[go: up one dir, main page]

WO2012124334A1 - Anticorps, composition pharmaceutique utilisés pour le traitement du cancer du sein, procédé de test de tumeur, et réactif pour le test de tumeur - Google Patents

Anticorps, composition pharmaceutique utilisés pour le traitement du cancer du sein, procédé de test de tumeur, et réactif pour le test de tumeur Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
epha10
antibody
cancer
tumor
breast cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2012/001802
Other languages
English (en)
Japanese (ja)
Inventor
角田　慎一
一也 長野
堤　康央
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institutes of Biomedical Innovation Health and Nutrition
Original Assignee
National Institutes of Biomedical Innovation Health and Nutrition
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institutes of Biomedical Innovation Health and Nutrition filed Critical National Institutes of Biomedical Innovation Health and Nutrition
Priority to JP2013504572A priority Critical patent/JP5956424B2/ja
Publication of WO2012124334A1 publication Critical patent/WO2012124334A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/5753—
    • G01N33/57535—
    • G01N33/57555—
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Oncology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hospice & Palliative Care (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un anticorps et une composition pharmaceutique, les deux étant efficaces pour le traitement du cancer du sein, en particulier un cas triple négatif de cancer du sein. L'invention concerne également un anticorps apte à se lier spécifiquement à un domaine extracellulaire de EphA10 pour agir. Un exemple du domaine extracellulaire est un domaine fibronectine. Le rapport positif EphA10 est d'environ 67% dans un cas triple négatif. Dans un échantillon biologique prélevé d'un corps vivant, EphA10 peut être détecté en tant que marqueur de tumeur pour le cancer de l'estomac, le cancer de la prostate ou le cancer colorectal.
PCT/JP2012/001802 2011-03-15 2012-03-14 Anticorps, composition pharmaceutique utilisés pour le traitement du cancer du sein, procédé de test de tumeur, et réactif pour le test de tumeur Ceased WO2012124334A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2013504572A JP5956424B2 (ja) 2011-03-15 2012-03-14 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

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011057029 2011-03-15
JP2011-057029 2011-03-15

Publications (1)

Publication Number Publication Date
WO2012124334A1 true WO2012124334A1 (fr) 2012-09-20

Family

ID=46830418

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/001802 Ceased WO2012124334A1 (fr) 2011-03-15 2012-03-14 Anticorps, composition pharmaceutique utilisés pour le traitement du cancer du sein, procédé de test de tumeur, et réactif pour le test de tumeur

Country Status (2)

Country Link
JP (1) JP5956424B2 (fr)
WO (1) WO2012124334A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014162739A (ja) * 2013-02-22 2014-09-08 National Institute Of Biomedical Innovation 二重特異性抗体及び医薬組成物
WO2023088482A1 (fr) * 2021-11-22 2023-05-25 China Medical University Anticorps mono- ou multi-spécifique du récepteur 10 à éphrine a, cellule t de récepteur antigénique chimérique l'exprimant et utilisations associées

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009087462A2 (fr) * 2007-12-24 2009-07-16 Oxford Biotherapeutics Ltd. Récepteur à éphrine de type a
JP2010216826A (ja) * 2009-03-13 2010-09-30 Japan Health Science Foundation 新規腫瘍マーカーを用いた乳癌の検査方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009087462A2 (fr) * 2007-12-24 2009-07-16 Oxford Biotherapeutics Ltd. Récepteur à éphrine de type a
JP2010216826A (ja) * 2009-03-13 2010-09-30 Japan Health Science Foundation 新規腫瘍マーカーを用いた乳癌の検査方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AASHEIM HANS-CHRISTIAN ET AL.: "Characterization of a novel Eph receptor tyrosine kinase, EphAlO, expressed in testis", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1723, 2005, pages 1 - 7 *
FOX BRIAN P. ET AL.: "Potential clinical relevance of Eph receptors and ephrin ligands expressed in prostate carcinoma cell lines", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 342, 2006, pages 1263 - 1272 *
SOICHIRO KANEZAKI ET AL.: "Triple negative Nyugan ni Okeru Ephrin receptor A10 no Soyaku Target to shite no Kanosei", ABSTRACTS OF 131ST ANNUAL MEETING OF PHARMACEUTICAL SOCIETY OF JAPAN 4, 5 March 2011 (2011-03-05), pages 294, 29P-0827 *
TAKANOBU WATANABE ET AL.: "Shinki Nyugan Bunshi Hyoteki Chiryoho no Kaihatsu o Mokuteki to shita Ephrin receptor A10 ni Taisuru scFv Kotai no Sosei", DRUG DELIVERY SYSTEM, vol. 25, no. 3, 2010, pages 343, P72 *
YUKA MAEDA ET AL.: "Nyugan ni Taisuru Shinki Soyaku Target Koho Ephrin receptor A10 no Kino Kaiseki", ABSTRACTS OF 131ST ANNUAL MEETING OF PHARMACEUTICAL SOCIETY OF JAPAN 4, 5 March 2011 (2011-03-05), pages 294, 29P-0826 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014162739A (ja) * 2013-02-22 2014-09-08 National Institute Of Biomedical Innovation 二重特異性抗体及び医薬組成物
WO2023088482A1 (fr) * 2021-11-22 2023-05-25 China Medical University Anticorps mono- ou multi-spécifique du récepteur 10 à éphrine a, cellule t de récepteur antigénique chimérique l'exprimant et utilisations associées

Also Published As

Publication number Publication date
JPWO2012124334A1 (ja) 2014-07-17
JP5956424B2 (ja) 2016-07-27

Similar Documents

Publication Publication Date Title
JP7479435B2 (ja) 癌治療および判断ターゲットとしてのｌｙ７５
JP2023040115A (ja) 葉酸受容体１の検出用の抗体及びアッセイ
US7521543B2 (en) A34 and A33-like 3 DNA, proteins, antibodies thereto and methods of treatment using same
JP6359461B2 (ja) Ｇｐｃ３標的治療剤療法が有効である患者に投与されるｇｐｃ３標的治療剤
US20120282177A1 (en) ROR1 as Therapeutic and Diagnostic Target
JP2017514143A (ja) メラノーマに使用するための抗ｄｌｌ３抗体および薬物コンジュゲート
US20230391886A1 (en) Compositions and methods for muc18 targeting
JP2006517109A (ja) アンフィレグリン抗体ならびに癌および乾癬を処置するためのその使用
CN104884617A (zh) 抗folr1抗体
US20030096285A1 (en) Identifying anti-tumor targets or agents by lipid raft immunization and proteomics
JP2006517109A5 (fr)
KR102766022B1 (ko) Cd37 의 검출을 위한 항체 및 검정
WO2015170480A1 (fr) Agent thérapeutique ciblant le gpc3 pour administration à des patients pour qui la thérapie par agent thérapeutique ciblant le gpc3 est efficace
WO2017062496A2 (fr) Anticorps anti-sas1b, procédés d'utilisation associés, et compositions et procédés de détection et de traitement du cancer
WO2019086480A1 (fr) Anticorps anti-cd5l et utilisations de celui-ci
US20130136689A1 (en) Proteins
TWI827421B (zh) 抗ｔｍｅｍ-１８０抗體及其用途
JP5956424B2 (ja) 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
TWI782000B (zh) 抗ｇｐｒ２０抗體、其製造方法及其應用
JP2025508984A (ja) 治療抗体
JP2022072531A (ja) 女性ホルモン依存性がんの悪性度及び予後の判定のためのバイオマーカー
US20150210770A1 (en) Therapeutic and diagnostic target
CA3215306A1 (fr) Anticorps specifiques d'il-38
CA3142238A1 (fr) Anticorps anti-adam8 et utilisations associees
JP2008540624A (ja) 癌の処置／診断のためのｐｓｋ−１及びそのモジュレーター

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12757605

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2013504572

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12757605

Country of ref document: EP

Kind code of ref document: A1