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WO2010090040A1 - Anticorps monoclonaux qui reconnaissent les épitopes des chaînes de sucres spécifiques d'un cholangiocarcinome - Google Patents

Anticorps monoclonaux qui reconnaissent les épitopes des chaînes de sucres spécifiques d'un cholangiocarcinome Download PDF

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WO2010090040A1
WO2010090040A1 PCT/JP2010/000708 JP2010000708W WO2010090040A1 WO 2010090040 A1 WO2010090040 A1 WO 2010090040A1 JP 2010000708 W JP2010000708 W JP 2010000708W WO 2010090040 A1 WO2010090040 A1 WO 2010090040A1
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Prior art keywords
cholangiocarcinoma
antibody
monoclonal antibody
cells
cca
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Japanese (ja)
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阪口薫雄
桑原一彦
荒木令江
坂本珠美
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Kumamoto University NUC
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Kumamoto University NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • 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
    • 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
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates to a monoclonal antibody that recognizes a cholangiocarcinoma-specific sugar chain epitope. More specifically, the present invention relates to a monoclonal antibody that recognizes a cholangiocarcinoma-specific sugar chain epitope, characterized by being able to diagnose cholangiocarcinoma using patient serum and inhibiting the proliferation of cholangiocarcinoma cells. .
  • Monoclonal antibodies are widely used for early diagnosis of cancer, specific diagnosis, screening, and identification of molecular targets for cancer treatment. Recently, humanization of monoclonal antibodies has become feasible, and various humanized monoclonal antibodies have been used in the treatment of malignant tumors and autoimmune diseases (for example, CD3 (Orthoclone OKT3), glycoprotein IIb / IIIa receptor ( ReoPro), ERBB2 (Herceptin), CD20 (Rituxan), CD25 (Zenapax and Simulect), RSVgpF (Synagis), TNF- ⁇ (Remicade), and IL-6R).
  • CD3 Orthoclone OKT3
  • ReoPro glycoprotein IIb / IIIa receptor
  • ERBB2 Herceptin
  • CD20 Raritan and Simulect
  • RSVgpF Sesarcomaset alpha-1
  • TNF- ⁇ Remicade
  • IL-6R IL-6R
  • Non-Patent Document 1 Non-Patent Document 1
  • Non-patent Document 2 Monoclonal antibodies can recognize any epitope on a glycoprotein, but the immune response is enhanced in peripheral lymphoid organs by the combination of T and B cells as proposed in the hapten-carrier model (Non-Patent Document 3). ). Antigen induces proliferation of antigen-specific B cells in the germinal center region of peripheral lymphoid organs (ie, spleen, lymph nodes, and Peyer's plaques).
  • Antigen-induced B cells undergo cell division and induce activation-induced cytidine deaminase AID through interaction with T cells that are specifically activated and surround the germinal center region (Non-patent Document 4).
  • B cells undergo somatic hypermutation of the immunoglobulin V region (Ig-V) gene by the action of AID causing transmutation from cytosine (C) to uridine (U) and guanine (G) to adenine (A). SHM), followed by G to A and A to G transmutation mutations by uracil DNA glycosylase (UNG) and an error-prone DNA polymerase (Non-patent Document 5).
  • Ig-V immunoglobulin V region
  • Non-Patent Document 6 This process of inducing Ig-V region SHM is dependent on T cells as affinity maturation of the IgV region in a T cell-dependent immune response.
  • Activation of T cells requires antigen-presenting cells such as dendritic cells and macrophages. These cells have the phagocytic action of foreign antigens, process the antigen and cleave it into small peptide pieces (10-12 amino acids), which ultimately result in the ⁇ and ⁇ chains of antigen-specific T cells. It is present in the class II molecule split of the major histocompatibility complex (MHC) for the T cell antigen receptor.
  • MHC major histocompatibility complex
  • T cell and B cell collaboration occurs for an antigen having an epitope of the B cell antigen receptor, allowing the T cell antigen receptor to be recognized.
  • antigen presenting cells do not present sugar epitopes on MHC class II molecules.
  • antigens do not elicit sufficient activation of T cells and cannot activate antigen-reactive B cell clones known as T cell-independent immune responses. Therefore, it is expected that a glycoprotein having a very large sugar chain part is very difficult to produce an excellent monoclonal antibody by a usual method.
  • GANP GC-associated nucleoprotein
  • SAC3 Saccharomyces mRNA transport molecule SAC3
  • MCM3-interaction / acetylated region Non-patent Document 9
  • RNA primase region Non-patent Document 10
  • Non-patent Document 11 Due to GANP deficiency, when immunized with T cell-dependent antigen (TD-Ag), a serious deficiency occurred in the generation of SHM in the Ig-V region (Non-patent Document 11).
  • GANP TG mice overexpression of GANP in mice (GANP TG mice) induced an increase in antibody SHM and affinity maturation (12).
  • GANP TG mice are useful as a system for generating high affinity monoclonal antibodies against peptide antigens under a T cell-dependent immune response.
  • Blood 95 (7) Blood 95 (7) -2328, 2000 Fischer, T., Straser, K., Racz, A., Rodriguez-Navarro, S., Oppizzi, M., Toochner, J. & Hurt, E.
  • the mRNA export machinery requires the no-veloc Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores.
  • EMBO J. 21 (21): 5843-5852, 2002 Takei, Y., Swietlik, M., Tanoue, A., Tsujimoto, G., Kouzarides, T. & Laskey, R. MCM3AP, a novel acetyltransferaseAPthat acetylates replication protein MCM3.
  • GNP Germinal center-associated nuclear protein
  • the present invention provides a novel monoclonal antibody that recognizes a bile duct cancer-specific sugar chain epitope in mucin MUC5AC and can detect MUC5AC specifically in cholangiocarcinoma serum. It was.
  • the present invention further includes a hybridoma producing the monoclonal antibody, a method for producing the monoclonal antibody, a diagnostic agent for cholangiocarcinoma containing the monoclonal antibody, a medicament for inhibiting and / or treating cholangiocarcinoma containing the monoclonal antibody, and It was an object to be solved to provide a method for detecting cholangiocarcinoma using the above monoclonal antibody.
  • the present inventors obtained an efficient and specific monoclonal antibody that recognizes a sugar chain epitope appearing on a glycoprotein having a large sugar chain region, using a GANP transgenic mouse (GANP TG mouse).
  • GANP TG mice were immunized with cholangiocarcinoma (CCA), and a monoclonal antibody that recognizes the sugar chain epitope of MUC5AC applicable for pharmaceutical purposes was obtained.
  • CCA cholangiocarcinoma
  • Cholangiocarcinoma is known to develop after hepatic dystomosis in northeastern Thailand and after prolonged inflammation due to sclerosing cholangitis (Sripa, B., Kaewkes, S., Sithithaworn, P., Mairiang, E., Laha, T., Smout, M., Pairojkul, C., Bhudhisawasdi, V., Tesana, S., Thinkamrop, B., Bethony, JM, Loukas, A., Brindley , PJ Liver fluke induces cholangiocarcinoma.
  • MUC5AC is known to increase in the serum of CCA patients, but many anti-MUC5AC monoclonal antibodies are specific for the polypeptide portion of MUC5AC, and their affinity is higher in cancer patients with serum ELISA Not enough to find with sensitivity and specificity. Therefore, it was necessary to obtain a high affinity monoclonal antibody that detects the level of cancer-specific MUC5AC in the serum of CCA patients.
  • AS1 and AS2 Monoclonal antibodies against AS1 and AS2 inhibited CCA cell proliferation in vitro.
  • AS1 and AS2 epitopes are useful for definitive clinical diagnosis using patient sera and are applicable to antibody therapy.
  • AS1 and AS2 epitopes are also useful as molecular targets for drug design. The present invention has been completed based on these findings.
  • a monoclonal antibody that recognizes a cholangiocarcinoma-specific sugar chain epitope of mucin MUC5AC in cholangiocarcinoma cells (2) Recognizes mucin MUC5AC in the serum of patients with cholangiocarcinoma but does not recognize mucin MUC5AC in the serum of healthy subjects, gastric cancer patients, pancreatic cancer patients, colon cancer patients and lung cancer patients, antibody. (3) The monoclonal antibody according to (1) or (2), which has an activity of inhibiting the growth of cholangiocarcinoma cells.
  • Collect antibody-producing cells from a GANP gene-transfected transgenic non-human mammal administered with an extract of cholangiocarcinoma tumor tissue, and select antibody-producing cells that produce antibodies reactive with cholangiocarcinoma tumor tissue The method for producing a monoclonal antibody according to any one of (1) to (5), further comprising producing the antibody in a selected antibody-producing cell.
  • a diagnostic agent for bile duct cancer comprising the monoclonal antibody according to any one of (1) to (5).
  • a medicament for suppressing and / or treating bile duct cancer, comprising the monoclonal antibody according to any one of (1) to (5).
  • the monoclonal antibody according to any one of (1) to (5) is contacted with a sample derived from a subject, and the presence or absence of a cholangiocarcinoma-specific sugar chain epitope of mucin MUC5AC in the sample is detected.
  • a method for detecting cholangiocarcinoma (11) The method for detecting bile duct cancer according to (10), wherein the subject-derived sample is serum or plasma.
  • the monoclonal antibody of the present invention recognizes a bile duct cancer-specific sugar chain epitope in mucin MUC5AC, and can detect MUC5AC specifically in the bile duct cancer patient's serum.
  • the monoclonal antibody of the present invention is useful as a diagnostic agent for cholangiocarcinoma and a medicament for suppressing and / or treating cholangiocarcinoma.
  • FIG. 1 shows an outline of establishment of monoclonal antibodies against CCA cells.
  • FIG. 2 shows the detection of tumor associated antigens in patient samples using monoclonal antibodies.
  • FIG. 3 shows an overview of a typical screening for antibodies.
  • FIG. 4 shows an overview of a sandwich ELISA for identifying cancer-specific epitopes on glycoproteins.
  • FIG. 5 shows the results of the sandwich ELISA.
  • FIG. 6 shows the results of a sandwich ELISA comparing CCA with other cancers.
  • FIG. 7 shows the results of examining the reactivity with individual serum samples by sandwich ELISA.
  • FIG. 8 shows the results of examining the specificity of monoclonal antibodies by immunohistochemistry using CCA tumor clinical samples.
  • FIG. 9 shows the results of examining the specificity of monoclonal antibodies by immunohistochemistry using CCA tumor clinical samples.
  • FIG. 10 shows the results of examining the molecular size of AS1 and AS2 antigens in pooled serum of CCA patients compared to normal serum.
  • FIG. 11 shows the results of identifying AS1 and AS2 using Alb / IgG-depleted serum.
  • FIG. 12 shows the results of identifying AS1 and AS2 using immunoprecipitation.
  • FIG. 13 shows the results of identifying AS1 and AS2 using the immunochromatography method.
  • FIG. 14 shows the results of AS1-CCA mass spectrometry.
  • FIG. 15 shows the results of AS1-CCA mass spectrometry.
  • FIG. 16 shows the results of AS2-CCA mass spectrometry.
  • FIG. 17 shows the results of AS2-CCA mass spectrometry.
  • FIG. 18 shows the results of AS2-CCA mass spectrometry.
  • FIG. 19 shows the results of identifying AS1 and AS2 using the immunochromatography method.
  • FIG. 20 shows an overview of immunoprecipitation of AS1, AS2, and MUC5AC.
  • FIG. 21 shows the immunoprecipitation of AS1, AS2, and MUC5AC detected with AS1.
  • FIG. 22 shows AS1 and AS2 characterization (epitope).
  • FIG. 23 shows enzymatic deglycosylation of patient serum treated with AS1.
  • FIG. 24 shows enzymatically deglycosylated M213A SFM treated with AS1.
  • FIG. 25 shows the prediction of AS1 antigen location.
  • FIG. 26 shows the prediction of AS1 antigen location.
  • FIG. 27 shows the structure of the core O-linked glycan.
  • FIG. 28 shows the in vitro effects of AS1 and AS2 on the growth of the M055 CCA cell line.
  • FIG. 29 shows the in vitro effects of AS1 and AS2 on the growth of M055 CCA cell line.
  • a ganp gene transgenic mouse (GANP TG mice: PCT / JP2003 / 014221 (WO 2004/040971)) that produces a high-affinity monoclonal antibody against a T cell-dependent antigen (TD-Ag) is used. They succeeded in establishing a monoclonal antibody that recognizes a cancer-specific epitope on the sugar chain component of glycoprotein in cholangiocarcinoma.
  • Surgically excised extracts of CCA samples were immunized into GANP TG mice, spleen cells were fused with X63 myeloma cells using polyethylene glycol and combined with thymic feeder cells in the presence of IL-6 in 96-well culture plates Incubated in medium. After selection of IMDM medium with HAT (hypoxanthine / aminopterin / thymidine), the supernatants of the growing clones were screened by ELISA using CCA extracts. Positive clones were further screened by immunostaining on cell lines. After cloning again, monoclonal antibodies designated AS1 and AS2 were selected as specific monoclonal antibodies against CCA cells.
  • the specificity of the monoclonal antibody was further investigated by immunohistochemical staining on clinical CCA tumor samples.
  • the monoclonal antibody AS1 specifically and strongly stained the whole tumor cell, and the monoclonal antibody AS2 selectively stained the tip region of the tumor cell. Neither antibody stained surrounding non-tumor cells or the tissues of the CCA patients tested.
  • monoclonal antibodies AS1 and AS2 recognized antigens in the pooled serum of CCA patients, but in pooled sera of healthy subjects, stomach cancer, pancreatic cancer, colon cancer and lung cancer. The antigen was not recognized.
  • the AS1 antigen is positive in about 54% of CCA cases, and the AS2 antigen is positive in about 27% of CCA cases, but the positive rate is very low in other malignant tumors and healthy cases.
  • Western blot analysis using sera from CCA patients showed that both monoclonal antibodies AS1 and AS2 recognize approximately 220-kDa broadband.
  • Proteomic analysis determined the target molecule containing the peptide backbone of MUC5AC. Further analysis by treatment with multiple glycosylation enzymes revealed that monoclonal antibodies AS1 and AS2 recognize different portions of the MUC5AC sugar chain secreted from CCA cells.
  • both monoclonal antibodies were found to inhibit cell growth of CCA cell lines expressing AS1 and AS2 epitopes on MUC5AC.
  • These findings indicate that tumor-specific modifications occur in the sugar chain component of MUC5AC in CCA cells, and the two monoclonal antibodies AS1 and AS2 of the present invention recognize these epitopes.
  • the tumor-specific monoclonal antibody of the present invention is useful for elucidation of a tumor-specific antigen epitope and application of cancer therapy as an antibody drug.
  • Monoclonal antibody of the present invention and production method thereof As an antigen for producing the monoclonal antibody of the present invention, a tumor tissue of bile duct cancer or an extract thereof is used.
  • a tumor tissue of cholangiocarcinoma a tissue surgically excised from a patient with cholangiocarcinoma can be used.
  • an extract for example, a supernatant obtained by homogenizing a collected tumor tissue in a buffer solution such as PBS Can be used.
  • the monoclonal antibody of the present invention means the whole antibody molecule or a fragment thereof (for example, Fab or F (ab ′) 2 fragment) capable of recognizing and binding to a cholangiocarcinoma-specific sugar chain epitope of mucin MUC5AC in cholangiocarcinoma cells. .
  • the monoclonal antibody of the present invention can be produced by various methods. Methods for producing such antibodies are well known in the art [see, eg, Sambrook, J et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)].
  • the monoclonal antibody of the present invention may be a human antibody or a human antibody.
  • the human antibody is, for example, a mouse-human chimeric antibody
  • the antibody gene is isolated from the mouse cell producing the monoclonal antibody of the present invention, and its H chain constant region is recombined with the human IgE H chain constant region gene. It can be prepared by introducing into myeloma cells.
  • human antibodies can be prepared by immunizing mice in which the immune system is replaced with humans with cholangiocarcinoma tumor tissue or an extract thereof as an antigen.
  • a tumor tissue of cholangiocarcinoma or an extract thereof is administered as an antigen to a mammal such as a rat, mouse, rabbit or the like.
  • the antigen can be administered to a transgenic non-human mammal into which a GANP gene has been introduced (for example, a transgenic mouse into which a GANP gene has been introduced).
  • TD-Ag T cell-dependent antigen
  • the ganp gene transgenic mouse is known and can be prepared by the method described in PCT / JP2003 / 014221 (WO 2004/040971), for example.
  • the dose of antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 ⁇ g when an adjuvant is used.
  • adjuvants include Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), and aluminum hydroxide adjuvant.
  • CFA complete adjuvant
  • IFA incomplete adjuvant
  • Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally.
  • the immunization interval is not particularly limited, and immunization is carried out 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks.
  • antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days after the last immunization day.
  • Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.
  • cell fusion between antibody-producing cells and myeloma cells is performed.
  • myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice can be used.
  • the cell line to be used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred.
  • myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.
  • the myeloma cell and the antibody-producing cell are fused.
  • Cell fusion is performed in animal cell culture media such as serum-free DMEM, RPMI-1640 medium, etc., and 1 ⁇ 10 6 to 1 ⁇ 10 7 cells / ml of antibody-producing cells and 2 ⁇ 10 5 to 2 ⁇ 10 6 cells / ml of myeloma cells.
  • a cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is carried out in the presence of a cell fusion promoter.
  • the cell fusion promoter polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
  • the target hybridoma is selected from the cells after cell fusion treatment.
  • a method after appropriately diluting the cell suspension with, for example, RPMI-1640 medium containing fetal bovine serum, sprinkle 3 ⁇ 10 5 cells / well on a microtiter plate, add selective medium to each well, and then select appropriately. Cultivate by changing the medium. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.
  • Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay (such as ELISA), radioimmunoassay, immunohistochemical analysis, or the like. Cloning of the fused cells is performed by limiting dilution or the like, and finally, a hybridoma that is a monoclonal antibody-producing cell can be established.
  • enzyme immunoassay such as ELISA
  • radioimmunoassay radioimmunoassay
  • immunohistochemical analysis or the like.
  • Hybridomas AS1 and AS2 have been obtained as hybridomas producing the monoclonal antibodies AS1 and AS2 of the present invention.
  • Hybridoma AS1 (indication for identification: Mouse-mouse hybridomas AS1) and hybridoma AS2 (indication for identification: Mouse-mouse hybridomas AS2) have receipt number FERM AP-21750 (accession number FERM P-21750) and receipt, respectively.
  • No. FERM AP-21751 Accession number FERM P-21751 As of December 17, 2008, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Higashi 1-chome, Tsukuba City, Ibaraki, Japan) It is deposited at the address No. 1 Chuo No. 6 (zip code 305-8666).
  • hybridomas AS1 and AS2 with the accession number FERM P-21750 and the accession number FERM P-21717 are dated December 14, 2009, respectively, under the Budapest Treaty as the accession number FERM BP-11204 or the accession number FERM BP-11205. It has been transferred to an international deposit.
  • the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium (for example, 37 ° C., 5% CO 2 concentration).
  • an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium (for example, 37 ° C., 5% CO 2 concentration).
  • hybridomas In the case of the ascites formation method, about 1 ⁇ 10 7 hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and a homologous animal, and the hybridomas are proliferated in large quantities. Ascites are collected after 1-2 weeks.
  • known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.
  • a diagnostic agent for bile duct cancer comprising the monoclonal antibody of the present invention, and a pharmaceutical agent for suppressing and / or treating bile duct cancer
  • the monoclonal antibody of the present invention suppresses and / or suppresses a diagnostic agent for bile duct cancer and bile duct cancer. Or it can be used as a medicament for treatment.
  • the diagnostic agent and medicament of the present invention include a pharmaceutically acceptable carrier and an excipient as necessary.
  • An agent, a diluent and the like can be appropriately contained.
  • As a medicament for suppressing and / or treating cholangiocarcinoma it can be formulated, for example, as an injection.
  • the dose of the drug for suppressing and / or treating cholangiocarcinoma depends on the symptom of the patient, age and body weight, administration method, etc., and the weight of the antibody as the active ingredient is usually about 10 ng to about The range is 100 mg / kg body weight.
  • the monoclonal antibody of the present invention that recognizes a cholangiocarcinoma-specific sugar chain epitope of mucin MUC5AC in a cholangiocarcinoma cell, a sample derived from a subject, Can be detected by detecting the presence or absence of a cholangiocarcinoma-specific sugar chain epitope of mucin MUC5AC in the sample.
  • the subject-derived sample used in the present invention includes tissue, serum, plasma, saliva, urine and the like obtained from the subject, with serum or plasma being particularly preferred.
  • the contact between the subject-derived sample and the antibody is not particularly limited, and may be carried out based on a method commonly used in this field.
  • For detection for example, after contacting the sample with the above antibody, specific binding of the mucin MUC5AC to an antibody against a cholangiocarcinoma-specific sugar chain epitope that may be present in the sample is labeled with a fluorescent substance, a luminescent substance, an enzyme, etc.
  • the detection can be performed quantitatively using a secondary antibody or the like.
  • the reaction for detection / diagnosis may be performed in a liquid phase such as a well, or may be performed on a solid support on which an antibody is immobilized.
  • the measured value is positive as cholangiocarcinoma by comparing with a standard value prepared in advance using a normal sample not affected by cholangiocarcinoma or a sample known to be cholangiocarcinoma. It can be determined whether or not there is.
  • the cut-off value can be set by measuring the amount of the cholangiocarcinoma-specific sugar chain epitope of mucin MUC5AC in the serum or plasma of many patients with bile duct cancer and healthy individuals.
  • a method for detecting the presence or absence of mucin MUC5AC cholangiocarcinoma-specific sugar chain epitope in a sample using the monoclonal antibody of the present invention include enzyme immunoassay (EIA) such as sandwich ELISA, or radioactive These techniques are well known to those skilled in the art, such as immunoassay (RIA).
  • EIA enzyme immunoassay
  • RIA immunoassay
  • an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, or the like may be appropriately selected according to the measurement method.
  • the enzyme include peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase and the like.
  • the detection sensitivity of the labeling substance can be improved.
  • the radioactive isotope include 125I
  • examples of the fluorescent compound include fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (RITC).
  • examples of chemiluminescent compounds include lophine, luminol, and lucigenin. Labeling of the antibody with the labeling substance can be performed according to a conventional method. Hereinafter, a labeled immunoassay method using a labeled antibody and an ELISA method will be described.
  • a measurement system for detecting the antibody of the present invention by a labeled immunoassay can be constructed using a non-competitive reaction system or a competitive reaction system.
  • a solid phase is required (solid phase method).
  • a competitive reaction system it is not always necessary to use a solid phase (liquid phase method), but it is preferable to use a solid phase because the measurement operation is simple.
  • the solid phase material include polystyrene, nylon, glass, silicon rubber, and cellulose.
  • Examples of the solid phase shape include a spherical shape, a well shape, a tube shape, and a sheet shape, but are not limited thereto.
  • a publicly known one used for labeled immunoassay can be arbitrarily used.
  • the antigen to be detected (a sample that may contain a mucin MUC5AC cholangiocarcinoma-specific sugar chain epitope) is immobilized and then reacted with the antibody of the present invention. Then, a prelabeled anti-immunoglobulin antibody (secondary antibody) is added to react with the antibody reacting with the immobilized antigen. By this secondary antibody labeling, the amount of antibody bound to the antigen can be detected. Since the detected amount of label is positively correlated with the amount of antigen to be detected, the amount of antigen can be determined from this.
  • an anti-immunoglobulin antibody secondary antibody
  • an anti-IgG antibody an anti-IgM antibody, etc.
  • antibody molecules may be used as they are, or Fab, Fab ′, F (ab ′) 2, which are antibody fragments containing an antigen binding site obtained by enzymatic treatment of an antibody, may be used.
  • a substance having a specific affinity for the antibody molecule for example, protein A having a specific affinity for IgG may be used.
  • the labeled immunoassay include an immunoassay using an enzyme as a label and an ELISA method.
  • a specimen or a diluted solution thereof is placed in a 96-well plate, and left at 4 ° C. to room temperature overnight or at 37 ° C. for about 1 to 3 hours to adsorb the sample to be detected and adsorbed to the solid phase.
  • the antibody of the present invention is reacted, and then an anti-immunoglobulin antibody (secondary antibody) conjugated with an enzyme in advance is reacted.
  • an appropriate chromogenic substrate that reacts with the enzyme for example, p-nitrophenyl phosphate if the enzyme is phosphatase
  • the antibody can be detected by this color development.
  • Bile Duct Cancer Samples Surgically excised samples were obtained from 100 patients with intrahepatic CCA approved at Srinagarind Hospital, Khon Kaen University, Thailand. Informed consent was obtained from all patients who participated in a project approved by the Human Research Ethics Committee at Khon Kaen University. Data including age, gender, histological grade, and involvement of lymph nodes, blood vessels and peri-neurons retrospectively based on medical records and pathology records from Khon Kaen University's Center for Liver Dystoma and Cholangiocarcinoma It was.
  • Immortalized human bile duct cells MMNK-1 (Maruyama, M., Kobayashi, N., Westerman, KA, Sakaguchi, M., Allain, JE, Totsugawa, T., Okitsu, T., Fukazawa, T., Weber, A., Stolz, DB, Leboulch, P. & Tanaka, N.
  • the breast cancer cell line MCF7 was maintained with RPMI-1640.
  • a novel nuclear phosphoprotein, GANP is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication.
  • Mice with higher antibody titer were further immunized, and after 3 days, spleen cells were collected and fused with mouse myeloma cell line X63 by the standard polyethylene glycol method (Kuwahara, K., Tomiyasu, S., Fujimura). , S., Nomura, K., Xing, Y., Nishiyama, N., Ogawa, M., Imajoh-Ohmi, S., Izuta, S.
  • GNP Germinal center-associated nuclear protein
  • HRP horseradish peroxidase
  • TMB horseradish peroxidase
  • the gel was transferred to a transfer buffer [40 mM Tris-HCl (pH 7.4), 20 mM sodium acetate, 2 mM EDTA, 20% (v / v) methanol and 0.05% SDS] After 15 minutes pre-equilibration in the protein, the proteins in the gel were electrotransferred to a nitrocellulose membrane. Membranes were blocked overnight at 4 ° C. with 1% (w / v) non-fat dry milk (PBS-T). The blot was incubated with primary antibody (established monoclonal antibody) for 1 hour at room temperature. HRP-conjugated goat anti-mouse IgM was used as a secondary antibody. Protein expression was detected with the Enhanced ChemiLuminescence kit (GE Healthcare).
  • GE Healthcare Enhanced ChemiLuminescence kit
  • Degraded peptides were dried, resolved with 2% acetonitrile / 0.1% trifluoroacetic acid and desalted with ZipTip (Millipore, Billerica, Mass.). Samples were repeatedly dried, resolved with 2% acetonitrile / 0.1% trifluoroacetic acid and subjected to liquid chromatography tandem mass spectrometry (LC / MS / MS). Known contamination peaks such as keratin and self-photolysis peaks were removed. LC / MS / MS results were compared with the NCBI database.
  • Germinal center-associated nuclear protein has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.Proc. Natl. Acad. Sci. USA 98 (18): 10279-10283, 2001) and fused with myeloma cell line X63 using polyethylene glycol. After selection in HAT medium, monoclonal antibodies against CCA cells were first selected by indirect ELISA using 96 well microculture plates pretreated with CCA extract (FIG. 1). Three independent fusions with myeloma cell lines further screened by ELISA using patient sera and immunohistochemical analysis on tissue sections, and the strategy shown in FIG. I found it.
  • FERM AP-21715 (Accession No. FERM P-21715) as of December 17, 2008 (National Institute of Advanced Industrial Science and Technology, Japan) It is deposited in 1st, 1-chome Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, Japan (zip code 305-8565).
  • a sandwich ELISA as shown in FIG. 4 was used. Microtiter plates were coated with soy agglutinin and pooled serum from CCA patients was applied. Monoclonal antibodies were screened using HRP-conjugated goat anti-mouse Ig antibody secondary antibody reagents to detect the presence of antigen in patient sera. The reactivity was measured by optical density at 450 nm (optical density: OD). Specificity was determined by subtracting the background OD. The reactivity with pooled serum is shown in FIG. Monoclonal antibody AS1 specifically reacted with pooled serum from CCA patients, but not with normal serum.
  • Monoclonal antibody AS2 had higher background reactivity against normal serum and blank samples, but was more reactive against CCA serum.
  • Monoclonal antibodies AS1 and AS2 were tested for reactivity using normal, CCA, liver cancer, pancreas, stomach, colon and benign tumors (FIG. 6).
  • Monoclonal antibody AS1 was very specific for CCA serum.
  • the monoclonal antibody AS2 is selective for CCA serum while showing higher background with other serum samples. A part of the result of examining the reactivity with each serum sample is shown in FIG. In terms of overall reactivity with 100 CCA serum samples, AS1 was positive in 54% of CCA serum and AS2 was positive in 27%.
  • affinity chromatography columns were prepared using AS1 and AS2 binding Sepharose beads. After the AS1 and AS2 antigens in CCA patient serum were purified in two steps, the antigens were subjected to mass spectrometry (FIG. 13). AS1 was hit with the peptide sequence of MUC5AC glycoprotein (FIGS. 14 and 15). AS2 hit with a high score against the peptide sequence of MUC5AC and multiple peptide sequences (FIGS. 16, 17, and 18). These results indicate that AS1 and AS2 recognize different epitopes of the large molecular size glycoprotein of 220-kDa. This glycoprotein was determined to be MUC5AC and MUC6 by mass spectrometry (FIG. 19).
  • the culture supernatant of M213A had low reactivity with the monoclonal antibody AS1. This was confirmed by immunoprecipitation and Western blot analysis of M213A cells. Monoclonal antibody AS1 was specific for M213 CCA tumor cells. Monoclonal antibody AS2 showed a Western blot profile similar to that when reacted with monoclonal antibody AS1, indicating that monoclonal antibody AS1 recognizes a different epitope of AS2 antigen, ie MUC5AC. A similar experiment was attempted using a commercially available anti-MUC5AC antibody.
  • the two monoclonal antibodies of the present invention were prepared by immunoprecipitation, Western blot analysis and ELISA. Showed strong reactivity to the MUC5AC antigen.
  • the antigenic epitope of AS1 is a sugar structure coated with sialic acid, and a part of this sugar structure can be cleaved with ⁇ -galactosidase, N-acetylglucosamidase, and O-glycanase (FIG. 25).
  • the antigenic epitope of AS1 is the sugar region, some of which is linked to Gal ⁇ (1-4) bonds.
  • GlcNAc ⁇ (1-6) has the former linked to the protein center of high molecular weight glycoprotein (MUC5AC) by O-bonding (see FIG. 27).
  • MUC5AC protein center of high molecular weight glycoprotein
  • the antigenic epitope of AS2 is presumed to be in the sugar component, most of which is linked to Gal ⁇ (1-4) bonds, which are linked to the polypeptide backbone of the high molecular weight glycoprotein (MUC5AC) by O-linkages. They are connected (FIGS. 26 and 27).
  • Epitopes specific or selective for cancer in glycoprotein sugar chains are useful as targets for drug delivery and antibody therapy. As shown in FIGS. 28 and 29, monoclonal antibodies AS1 and AS2 inhibited the growth of CCA cell lines in vitro. This result indicates that this sugar chain epitope can be a target for medical treatment.
  • the method for producing a monoclonal antibody of the present invention is an efficient method for producing a specific monoclonal antibody against a sugar chain epitope, which has been difficult to produce by conventional techniques. Screening focused on the establishment of monoclonal antibodies against glycoproteins revealed that most of the specific epitopes were in the sugar chain region of MUC5AC. GANP TG mice were able to produce antibodies against cancer-specific carbohydrate epitopes with high efficiency.
  • Monoclonal antibodies AS1 and AS2 are useful for studying cancer-specific epitopes and are excellent probes for identifying cancer-specific epitopes in various glycoproteins.
  • the information of the monoclonal antibody of the present invention can also be applied to design a tertiary structure molecular probe based on the crystal information of the antigen binding site created by the heavy and light chain Ig-V regions.

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Abstract

La présente invention concerne de nouveaux anticorps monoclonaux qui reconnaissent des épitopes de chaînes de sucres spécifiques de cholangiocarcinomes dans la mucine MUC5AC et qui peuvent permettre la détection spécifique d'un cholangiocarcinome de MUC5AC dans le sérum de patients atteints d'un cholangiocarcinome. Les anticorps monoclonaux décrits reconnaissent les épitopes des chaînes de sucres spécifiques d'un cholangiocarcinome de la mucine MUC5AC dans les cellules de cholangiocarcinome.
PCT/JP2010/000708 2009-02-06 2010-02-05 Anticorps monoclonaux qui reconnaissent les épitopes des chaînes de sucres spécifiques d'un cholangiocarcinome Ceased WO2010090040A1 (fr)

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WO2005023301A1 (fr) * 2003-09-04 2005-03-17 Chugai Seiyaku Kabushiki Kaisha Remede et medicament de detection pour le cancer des voies biliaires
WO2008023947A1 (fr) * 2006-08-23 2008-02-28 Korea Research Institute Of Bioscience And Biotechnology Composition pharmaceutique pour le traitement de cholangiocarcinome, procédé d'inhibition de croissance ou d'invasion de cholangiocarcinome, et procédé de traitement de cholangiocarcinome

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JP2016199554A (ja) * 2016-06-01 2016-12-01 株式会社トランスジェニック 変異型α−アクチニン−４に対する抗体

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