塩化ベンゼトニウム、フェノール、ブチル若しくはベンジルアルコール、メチル若しくはプロピルパラベン等のアルキルパラベン、カテコール、レゾルシノール、シクロヘキサノール、３-ペンタノール、及びｍ-クレゾール等である。緩衝剤は、リン酸、クエン酸、及びその他の有機酸である。 The pharmaceutical composition may also include a preservative that inhibits the growth of microorganisms or a buffer that helps maintain the pH within an acceptable range. Preservatives include sodium azide, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, alkyl paraben such as butyl or benzyl alcohol, methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol and the like. Buffering agents are phosphoric acid, citric acid and other organic acids.
　また、医薬組成物は、例えば、賦形剤、安定剤、ＥＤＴＡ等のキレート化剤、塩、又は抗菌剤を含んでもよい。他にも、アスコルビン酸及びメチオニン等の酸化防止剤、ポリペプチド、血清アルブミン、ゼラチン、若しくは非特異的免疫グロブリン等のタンパク質、ポリビニルピロリドン等の親水性ポリマー、グリシン、グルタミン、アスパラギン、ヒスチジン、アルギニン、若しくはリジン等のアミノ酸、グルコース、マンノース、若しくはデキストリン等の単糖類、二糖類、及びその他の炭水化物、スクロース、マンニトール、トレハロース、若しくはソルビトール等の糖類を含むことが可能である。 The pharmaceutical composition may also contain, for example, an excipient, a stabilizer, a chelating agent such as EDTA, a salt, or an antibacterial agent. In addition, antioxidants such as ascorbic acid and methionine, polypeptides, proteins such as serum albumin, gelatin, or nonspecific immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, glycine, glutamine, asparagine, histidine, arginine, Alternatively, it is possible to include amino acids such as lysine, monosaccharides such as glucose, mannose or dextrin, disaccharides, and other carbohydrates and saccharides such as sucrose, mannitol, trehalose or sorbitol.
　（腫瘍の検査方法及び腫瘍検査用試薬）
　本実施形態に係る腫瘍検査用試薬は、胃がん、前立腺がん、又は大腸がんの検査に使用されるものであり、EphA10を腫瘍マーカーとして検出する。腫瘍検査用試薬の判定手法は、生体検体中のEphA10を確認可能なものであれば良く、定性的であってもよいし、定量的であってもよい。例えば、単にEphA10の有無を検出するものであってもよく、またEphA10の発現量を相対的若しくは絶対的に決定するものでもよい。 (Test method of tumor and reagent for tumor test)
The reagent for tumor examination according to the present embodiment is used for examination of stomach cancer, prostate cancer or colon cancer, and detects EphA10 as a tumor marker. The determination method of the reagent for tumor examination may be any one that can confirm EphA10 in a biological sample, and may be qualitative or quantitative. For example, it may simply detect the presence or absence of EphA10, and may relatively or absolutely determine the expression level of EphA10.
　生体検体は、乳がん患者若しくは乳がん発症の可能性のある患者の血液や乳がん組織に由来するものであり、好ましくは乳がん細胞を含む検体であれば良い。生体検体は、患者から採取された検体であり、本発明の検査方法のために取得した検体であってもよいが、他の検査に供するために取得した検体や、手術により採取した検体であってもよい。例えば検体を免疫組織染色検査に供する場合、検査に供する試料として、患者から得られた検体から調製したパラフィン切片を用いることができる。また、例えば検体をWestern Blot法又はRT-PCR法に供する場合、試験に供する試料として、患者から得られた検体から調製したタンパク質抽出液、又はｍＲＮＡ抽出液を用いることができる。 The biological sample is derived from the blood or breast cancer tissue of a breast cancer patient or a patient having a possibility of developing breast cancer, and may preferably be a sample containing breast cancer cells. The biological sample is a sample collected from a patient and may be a sample acquired for the examination method of the present invention, but is a sample acquired for use in other examinations or a sample collected by surgery May be For example, when a sample is subjected to an immunohistological staining test, paraffin sections prepared from a sample obtained from a patient can be used as a sample to be subjected to the test. For example, when subjecting a sample to Western Blot or RT-PCR, a protein extract or mRNA extract prepared from a sample obtained from a patient can be used as a sample to be subjected to the test.
　免疫学的手法によりEphA10の検査を行う場合には、抗EphA10抗体が検査用試薬に含まれる。ｍＲＮＡレべルでEphA10の発現を検出する場合は、例えばRT-PCRを用いる場合は、EphA10のｍＲＮＡを増幅可能なプライマーが検査用試薬に含まれる。例えば、EphA10が、各遺伝子の発現を制御する遺伝子の突然変異等に起因して、各タンパク質の発現が影響を受けるのであれば、各遺伝子の発現を制御する遺伝子について、遺伝子レベルで野生型や突然変異型を確認するものでもよい。 When EphA10 is examined by immunological techniques, an anti-EphA10 antibody is included in the test reagent. When detecting expression of EphA10 at mRNA level, for example, when using RT-PCR, a primer capable of amplifying EphA10 mRNA is included in the test reagent. For example, if EphA10 is affected by the mutation of a gene that controls the expression of each gene, the expression of each protein is affected. The mutant form may be confirmed.
　EphA10の発現のタンパク質レベルでの検出は、免疫学的手法によるのが好適である。例えば、免疫染色法（蛍光抗体法、酵素抗体法、重金属標識抗体法、放射性同位元素標識抗体法を含む）、電気泳動法による分離と蛍光、酵素、放射性同位元素等による検出方法との組み合わせ（Western Blot法、蛍光二次元電気泳動法を含む）、酵素免疫測定吸着法（ELISA）、ドット・ブロッティング法等により行うことができる。そして、EphA10の発現が認められる場合に、胃がん細胞、前立腺がん細胞、及び大腸がん細胞であると判断する。 Detection of EphA10 expression at the protein level is preferably by immunological techniques. For example, a combination of an immunostaining method (including a fluorescent antibody method, an enzyme antibody method, a heavy metal labeling antibody method, a radioactive isotope labeling antibody method), a separation by electrophoresis and a detection method by fluorescence, an enzyme, a radioactive isotope, etc. Western blot, fluorescent two-dimensional electrophoresis, and enzyme-linked immunosorbent assay (ELISA), dot blotting, and the like can be used. Then, when expression of EphA10 is observed, the cells are judged to be gastric cancer cells, prostate cancer cells, and colon cancer cells.
　（抗EphA10EXモノクローナル抗体）
　ヒトEphA10の細胞外ドメイン（EphA10EX）とヒトIgG1Fcドメインの融合蛋白質（EphA10EX-Fcキメラ）を抗原としてマウスに免疫し、抗体価上昇がみられたマウスの脾細胞から常法によってハイブリドーマを作製した。図１に示すように、ELISAにてスクリーニングし、得られたハイブリドーマの中から、EphA10EXに対する結合性に基づき、ネガティブコントロールとしてのDR5-Fcには結合せず、EphA10EXに親和性を有する４クローンを選出した。 (Anti-EphA10 EX monoclonal antibody)
The fusion protein (EphA10 EX -Fc chimera) of the extracellular domain (EphA10 EX ) of human EphA10 and the human IgG1 Fc domain is used as an antigen to immunize mice as an antigen, and hybridomas are prepared from spleen cells of mice with elevated antibody titer by a conventional method did. As shown in FIG. 1, it was screened by ELISA, from among the obtained hybridomas based on the binding to EphA10 EX, not bind to DR5-Fc as a negative control, 4 having affinity for EphA10 EX I picked a clone.
　次に、抗体遺伝子のシーケンス解析を行った。抗体の重鎖がIgG2bであり、軽鎖がκであることを確認し、ハイブリドーマｍＲＮＡから5’-RACE法により、ＶＨとＶＬ領域のＤＮＡ／アミノ酸配列を決定した。ＶＨのＣＤＲは、DYFIT（ＣＤＲ１）、EIYPGSGSIYYNENFKG（ＣＤＲ２）及びSWVPYAMDY（ＣＤＲ３）であった。また、ＶＬのＣＤＲは、KASQSVSKDVA（ＣＤＲ１）、SASKRYT（ＣＤＲ２）及びQQDYSSPRT（ＣＤＲ３）であった。ＶＨのＦＲは、QVLLQQSGPELVKPGASVKMSCKASGYTFT（ＦＲ１）、WVSQRTGQGLEWIG（ＦＲ２）、KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR（ＦＲ３）及びWGQGTSVTVSS（ＦＲ４）であった。また、ＶＬのＦＲは、SFVMTQTPKFLLVSAGDRITITC（ＦＲ１）、WYQQKPGQSPKLLIY（ＦＲ２）、GVPDRFIGSGYGTDFTFTISTVQAEDLAVYFC（ＦＲ３）及びFGGGTKLEIKRA（ＦＲ４）であった。３つのクローン（クローン１，２，４）が可変領域の配列が同一の抗体を産生していることが確認された。 Next, sequence analysis of antibody genes was performed. It was confirmed that the heavy chain of the antibody was IgG2b and the light chain was kappa, and the DNA / amino acid sequences of the VH and VL regions were determined from the hybridoma mRNA by the 5'-RACE method. The CDRs of VH were DYFIT (CDR1), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3). Moreover, CDRs of VL were KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3). The FR of VH was QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4). In addition, FRs of VL were SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDRIGSGYGTDFTFTISTVQAEDLAVYFC (FR3) and FGGGTKLEIKRA (FR4). It was confirmed that three clones ( clone 1, 2 and 4) produce an antibody whose variable region sequence is identical.
　次に、上述の抗EphA10EXモノクローナル抗体のEphA10への結合性を確認した。EphA10－pIRES2-DsRedVectorを293T細胞へトランスフェクションしてフローサイトメトリーによって選別した。結果を図２に示す。フローサイトメトリー解析の結果、作製したモノクローナル抗体は、細胞に発現しているEphA10を認識できることが確認された。 Next, the binding of the above-mentioned anti-EphA10 EX monoclonal antibody to EphA10 was confirmed. EphA10-pIRES2-DsRedVector was transfected into 293T cells and sorted by flow cytometry. The results are shown in FIG. As a result of flow cytometry analysis, it was confirmed that the prepared monoclonal antibody can recognize EphA10 expressed in cells.
　次に、Ephrin receptor typeAファミリー（EphA1～A8,A10）のうち、リコンビナント蛋白質（EphA1、EphA2、EphA5、EphA6、EphA10）に対する特異性をELISAで評価した。図３に示すように、0.75μg/mL、0.25μg/mL、0.083μg/mLのいずれの濃度においても、作製したモノクローナル抗体はEphA10に対して特異的に認識し、他のファミリー分子は認識しなかった。 Next, among the Ephrin receptor type A family (EphA1 to A8, A10), the specificity for recombinant proteins (EphA1, EphA2, EphA5, EphA6, EphA10) was evaluated by ELISA. As shown in FIG. 3, at any concentration of 0.75 μg / mL, 0.25 μg / mL, 0.083 μg / mL, the prepared monoclonal antibody specifically recognizes EphA10, and other family molecules recognize It was not.
　次に、抗EphA10EXモノクローナル抗体の結合活性を調べた。Biacore3000（GEヘルスケアバイオサイエンス社製）を用い、表面プラズモン共鳴法（SPR法）にて結合活性測定を行った。添付のプロトコルに従い、20μg/mLのEphA10EX-FcをCM5センサーチップに固層化した。装置添付の解析ソフトを用いて解析し、結合速度定数及び解離速度定数を算出した。その結果、モノクローナル抗体の解離定数ＫＤ値は、３．９×１０－９Ｍであり、一般に抗体医薬に使用可能なレベルの結合親和性を有していることがわかった。 Next, the binding activity of the anti-EphA10 EX monoclonal antibody was examined. The binding activity was measured by surface plasmon resonance (SPR method) using Biacore 3000 (manufactured by GE Healthcare Biosciences). According to the attached protocol, 20 μg / mL of EphA10 EX -Fc was immobilized on a CM5 sensor chip. The binding rate constant and dissociation rate constant were calculated using analysis software attached to the device. As a result, it was found that the dissociation constant KD value of the monoclonal antibody is 3.9 × 10 −9 M, and in general, it has a binding affinity that can be used for antibody drugs.
　次に、抗EphA10EXモノクローナル抗体の認識ドメインの解析を行った。ヒトEphA10EXは、他のEphファミリー分子との遺伝子相同性をもとに、N末端からLigandbinding domain（以後、LBD）と２つのFibronectindomain（N末側をFD1、C末側をFD2と表記）から構成される。まず全長及び各ドメインを欠失させたEphA10（下記（ａ）～（ｂ））の遺伝子発現ベクターを作製した。
（ａ）EphA10-Full（A.A.34～1008,109kDa）；全長のEphA10
（ｂ）EphA10-△LBD（A.A.212～1008,86kDa）；LBD欠失
（ｃ）EphA10-△LBD-FD1（A.A.454～1008,61kDa）；LBD、FD1が欠失
（ｄ）EphA10-△LBD-FD1-FD2（A.A.566～1008,48kDa）；LBD、FD1、FD2欠失
　pIRES2-DsRedExpress（Takara Bio）にEphA10のシグナル配列（A.A.1～33）、続いて上記全長及び部分欠失EphA10のcDNAを挿入することでほ乳類発現ベクターを作製した。ヒト胎児由来腎細胞株293T細胞を１２wellプレートに２×１０５cells/1 mL/wellで播種し、37℃で培養した。１２時間後、上記（ａ）～（ｄ）の遺伝子発現ベクターとコントロールとしてEmptyベクター各1ｍｇずつをトランスフェクション試薬FuGENE HD（Roche）とともに細胞に作用させた。その２４時間後、蛍光たんぱく質DsRedの発現によって各発現ベクターの導入を確認したうえで細胞を可溶化し、lysateをSDS-PAGE、さらに抗EphA10EXモノクローナル抗体を一次抗体として、Western Blotを行うことで、発現した部分欠失EphA10たんぱく質を抗EphA10EXモノクローナル抗体が認識できるかどうかを解析した。 Next, analysis of the recognition domain of anti-EphA10 EX monoclonal antibody was performed. Human EphA10 EX is based on the gene homology with other Eph family molecules, and from the N-terminus, the Ligandbinding domain (hereinafter LBD) and two Fibronectin domains (the N-terminal side is denoted as FD1 and the C-terminal side as FD2) Configured First, a gene expression vector of EphA10 (the following (a) to (b)) in which the full length and each domain were deleted was prepared.
(A) EphA10-Full (AA 34 to 1008, 109 kDa); full-length EphA10
(B) EphA10-ΔLBD (AA 212 to 1008, 86 kDa); LBD deleted (c) EphA10-Δ LBD-FD1 (AA 454 to 1008, 61 kDa); LBD, FD1 deleted (d) EphA 10-Δ LBD-FD 1 LBD, FD1, FD2-deleted pIRES2-DsRedExpress (Takara Bio) insert the EphA10 signal sequence (AA1-33), followed by the full-length and partial deletion EphA10 cDNA A mammalian expression vector was constructed. Human fetal kidney cell line 293T cells were seeded at 2 × 10 5 cells / 1 mL / well in 12-well plates and cultured at 37 ° C. After 12 hours, the gene expression vectors of (a) to (d) and 1 mg of the Empty vector as a control were allowed to act on the cells together with the transfection reagent FuGENE HD (Roche). After 24 hours, the introduction of each expression vector was confirmed by the expression of the fluorescent protein DsRed, and then the cells were solubilized, and the lysate was subjected to SDS-PAGE, and further Western blot was performed using the anti-EphA10 EX monoclonal antibody as the primary antibody. Then, it was analyzed whether the expressed partially deleted EphA10 protein could be recognized by the anti-EphA10 EX monoclonal antibody.
　図４に示すWestern blotの結果、EphA10-Full（ａ）、EphA10-ΔLBD（ｂ）、EphA10-ΔLBD-FD1(ｃ)発現細胞のlysateでは、予測される分子量の位置（各々109kDa,86kDa, 61kDa）にバンドが観察された。一方、細胞外ドメインを全て欠失したEphA10-ΔLBD-FD1-FD2(ｄ)、及びEmptyベクターを発現させた細胞lysateではバンドが検出されなかった。以上の結果から、本発明の抗EphA10EXモノクローナル抗体は、ヒトEphA10細胞外部分、さらには膜貫通ドメイン近位のFibronectinIII domain（FD2）部分を認識していることが判明した。 As a result of Western blot shown in FIG. 4, in the lysate of EphA10-Full (a), EphA10-ΔLBD (b) and EphA10-ΔLBD-FD1 (c) expressing cells, predicted molecular weight positions (109 kDa, 86 kDa, 61 kDa, respectively) A band was observed in On the other hand, no band was detected in EphA10-ΔLBD-FD1-FD2 (d) in which the extracellular domain was completely deleted, and in the lysate of cells expressing the Empty vector. From the above results, it was revealed that the anti-EphA10 EX monoclonal antibody of the present invention recognizes the human EphA10 extracellular portion, and furthermore, the Fibronectin III domain (FD2) portion near the transmembrane domain.
　次に、抗EphA10EXモノクローナル抗体による乳がん細胞増殖抑制作用の評価を行った。トリプロネガティブ乳がん細胞であるMDA-MB-468（EphA10も発現）を９６wellプレートに、５×１０３cells/50mL/wellで播種し、一晩37℃で培養した。１２時間後、80mg/mLに調製した抗EphA10EXモノクローナル抗体を25mL/wellで添加（終濃度；20mg/mL）し、その１５分後、16mg/mLに調製したEphrin-A5-Fcキメラたんぱく質（R&D）を25mL/wellで添加（終濃度；4mg/mL）した。37℃で６日間培養した後、細胞数を細胞内ATP含量を指標に評価した（CellTiter-GloTM Luminescent CellViability Assay, Promega）。 Next, the breast cancer cell growth inhibitory effect was evaluated by anti-EphA10 EX monoclonal antibody. The tripro negative breast cancer cells MDA-MB-468 (also expressing EphA10) were seeded at 5 × 10 3 cells / 50 mL / well in 96 well plates and cultured overnight at 37 ° C. After 12 hours, the anti-EphA10 EX monoclonal antibody prepared at 80 mg / mL was added (final concentration; 20 mg / mL) at 25 mL / well, and after 15 minutes, the Ephrin-A5-Fc chimeric protein (prepared at 16 mg / mL) R & D was added at 25 mL / well (final concentration; 4 mg / mL). After culturing at 37 ° C. for 6 days, the number of cells was evaluated using intracellular ATP content as an index (CellTiter-GloTM Luminescent CellViability Assay, Promega).
　抗EphA10EXモノクローナル抗体の乳がん治療薬としての可能性を検証するため、EphA10が発現しているヒトトリプルネガティブ細胞株MDA-MB-468に、EphA10のリガンド候補Ephrin-A5のFcキメラと抗EphA10EXモノクローナル抗体を共作用させ、抗体による増殖抑制作用を評価した。その結果、図５に示すように、Ephrin-A5-Fcの添加によって促進された乳がん細胞の増殖が、抗EphA10EXモノクローナル抗体の添加によって完全に抑制されたことが実証された。 In order to test the potential of anti-EphA10 EX monoclonal antibody as a therapeutic agent for breast cancer, EphA10 expressing human triple negative cell line MDA-MB-468, EphA10 ligand candidate Ephrin-A5 Fc chimera and anti-EphA10 EX The monoclonal antibodies were allowed to react with each other to evaluate their antiproliferative activity. As a result, as shown in FIG. 5, it was demonstrated that the growth of breast cancer cells promoted by the addition of Ephrin-A5-Fc was completely suppressed by the addition of the anti-EphA10 EX monoclonal antibody.
　次に、ヒトEphA10を安定発現させたMDA-MB-435EphA10細胞をEphA10発現トリプルネガティブ細胞モデルとし、BALB/c-nu/nuマウスのFat Pad（乳線脂肪組織）に５×１０６cells移植することにより胆癌マウスを作製した。腫瘍体積が約100 mm３程度になった時点を初日（Day0）とし、抗EphA10EXモノクローナル抗体(200μg/mouse)あるいは、対照として生理食塩水を４回(Day0、Day3、Day7、Day10)静脈内投与した。腫瘍体積は下記の式により算出した。 Next, MDA-MB-435 EphA10 cells stably expressing human EphA10 were used as EphA10 expressing triple negative cell model, and 5 × 10 6 cells were transplanted into Fat pad (milk fat tissue) of BALB / c-nu / nu mice. By doing this, a biliary cancer mouse was produced. The first day (Day 0) is when the tumor volume reaches approximately 100 mm 3 and anti-EphA10 EX monoclonal antibody (200 μg / mouse) or saline as a control 4 times (Day 0, Day 3, Day 7, Day 10) intravenously Administered. Tumor volume was calculated by the following equation.
　腫瘍体積＝(腫瘍長径×短径２)×1/2
　図６は、MDA-MB-435EphA10細胞を使用した場合において、本モノクローナル抗体が、トリプルネガティブ症例に対する治療薬として有用であることを示す図である。縦軸は、初日の腫瘍体積に対する各日数における腫瘍体積の割合（％）を示す。図６に示されるように、抗EphA10EXモノクローナル抗体投与群では、対照群と比較して有意な腫瘍増殖抑制効果が認められた。従って、抗EphA10EXモノクローナル抗体、あるいはそのヒト型化抗体は、EphA10陽性トリプルネガティブ乳がんに対して有効な治療薬となる可能性が示された。 Tumor volume = (tumor major axis x minor axis 2 ) x 1/2
FIG. 6 shows that this monoclonal antibody is useful as a therapeutic agent for triple negative cases when using MDA-MB-435 EphA10 cells. The vertical axis shows the percentage (%) of tumor volume on each day to the tumor volume on the first day. As shown in FIG. 6, in the anti-EphA10 EX monoclonal antibody-administered group, a significant tumor growth inhibitory effect was observed as compared to the control group. Therefore, it has been shown that anti-EphA10 EX monoclonal antibody or its humanized antibody may be an effective therapeutic agent for EphA10 positive triple negative breast cancer.
　本結果から、EphA10EXに対するモノクローナル抗体は、トリプルネガティブ症例を含む乳がんに対する新規治療薬として有用であることが示された。トリプルネガティブ症例は、腫瘍が侵攻性でありそのため転移の可能性が高く、標的治療法としての、HER-2陽性の乳がんに処方されるトラスツズマブや、ホルモン受容体陽性の乳がんに処方されるタモキシフェンやアロマターゼ阻害剤が効かないため、乳がんのなかでもたちが悪いとされており、しかもトリプルネガティブ症例に対するトラスツズマブのような標的治療薬はいまだ存在しないとされていたが、本発明に係る抗EphA10EXモノクローナル抗体によれば、大半のトリプルネガティブ症例に対して標的治療が可能となり、乳がん治療に対して極めて大きな貢献をもたらすことになる。トリプルネガティブ症例に対する従来の抗がん剤による治療では効果が不十分であるため治療期間が長期化し患者の精神的・肉体的・経済的負担は著しいものであったが、本発明に係る抗EphA10EXモノクローナル抗体によれば短期間にて治癒することができ、更に完治して社会復帰することにより人材の社会損失を避けることができる。 The present results indicate that the monoclonal antibody against EphA10 EX is useful as a novel therapeutic agent for breast cancer including triple negative cases. In triple negative cases, the tumor is aggressive and there is a high possibility of metastasis, and trastuzumab prescribed for HER-2 positive breast cancer or tamoxifen prescribed for hormone receptor positive breast cancer as targeted treatment Although it has been regarded as poor in breast cancer because aromatase inhibitors do not work, and there has been no target therapeutic agent such as trastuzumab for triple negative cases, the anti-EphA10 EX monoclonal according to the present invention Antibodies allow targeted treatment of most triple-negative cases and make a significant contribution to breast cancer treatment. Although the conventional anticancer agent treatment for triple negative cases is not effective enough, the treatment period is prolonged and the patient's mental, physical and economic burden is significant. However, anti-EphA10 according to the present invention According to the EX monoclonal antibody, it can be cured in a short period of time, and complete recovery and reintegration can avoid social loss of human resources.
　（組織マイクロアレイ解析）
　次に、摘出検体組織としての乳がん組織を、ホルムアルデヒドを使用して固定した。固定された乳がん組織は十分に水洗され、その後、低濃度アルコールからしだいに濃度を上げて無水アルコールまで順次移していき、十分に脱水された。次にキシロールを用いてアルコールを抜き、固定した乳がん組織を融解している液状パラフィンの中に入れて充分浸し、その後徐々に冷却して、パラフィンブロックに包埋した。パラフィンブロックから直径０．６～２ｍｍ大の円筒形状にくりぬき、その円筒形状のくりぬいた被検組織を多数集めて、新たなパラフィンブロックに埋め込み、その後薄切し、組織切片が配置されたシート状の薄片切片試料をスライドグラス上に載せて乳がん組織の組織マイクロアレイを作成した。 (Tissue microarray analysis)
Next, breast cancer tissue as a removed sample tissue was fixed using formaldehyde. The fixed breast cancer tissues were thoroughly washed with water, and then gradually increased in concentration from low concentration alcohol and sequentially transferred to absolute alcohol and sufficiently dehydrated. The alcohol was then removed using xylol and the fixed breast cancer tissue was placed in the melted liquid paraffin and thoroughly soaked, and then gradually cooled and embedded in a paraffin block. A cylindrical block of 0.6 to 2 mm in diameter is cut out of a paraffin block, and a large number of cylindrical test structures are collected, embedded in a new paraffin block, and then sliced and a sheet of tissue sections is arranged. The sectioned slice samples were placed on a slide glass to prepare a tissue microarray of breast cancer tissue.
　そして抗EphA10EXモノクローナル抗体を使用して免疫染色を行った。免疫染色は、DakoCytomation Envision（登録商標）＋system-HRP（ダコ社製）を用いて行った。抗体の浸透性を高めるために耐熱ガラス容器に入れたｐＨ６．０のクエン酸緩衝液に漬け、オートクレーブにて121℃で２０分間熱処理し抗原を賦活化させた。室温まで放置して冷却し、流水で緩衝液を水洗後、３％過酸化水素加メチルアルコールに１０分間インキュベートして内因性ペルオキシダーゼ活性をブロッキングした。更にこの細胞を１０％BSA中で３０分間インキュベートすることにより非特異的なタンパク質の結合をブロックした。その後、一次抗体溶液として抗EphA10EXモノクローナル抗体（5μg/mL）を滴下し常温で３０分間反応させた。PBSにて洗浄後、余分な試薬を取り除き二次抗体としてポリマー試薬（Envision+，DAKO社）を滴下し反応させた。PBSで洗浄後、DAB溶液を滴下し発色を行った。流水にて洗浄後、ヘマトキシリン液にて染色させた。流水にて水洗後、アルコール溶液、次いでキシレン溶液をくぐらせ脱水し、封入剤を数秒間アプライ（VectaMount、ベクターラボラトリーズ社製）して、カバーグラスを被せて共焦点レーザ走査型顕微鏡（MRC-2000、バイオラッド社製）にて観察した。図７は、乳がん組織の組織マイクロアレイである。図７に示すように、トリプルネガティブ症例におけるEphA10陽性は１０／１５であり、陽性率は約６７％であった。 Then, immunostaining was performed using an anti-EphA10 EX monoclonal antibody. Immunostaining was performed using DakoCytomation Envision (registered trademark) + system-HRP (manufactured by Dako). In order to increase the permeability of the antibody, it was immersed in a citrate buffer solution of pH 6.0 contained in a heat-resistant glass container, and heat-treated at 121 ° C. for 20 minutes in an autoclave to activate the antigen. After cooling to room temperature, the buffer solution was washed with running water and then incubated in 3% hydrogen peroxide methyl alcohol for 10 minutes to block the endogenous peroxidase activity. Furthermore, nonspecific protein binding was blocked by incubating the cells in 10% BSA for 30 minutes. Thereafter, anti-EphA10 EX monoclonal antibody (5 μg / mL) was dropped as a primary antibody solution, and reacted at room temperature for 30 minutes. After washing with PBS, excess reagent was removed and a polymer reagent (Envision +, manufactured by DAKO) was dropped and reacted as a secondary antibody. After washing with PBS, the DAB solution was added dropwise to develop color. After washing with running water, the cells were stained with hematoxylin solution. After washing with running water, it is dehydrated by passing through an alcohol solution and then a xylene solution, and the mounting agent is applied (VectaMount, manufactured by Vector Laboratories) for a few seconds, covered with a cover glass, and a confocal laser scanning microscope (MRC-2000) , Manufactured by Bio-Rad, Inc.). FIG. 7 is a tissue microarray of breast cancer tissue. As shown in FIG. 7, EphA10 positive in the triple negative case was 10/15, and the positive rate was about 67%.
　次に、上述の手法と同様にして、生検組織検体から摘出された標本を用いて胃がん組織、前立腺がん組織、及び大腸がん組織の組織マイクロアレイを作製し、抗EphA10EXモノクローナル抗体を使用して免疫染色を行った。結果を図８に示す。図８に示すように、EphA10は、胃がん、前立腺がん、及び、大腸がんにおいても発現していることが判明した。 Next, in the same manner as described above, a tissue microarray of stomach cancer tissue, prostate cancer tissue, and colon cancer tissue is prepared using specimens removed from a biopsy tissue sample, and an anti-EphA10 EX monoclonal antibody is used. Immunostaining. The results are shown in FIG. As shown in FIG. 8, it was found that EphA10 is also expressed in gastric cancer, prostate cancer, and colon cancer.
　次に、乳がん細胞及び前立腺がん細胞において、EphA10遺伝子発現を調べた。乳がん細胞として、MDA-MB-468、MDA-MB-231、MDA-MB-435s、MCF-7、SKBR3、MDA-MB-435を使用した。前立腺がん細胞として、アンドロゲン依存性のLNCaP、アンドロゲン非依存性のPC-3、DU-145を使用した。下記プライマーを用いてRT-PCRを行った。
Forward：CCAAGTGTGCCCTGACTACCTGTC
Reverse：GTTCAGCCAAAGAGATGCCTAGGCTCAC
　アニ―リングを57℃で1分、伸長反応を68℃で1分に設定し、ポリメラーゼはAmpliTaq Gold（Applied biosystems）を用いてPCR反応を35サイクル行い、PCR産物を１．５％TAEアガロースゲルを用いて電気泳動した。 Next, EphA10 gene expression was examined in breast cancer cells and prostate cancer cells. As breast cancer cells, MDA-MB-468, MDA-MB-231, MDA-MB-435s, MCF-7, SKBR3, and MDA-MB-435 were used. Androgen-dependent LNCaP, androgen-independent PC-3 and DU-145 were used as prostate cancer cells. RT-PCR was performed using the following primers.
Forward: CCAAGTGTGCCCTGACTACCTGTC
Reverse: GTTC AGCCAAAGAGATGCCTAGGCTCAC
The annealing is set at 57 ° C. for 1 minute, the extension reaction at 68 ° C. for 1 minute, the polymerase is 35 cycles of PCR using AmpliTaq Gold (Applied biosystems), and the PCR product is 1.5% TAE agarose gel Electrophoresis was performed using
　結果を、図９に示す。図９に示すように、EphA10は乳がん細胞に発現するのみならず、前立腺がん細胞においても発現することが認められた。 The results are shown in FIG. As shown in FIG. 9, EphA10 was found to be expressed not only in breast cancer cells but also in prostate cancer cells.
　また、上述の手法と同様にして、各種組織の組織マイクロアレイを作製し、EphA10による発現の有無を試験した。表１に示すように、EphA10は、精巣を例外として主要な正常組織中には発現が認められなかった。これによりEphA10はがん組織に特異性があることが実証された。 Further, in the same manner as the above-mentioned method, tissue microarrays of various tissues were prepared, and the presence or absence of expression by EphA10 was tested. As shown in Table 1, EphA10 was not expressed in major normal tissues except testis. This demonstrated that EphA10 has specificity to cancer tissues.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　Her-2陰性症例やトリプルネガティブ症例であっても利用できるので、今まで治療が困難又は不可能な症例に適用できる。また、胃がん、前立腺がん、又は大腸がんの外科的手術の場合に、組織の切除範囲の決定の際に利用でき、また術前後の化学療法、放射線療法、免疫療法等における治療方法の選択に利用できる。 Since even Her-2 negative cases and triple negative cases can be used, it can be applied to cases where treatment has been difficult or impossible until now. In addition, in the case of surgery for gastric cancer, prostate cancer, or colon cancer, it can be used to determine the resecting range of tissue, and also the selection of treatment methods for chemotherapy, radiation therapy, immunotherapy, etc. before and after surgery. Available to
　配列番号１５～１６：プライマー Sequence number 15-16: Primer

Claims (9)

  1. 　EphA10の細胞外ドメインに特異的に結合する抗体。 An antibody that specifically binds to the extracellular domain of EphA10.
  2. 　EphA10のフィブロネクチンドメインに結合する請求項１に記載の抗体。 The antibody according to claim 1, which binds to the fibronectin domain of EphA10.
  3. 　重鎖可変ドメインが、DYFIT（ＣＤＲ１）、EIYPGSGSIYYNENFKG（ＣＤＲ２）及びSWVPYAMDY（ＣＤＲ３）により規定される相補性決定領域（ＣＤＲ）を含む請求項２に記載の抗体。 The antibody according to claim 2, wherein the heavy chain variable domain comprises complementarity determining regions (CDRs) defined by DYFIT (CDR1), EIYPGSGSIYYNENFKG (CDR2) and SWVP YAMDY (CDR3).
  4. 　軽鎖可変ドメインが、KASQSVSKDVA（ＣＤＲ１）、SASKRYT（ＣＤＲ２）及びQQDYSSPRT（ＣＤＲ３）により規定される相補性決定領域（ＣＤＲ）を含む請求項２に記載の抗体。 The antibody according to claim 2, wherein the light chain variable domain comprises complementarity determining regions (CDRs) defined by KASQSVSKDVA (CDR1), SASKRYT (CDR2) and QQDYSSPRT (CDR3).
  5. 　重鎖可変ドメインが、QVLLQQSGPELVKPGASVKMSCKASGYTFT（ＦＲ１）、WVSQRTGQGLEWIG（ＦＲ２）、KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR（ＦＲ３）及びWGQGTSVTVSS（ＦＲ４）により規定されるフレームワーク領域（ＦＲ）を含む請求項２に記載の抗体。 3. The antibody according to claim 2, wherein the heavy chain variable domain comprises a framework region (FR) defined by QVLLQQSGPELVKPGASVKMSCKASGYTFT (FR1), WVSQRTGQGLEWIG (FR2), KATLTADKSSNTAYMQLNSLTSEDSAVYFCAR (FR3) and WGQGTSVTVSS (FR4).
  6. 　軽鎖可変ドメインが、SFVMTQTPKFLLVSAGDRITITC（ＦＲ１）、WYQQKPGQSPKLLIY（ＦＲ２）、GVPDRFIGSGYGTDFTFTISTVQAEDLAVYFC（ＦＲ３）及びFGGGTKLEIKRA（ＦＲ４）により規定されるフレームワーク領域（ＦＲ）を含む請求項２に記載の抗体。 The antibody according to claim 2, wherein the light chain variable domain comprises a framework region (FR) defined by SFVMTQTPKFLLVSAGDRITITC (FR1), WYQQKPGQSPKLLIY (FR2), GVPDFIGSGYGTDFTFTISTVQAEDLAVYFC (FR3) and FGGGTKLEIKRA (FR4).
  7. 　請求項１乃至６の何れか１項に記載の抗体を有効成分として含有することを特徴とする乳がんの治療に用いられる医薬組成物。 A pharmaceutical composition used for treating breast cancer comprising the antibody according to any one of claims 1 to 6 as an active ingredient.
  8. 　生体から分離された生体検体について、EphA10を、胃がん、前立腺がん、及び大腸がんの腫瘍マーカーとして検出することを特徴とする腫瘍検査方法。 A tumor examination method characterized by detecting EphA10 as a tumor marker of stomach cancer, prostate cancer, and colon cancer about a living body sample separated from a living body.
  9. 　生体検体中のEphA10を検出するための、抗EphA10抗体又はEphA10のｍＲＮＡを増幅可能なプライマーを含む、胃がん、前立腺がん、及び大腸がんの検査方法に用いる腫瘍検査用試薬。 The reagent for tumor examinations used for the examination method of stomach cancer, a prostate cancer, and a colon cancer containing the primer which can amplify EphA10 antibody or EphA10 mRNA in order to detect EphA10 in a biological sample.
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WO2023088482A1 (en) * 2021-11-22 2023-05-25 China Medical University Antibody mono-or multi-specific to ephrin type-a receptor 10, chimeric antigen receptor t-cell expressing the same and uses thereof

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