WO2011052753A1 - Antibody binding to mansc1 protein and having anticancer activity - Google Patents

Antibody binding to mansc1 protein and having anticancer activity Download PDF

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WO2011052753A1
WO2011052753A1 PCT/JP2010/069377 JP2010069377W WO2011052753A1 WO 2011052753 A1 WO2011052753 A1 WO 2011052753A1 JP 2010069377 W JP2010069377 W JP 2010069377W WO 2011052753 A1 WO2011052753 A1 WO 2011052753A1
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amino acid
acid sequence
seq
variable region
antibody
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PCT/JP2010/069377
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French (fr)
Japanese (ja)
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益紀 梶川
雅仁 杉浦
和之 新
絵美 清水
千恵美 松見
由紀恵 斎藤
二三子 鳥羽
あや子 中村
建行 村井
文子 大脇
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株式会社ACTGen
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    • 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
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an antibody having anticancer activity and use thereof.
  • Cancer is a disease that occupies the top cause of death in Japan. According to statistics from the National Cancer Center's Cancer Information Center, there were approximately 329 thousand people who died from cancer in 2006. By region, the lungs (23%), stomach (17%), Liver (11%), colon (7%, 11% when combined with large intestine), pancreas (6%), followed by stomach (13%), lung (13%), colon (10%) When combined with the large intestine, 14%), breast (9%), liver (8%). The number of cancer patients is increasing year by year, and the development of highly effective and safe drugs and treatment methods is strongly desired.
  • Gastric cancer is one of the cancers with very high morbidity and mortality in Japan, but it is now relatively easy to cure due to advances in diagnostic methods and treatment methods such as surgical excision and chemotherapy. It is also one of them.
  • Skills gastric cancer it is regarded as one of very high-grade gastric cancer that is difficult to treat.
  • Skills gastric cancer has cancer cells that do not appear on the mucosal surface and diffusely infiltrate the entire stomach wall or half to more than 1/3, resulting in thickening and hardening of the stomach wall without forming a macroscopically obvious tumor. It has the feature that the boundary with the surrounding mucosa is unclear.
  • Skills gastric cancer has a lower age of onset, progresses faster, and is difficult to diagnose than normal gastric cancer. At the time of diagnosis, 60% of the patients were already inoperable due to peritoneal dissemination and metastasis, and even if resected by surgery, the 5-year survival rate is only 15-20%.
  • antibodies as anticancer agents is gaining importance as an approach in the treatment of various disease states (cancer types).
  • cancer types cancer types
  • an antibody is targeted to a tumor-specific antigen
  • the administered antibody is presumed to accumulate in the tumor, so complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) can be expected to attack cancer cells via the immune system.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • the bound drug can be efficiently delivered to the tumor site.
  • the amount of drug reaching the other tissue can be reduced, and as a result, side effects can be reduced.
  • the tumor-specific antigen has activity to induce cell death, administer an antibody with agonistic activity, and if the tumor-specific antigen is involved in cell growth and survival, neutralize activity
  • administering an antibody having a tumor tumor growth arrest or regression can be expected due to the accumulation of antibody specific to the tumor and the activity of the antibody. From such characteristics, the antibody is considered suitable for application as an anticancer agent.
  • the antibody drugs marketed so far are leukemia and lymphoma targeting CD20 targeting rituximab (trade name rituxan) and iburitumomab ozogamicin (trade name Zevailn), and CD33 targeting gemutuzumab ozogamitin (trade name Mylotarg) Etc. are being developed.
  • rituximab trade name rituxan
  • iburitumomab ozogamicin trade name Zevailn
  • CD33 gemutuzumab ozogamitin
  • VEGF gemutuzumab ozogamitin
  • trastuzumab (trade name Herceptin) targeting Her2 / neu
  • bevacizumab (trade name Avastin) targeting VEGF
  • other human diseases such as tocilizumab (trade name Actemula), which is a human IL-6 receptor antibody, have been developed for rheumatoid arthritis and Castleman's disease.
  • MANSC1 Homo sapiens MANSC domain containing 1
  • MANSC is a protein that is present in the cell membrane and has a motif containing seven highly conserved cysteine sequences on the N-terminal side of the extracellular domain.
  • MANSC is an abbreviation for motif at N terminus with seven cysteines.
  • the MANSC domain having this motif has been clarified to be highly conserved not only in higher vertebrates but also in multicellular organisms from molluscs and chordae by analysis using TBLASTN for EST.
  • the MANSC domain is also present in HAI-1, an activator inhibitor of hepatocyte growth factor HGF, and LRP-11, a low-density lipoprotein receptor-related factor.
  • Non-patent Document 1 Non-patent Document 2
  • Patent Document 1 Non-patent Document 2
  • Patent Document 1 Non-patent Document 3
  • these documents do not disclose the detailed behavior and function of the MANSC1 molecule itself.
  • the present invention has been made in view of such a situation, and an object thereof is to provide a novel antibody having excellent anticancer activity.
  • a further object of the present invention is to provide an anticancer agent comprising such an antibody as an active ingredient.
  • the present inventors first prepared a cDNA library derived from a cancer cell line, GCIY cell, and expressed it on the cell surface or secreted from the cell by the SST-REX method. The ones that code are selected. Subsequently, monoclonal antibodies against the protein encoded by the selected cDNA were prepared and examined for binding to various cancer cell lines and in vitro and in vivo anticancer activity. As a result, it was found that the obtained monoclonal antibody binds to the MANSC1 protein and has excellent anticancer activity in vitro and in vivo. Furthermore, the present inventor succeeded in identifying the region containing the epitope of these antibodies in MANSC1 protein and determining the structure of the variable region of the light chain and heavy chain of this antibody, thereby completing the present invention. It came.
  • the present invention relates to a monoclonal antibody that binds to MANSC1 protein and has anticancer activity, and an anticancer agent comprising the antibody as an active ingredient, more specifically, (1) an antibody that binds to a human-derived MANSC1 protein and has anticancer activity; (2) The antibody according to (1), which binds to an extracellular region of a human-derived MANSC1 protein, (3) The antibody according to (1), wherein the cancer is gastric cancer, glioma, or breast cancer, (4) The antibody according to claim 1 having the characteristics described in the following (a) or (b): (a) the amino acid sequence according to SEQ ID NO: 3 to 5 or at least one of the amino acid sequences, In the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and at least one of the amino acid sequences set forth in SEQ ID NOs: 6 to 8 or the amino acid sequences, one or more (B) the amino acid sequence
  • a light chain variable region comprising the determined amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence from which the signal sequence has been removed, or at least one of these amino acid sequences, Retains a heavy chain variable region comprising an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted;
  • the antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 33 to 35 or at least one of the amino acid sequences, In the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 36 to 38 or at least one of the amino acid sequences, one or more (B) the amino acid sequence set forth in SEQ ID NO: 40 or the signal sequence was removed
  • one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, an amino acid sequence set forth in SEQ ID NO: 42, an amino acid sequence in which a signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, (6)
  • the antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 43 to 45 or at least one of the amino acid sequences, One or more of the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 46 to 48 or at least one of the amino acid sequences (B) the amino acid sequence shown in
  • one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 52, an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, (7)
  • the antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 53 to 55 or at least one of the amino acid sequences, One or more of the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 56 to 58 or at least one of the amino acid sequences (B) the amino acid sequence set
  • one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 62, an amino acid sequence in which a signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, (8)
  • the antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 63 to 65 or at least one of the amino acid sequences, In the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and at least one of the amino acid sequences set forth in SEQ ID NOs: 66 to 68 or the amino acid sequences, one or more (B)
  • one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, an amino acid sequence set forth in SEQ ID NO: 72, an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, (9)
  • the antibody according to claim 1 having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 73 to 75 or at least one of the amino acid sequences, A light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 76 to 78 or at least one of the amino acid sequences.
  • the amino acid sequence or at least one of these amino acid sequences one or more amino acids are substituted, deleted, added and / or Or at least one of the light chain variable region comprising the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 92, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Or a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, (11)
  • the antibody according to claim 1 having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 93 to 95 or at least one of the amino acid sequences, One or more of the light chain variable region comprising an amino acid sequence in which
  • amino acid sequence or at least one of these amino acid sequences one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 104, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Or a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, (12) The antibody according to (1), which binds to a peptide sequence consisting of amino acid sequences at positions 61 to 153 of a human-derived MANSC1 protein, (13) The antibody according to (1), which binds to the epitope of the antibody according to any one of the following (a) to (h) in a human-derived MANSC1 protein (a) according to SEQ ID NO: 10 An antibody having a light chain variable region comprising the amino acid sequence and a heavy chain variable region comprising the amino acid
  • an antibody that binds to a human-derived MANSC1 protein and has excellent anticancer activity in vitro and in vivo is provided.
  • Use of the antibody of the present invention makes it possible to treat or prevent cancer.
  • the antibody of the present invention is particularly effective in suppressing the growth of gastric cancer cells, gliomas, or breast cancer cells.
  • Cancer cell lines include bladder cancer cell lines (T24), prostate cancer cell lines (PC3, Du145), pancreatic cancer cell lines (BxPC3, AsPC1), glioma cell lines (U251, U87MG, T98G), gastric cancer cell lines (MKN1, GCIY) was used.
  • the left figure shows a nuclear stained image using Hoechst33342, the figure shows an antibody-stained image, and the right figure shows an image obtained by superimposing a nuclear stained image using Hoechst33342 and an antibody-stained image. It is a microscope picture which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and various cultured cancer cells which performed the cell fixation and the membrane permeation process by cell staining.
  • Cancer cell lines include bladder cancer cell lines (T24), prostate cancer cell lines (PC3, Du145), pancreatic cancer cell lines (BxPC3, AsPC1), glioma cell lines (U251, U87MG, T98G), gastric cancer cell lines (MKN1, GCIY) was used.
  • the left figure shows a nuclear stained image using Hoechst33342, the figure shows an antibody-stained image, and the right figure shows an image obtained by superimposing a nuclear stained image using Hoechst33342 and an antibody-stained image. It is an electrophoresis photograph showing the result of immunoprecipitation using the ACT35-51_1B4A7D antibody on the culture supernatant of 293T cells expressing the MANSC1 gene.
  • mice As negative control cells, 293T cells (mock) into which only the vector was introduced were used. Moreover, mouse IgG2a was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1. Bladder cancer cell lines (T24) and prostate cancer cell lines (PC3, Du145) were used as the target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody.
  • the vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1.
  • Glioma cell lines U251, U87MG, T98G were used as the target cancer cell lines.
  • Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the tumor volume transition in the mouse
  • Saline was used as a control and Taxotere was used as a positive control. It is a figure which shows the body weight transition in the mouse
  • ACT35-51_1B4A7D antibody against Ba / F3 cells expressing genes corresponding to 60, 153, 186, 219, 250, 280, and 385 amino acids from the N-terminus of the antigen MANSC1 molecule and as a control
  • the reactivity of the MPL antibody was analyzed with a flow cytometer.
  • the filled histogram portion of each flow cytometer data shows the reaction with the antibody against each MANSC1 molecule, and the white histogram portion shows the response of mouse IgG2a (Beckman Coulter, # 731589) used as a control.
  • the filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control.
  • An anti-MPL antibody was used as a positive control. It is a figure which shows the reactivity of ACT35-51_5D6C11 antibody and the Ba / F3 cell which expresses a MANSC1 gene.
  • An anti-MPL antibody was used as a positive control. It is a figure which shows the reactivity of ACT35-51_7D9C8 antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene was analyzed with a flow cytometer.
  • the filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control.
  • An anti-MPL antibody was used as a positive control. It is a figure which shows the reactivity of ACT35-51_8G11B7 antibody and Ba / F3 cell which expresses a MANSC1 gene.
  • An anti-MPL antibody was used as a positive control. It is a figure which shows the reactivity of ACT35-51_9G12F12C antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene was analyzed with a flow cytometer.
  • the filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control.
  • An anti-MPL antibody was used as a positive control. It is a figure which shows the reactivity of ACT35-51_9G12F3E antibody and the Ba / F3 cell which expresses a MANSC1 gene.
  • An anti-MPL antibody was used as a positive control. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_3C3F3 monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_5D6C11 monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_5E2H6 monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_7D9C8 monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_8G11B7 monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG2b (MBL, # M077-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_2C4E8C monoclonal antibody has on the proliferation of cancer cells.
  • the vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1.
  • a gastric cancer cell line (GCIY) was used as the target cancer cell line.
  • Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1).
  • OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG3 (MBL, # M078-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_5C4F monoclonal antibody has on the proliferation of cancer cells.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1).
  • OD (OD450nm-OD630nm) value is shown.
  • Mouse IgM (MBL, # M079-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_6F6C monoclonal antibody has on the proliferation of cancer cells.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1).
  • OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG1 (MBL, # M075-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_8F7C monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1).
  • OD (OD450nm-OD630nm) value is shown.
  • Mouse IgG2b (MBL, # M077-3) was used as a negative control antibody. It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_1A7H7 monoclonal antibody.
  • Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines.
  • the vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1).
  • OD (OD450nm-OD630nm) value is shown.
  • Mouse IgM (MBL, # M079-3) was used as a negative control antibody. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_3C3F3 antibody.
  • the result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_5D6C11 antibody.
  • the result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by solid lines. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_5E2H6 antibody.
  • the result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_7D9C8 antibody.
  • the result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by solid lines. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_8G11B7 antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_2C4E8C antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line.
  • the present invention provides an antibody that binds to a human-derived MANSC1 protein and has anticancer activity.
  • the “antibody” in the present invention includes all classes and subclasses of immunoglobulins.
  • “Antibody” includes polyclonal antibodies and monoclonal antibodies, and also includes forms of functional fragments of antibodies.
  • Polyclonal antibodies are antibody preparations comprising different antibodies directed against different epitopes.
  • the “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population. In contrast to polyclonal antibodies, monoclonal antibodies are those that recognize a single determinant on an antigen.
  • the antibody of the present invention is preferably a monoclonal antibody.
  • the antibodies of the invention are antibodies that have been separated and / or recovered (ie, isolated) from components of the natural environment.
  • the “human-derived MANSC1 protein (NCBI Reference Sequence: NM_018050.2)” to which the antibody of the present invention binds is present in the cell membrane and contains seven highly conserved cysteine sequences on the N-terminal side of the extracellular domain. A protein having a motif.
  • the human-derived MANSC1 protein is a protein consisting of a 431 amino acid sequence, of which the 26 amino acid portion from the N-terminal is a signal sequence, the 27th to 385th extracellular region, the 386th to 408th is a transmembrane region, It is presumed to be a transmembrane protein having the 409th and subsequent regions in the intramembrane region.
  • the amino acid sequence of a typical human-derived MANSC1 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the MANSC1 gene is shown in SEQ ID NO: 1.
  • a human-derived MANSC1 protein may naturally have a mutated amino acid in addition to such a typical amino acid sequence. Therefore, the “human-derived MANSC1 protein” in the present invention is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, but in addition to that, in the amino acid sequence represented by SEQ ID NO: 2, Or what consists of an amino acid sequence by which several amino acid was substituted, deleted, inserted, or added is also contained.
  • the substitution, deletion, insertion or addition of the amino acid sequence is generally within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 1 amino acid).
  • anticancer activity means an activity of suppressing the growth of cancer cells in vitro and / or in vivo.
  • the anticancer activity can be evaluated, for example, by analysis using the MTT assay described in Example 8 or the cancer-bearing model described in Example 9.
  • a preferred embodiment of the antibody of the present invention shows that when the MTT assay described in Example 8 is performed, the growth of a gastric cancer cell line (eg, GCIY) is 50% compared to the control after 72 hours of antibody addition.
  • a gastric cancer cell line eg, GCIY
  • An antibody that suppresses the above for example, 60% or more, 70% or more).
  • Another preferred embodiment of the antibody of the present invention is that when the MTT assay described in Example 8 is performed, the proliferation of a glioma cell line (for example, T98G) is 72 hours after the addition of the antibody compared to the control. It is an antibody that suppresses 50% or more (for example, 60% or more, 70% or more, 80% or more, 90% or more).
  • the analysis using the cancer-bearing model described in Example 9 shows that the tumor volume is 30% or more (for example, 35 % Or more, 40% or more, 45% or more, 50% or more, 55% or more).
  • an analysis using the cancer-bearing model described in Example 9 shows that the weight of the excised tumor is 20% or more (for example, 25% or more, 30% or more, 35% or more).
  • these antibodies preferably further have the property of not reducing the body weight of the administration subject. It is particularly preferred that the antibody of the present invention has a plurality of the above activities.
  • Another preferred embodiment of the antibody of the present invention is an antibody having a high affinity for human-derived MANSC1 protein, and the surface plasmon resonance (SPR) method described in Example 12 using purified MANSC1 protein.
  • the value of the dissociation constant obtained by is preferably 10 ⁇ 9 or less, more preferably 5 ⁇ 10 ⁇ 10 or less.
  • a light chain variable region comprising light chains CDR1 to CDR3 (amino acid sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 5) and heavy chain CDR1 to CDR3 (SEQ ID NO: 6).
  • An antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8), Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 33 to SEQ ID NO: 35) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 36 to SEQ ID NO: 38)
  • An antibody having a heavy chain variable region comprising, Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 43 to SEQ ID NO: 45) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 46 to SEQ ID NO: 48)
  • An antibody having a heavy chain variable region comprising, Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 53 to SEQ ID NO: 55) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 56 to SEQ ID NO
  • the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 10 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 12 (or An amino acid sequence in which a signal sequence is removed from the amino acid sequence),
  • the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 40 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 42 (or the amino acid).
  • the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 50 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 52 (or the amino acid).
  • An amino acid sequence in which the signal sequence is removed from the sequence) The light chain variable region is composed of the amino acid sequence set forth in SEQ ID NO: 60 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 62 (or the amino acid).
  • the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 70 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 72 (or the amino acid).
  • An amino acid sequence in which the signal sequence is removed from the sequence) The light chain variable region is composed of the amino acid sequence set forth in SEQ ID NO: 80 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 82 (or the amino acid).
  • the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 90 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 92 (or the amino acid).
  • An amino acid sequence in which the signal sequence is removed from the sequence The light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 102 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 104 (or the amino acid).
  • An antibody consisting of an amino acid sequence in which the signal sequence is removed from the sequence).
  • an antibody comprising the light chain variable region and the heavy chain variable region those skilled in the art specify a peptide region (epitope) on the human-derived MANSC1 protein recognized by the antibody, Various antibodies that bind to the region and show anticancer activity can be produced.
  • the epitope of the antibody can be determined by a known method such as examining the binding to an overlapping synthetic oligopeptide obtained from the amino acid sequence of MANSC1 protein derived from human (for example, Ed Harlow and D. Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, US Pat. No. 4,088,871).
  • a peptide library by phage display can also be used for epitope mapping.
  • the peptide region on the MANSC1 protein recognized by the antibody of the present invention is preferably an extracellular region of the MANSC1 protein.
  • the extracellular region of the MANSC1 protein recognized by the antibody of the present invention is preferably a region within the range of positions 61 to 153 of the amino acid sequence of the MANSC1 protein.
  • the antibodies of the present invention include mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, and functional fragments of these antibodies.
  • a human as a pharmaceutical
  • a chimeric antibody, a humanized antibody, or a human antibody is desirable from the viewpoint of reducing side effects.
  • a “chimeric antibody” is an antibody in which a variable region of a certain antibody is linked to a constant region of a heterogeneous antibody.
  • a chimeric antibody for example, immunizes a mouse with an antigen, cuts out an antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, and binds to a human bone marrow-derived antibody constant region (constant region) gene.
  • Can be obtained by incorporating it into an expression vector and introducing it into a host for production for example, Japanese Patent Application Laid-Open No. 8-280387, US Pat. No. 4816397, US Pat. No. 4,816,567, US Pat. No. 5807715).
  • the “humanized antibody” is an antibody obtained by transplanting the gene sequence of the antigen-binding site (CDR) of a non-human-derived antibody to a human antibody gene (CDR grafting), and its production method is publicly known. (See, for example, EP239400, EP125023, WO90 / 07861, WO96 / 02576).
  • a “human antibody” is an antibody derived from all regions. In the production of human antibodies, a method for screening production of active antibodies from human B cells, a phage display method, and a transgenic animal (for example, a mouse) capable of producing a repertoire of human antibodies by immunization. It can be used.
  • the “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a human-derived MANSC1 protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.
  • Fab means a monovalent antigen-binding fragment of an immunoglobulin composed of one light chain and part of a heavy chain. It can be obtained by papain digestion of antibodies and by recombinant methods. “Fab ′” differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the hinge region of the antibody. “F (ab ') 2” means a divalent antigen-binding fragment of an immunoglobulin that consists of both light chains and parts of both heavy chains.
  • “Variable region fragment (Fv)” is the smallest antibody fragment with complete antigen recognition and binding sites. Fv is a dimer in which a heavy chain variable region and a light chain variable region are strongly linked by a non-covalent bond. “Single-chain Fv (scFv)” comprises the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain. “Sc (Fv) 2” is a chain formed by joining two heavy chain variable regions and two light chain variable regions with a linker or the like.
  • a “diabody” is a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, each region comprising a separate It forms a pair with the complementary region of the strand.
  • a “multispecific antibody” is a monoclonal antibody that has binding specificities for at least two different antigens. For example, it can be prepared by co-expression of two immunoglobulin heavy / light chain pairs where the two heavy chains have different specificities.
  • the present invention provides a peptide consisting of the light chain or heavy chain of an antibody comprising the CDR identified in the present invention, or a variable region thereof.
  • a preferred peptide is a peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 3 to 5 or a variable region thereof, A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence of SEQ ID NOs: 33 to 35 or a variable region thereof, A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 43 to 45 or a variable region thereof, A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence of SEQ ID NOs: 53 to 55 or a variable region thereof, A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 63 to 65 or a variable region thereof; A peptide
  • Another preferred peptide is a peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 8, or a variable region thereof, A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 36 to 38 or a variable region thereof, A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 46 to 48 or a variable region thereof, A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 56 to 58 or a variable region thereof, A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 66 to 68 or a variable region thereof, A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 76 to 78 or a
  • functional antibodies can be produced by linking these peptides with, for example, a linker.
  • the antibody of the present invention includes an antibody whose amino acid sequence has been modified without reducing the desired activity (antigen binding activity, anticancer activity, and / or other biological properties).
  • An amino acid sequence variant of the antibody of the present invention can be prepared by introducing a mutation into DNA encoding the antibody chain of the present invention or by peptide synthesis. Such modifications include, for example, residue substitutions, deletions, additions and / or insertions within the amino acid sequences of the antibodies of the invention.
  • the site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR).
  • Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen.
  • the amino acid of the CDR is modified to screen for antibodies with increased affinity for the antigen.
  • Methods are known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002/051870, J. Biol. Chem., 280: 24880-24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).
  • the number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid).
  • the amino acid modification is preferably a conservative substitution.
  • conservative substitution means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs.
  • acidic amino acids for example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan).
  • basic amino acids lysine, arginine, histidine
  • neutral amino acids amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cystein
  • “having equivalent activity” means that the antigen binding activity or anticancer activity is equivalent to the target antibody (typically, ACT35-51_1B4A7D antibody) (eg, 70% or more, preferably 80% or more, More preferably 90% or more).
  • the binding activity to the antigen can be evaluated, for example, by preparing Ba / F3 cells expressing the antigen and analyzing the reactivity with the antibody sample with a flow cytometer (Examples 4 and 11). Further, as described above, the anticancer activity can be evaluated by, for example, analysis using the MTT assay described in Example 8 or the cancer-bearing model described in Example 9.
  • the modification of the antibody of the present invention may be modification of a post-translational process of the antibody, for example, changing the number or position of glycosylation sites.
  • the ADCC activity of the antibody can be improved.
  • Antibody glycosylation is typically N-linked or O-linked.
  • Antibody glycosylation is highly dependent on the host cell used to express the antibody.
  • the glycosylation pattern can be modified by a known method such as introduction or deletion of a specific enzyme involved in sugar production (JP 2008-113663, US Pat. No. 5,473,335, US Pat. No. 5,510,261, US Pat. No. 5278299, International Publication No. 99/54342).
  • deamidation is suppressed by substituting an amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be.
  • glutamic acid can be substituted with other amino acids to increase antibody stability.
  • the present invention also provides the antibody thus stabilized.
  • the antibody of the present invention is a polyclonal antibody
  • an immunized animal is immunized with an antigen (such as a human-derived MANSC1 protein, a partial peptide thereof, or a cell expressing these), and the conventional means (for example, Salting out, centrifugation, dialysis, column chromatography, etc.).
  • Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.
  • the hybridoma method typically includes the Kohler and Milstein method (Kohler & Milstein, Nature, 256: 495 (1975)).
  • the antibody-producing cells used in the cell fusion step in this method are animals (eg, mice, rats, hamsters, rabbits) immunized with antigens (human-derived MANSC1 protein, partial peptides thereof, or cells expressing these). Monkeys, goats) spleen cells, lymph node cells, peripheral blood leukocytes and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal in a medium.
  • the myeloma cells various known cell lines can be used.
  • the antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species.
  • Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with antigen and mouse myeloma cells, and subsequent screening produces monoclonal antibodies specific for human-derived MANSC1 protein. Hybridomas can be obtained.
  • Monoclonal antibodies against human-derived MANSC1 protein can be obtained by culturing hybridomas or from ascites of mammals to which hybridomas have been administered.
  • a DNA encoding the antibody or peptide of the present invention is cloned from a hybridoma, a B cell or the like and incorporated into an appropriate vector, which is then introduced into a host cell (eg, a mammalian cell line, E. coli, yeast cell, insect). Cells, plant cells, etc.) and a method for producing the antibody of the present invention as a recombinant antibody (for example, PJDelves, Antibody Production: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem.
  • a host cell eg, a mammalian cell line, E. coli, yeast cell, insect. Cells, plant cells, etc.
  • a method for producing the antibody of the present invention as a recombinant antibody for example, PJDelves, Antibody Production: Essential Technologies, 1997 WILEY, P
  • DNA encoding the heavy chain or the light chain may be separately incorporated into an expression vector to transform the host cell.
  • Host cells may be transformed into a single expression vector (see WO94 / 11523).
  • the antibody of the present invention can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. For the separation and purification of the antibody, the methods used in the usual purification of polypeptides can be used.
  • transgenic animals such as cows, goats, sheep or pigs
  • transgenic animal production technology a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.
  • the present invention provides a DNA encoding the antibody or peptide of the present invention, a vector containing the DNA, a host cell holding the DNA, and a method for producing the antibody comprising culturing the host cell and recovering the antibody Is also provided.
  • the present invention is a cancer treatment comprising the steps of administering an anticancer agent comprising the antibody of the present invention as an active ingredient and a therapeutically or prophylactically effective amount of the antibody of the present invention to mammals including humans. It also provides a preventive method.
  • the treatment or prevention method of the present invention can be applied to various mammals including dogs, cats, cows, horses, sheep, pigs, goats, rabbits and the like, in addition to humans.
  • the antibodies of the present invention strongly suppressed the growth of gastric cancer cells, glioma cells, and breast cancer cells, among other cancers. It is particularly effective.
  • the anticancer agent comprising the antibody of the present invention as an active ingredient can be used in the form of a composition containing the antibody of the present invention and an optional component such as physiological saline, sucrose aqueous solution or phosphate buffer.
  • the anticancer agent of the present invention may be formed into a liquid or lyophilized form as necessary, and optionally a pharmaceutically acceptable carrier or medium, such as a stabilizer, preservative, isotonic agent, etc. Can also be included.
  • the pharmaceutically acceptable carrier examples include mannitol, lactose, saccharose, human albumin and the like in the case of a lyophilized preparation.
  • physiological saline, water for injection, phosphoric acid, etc. examples thereof include, but are not limited to, a salt buffer and aluminum hydroxide.
  • the administration method of the anticancer agent varies depending on the age, weight, sex, health status, etc. of the administration subject, but it should be administered by any of the administration routes of oral administration or parenteral administration (eg, intravenous administration, arterial administration, local administration). Can do.
  • a preferred method of administration is parenteral administration.
  • the dose of an anticancer drug may vary depending on the patient's age, weight, sex, health status, degree of cancer progression, and the components of the anticancer drug to be administered.
  • the daily dose is 0.1 to 1000 mg, preferably 1 to 100 mg.
  • the antibody of the present invention can be applied not only to cancer treatment and prevention but also to cancer diagnosis.
  • the antibody of the present invention may be labeled.
  • a label for example, a radioactive substance, a fluorescent dye, a chemiluminescent substance, an enzyme, and a coenzyme can be used.
  • radioisotope, fluorescein, rhodamine, dansyl chloride, luciferase, peroxidase, alkaline phosphatase examples include lysozyme and biotin / avidin.
  • the antibody of the present invention can be obtained in any dosage form by employing any suitable means.
  • the antibody titer of a purified antibody can be measured and appropriately diluted with PBS (Phosphate buffer saline, phosphate buffer containing physiological saline) or the like, and then 0.1% sodium azide or the like can be added as a preservative.
  • PBS Phosphate buffer saline, phosphate buffer containing physiological saline
  • 0.1% sodium azide or the like can be added as a preservative.
  • the antibody titer of a substance obtained by adsorbing the antibody of the present invention on latex or the like can be obtained, diluted appropriately, and added with a preservative.
  • MANSC1 protein or a partial peptide thereof can be administered as a cancer vaccine to mammals including humans (for example, (See JP 2007-277251, JP 2006-052216).
  • the present invention also provides a cancer vaccine composition containing MANSC1 protein or a partial peptide thereof used for such cancer vaccine applications.
  • a pharmaceutically acceptable carrier or medium such as a stabilizer, preservative, isotonic agent and the like can be contained in the same manner as the anticancer agent of the present invention.
  • Example 1 Implementation of SST-REX SST-REX was performed in order to obtain comprehensive information on membrane or secretory genes expressed on the cell surface of Skills gastric cancer cell line GCIY cells.
  • RNA was dissolved in 100 ⁇ l of water, and 3 ⁇ g of mRNA was obtained using FastTrack 2.0 mRNA Isolation kit (invitrogen, # K1593-02). Using the SuperScript TM Choice System (invitorgen, # 18090-019), double-stranded cDNA was prepared from the obtained mRNA.
  • GCIY cells are a gastric cancer cell line established from Borrman IV gastric cancer and peritoneal disseminated metastasis of ascites from a woman who suffered from peritoneal disseminated metastasis, and expressed multidrug resistance gene (mdr-1). , And poorly differentiated adenocarcinoma cells in which secretion of CEA, CA19-9, and ⁇ FP is observed.
  • Double-stranded cDNA was dissolved in an aqueous solution of Bst XI Adapter in which 9 ⁇ g of Bst XI Adapter (invitorgen, # N408-18) was dissolved in 10 ⁇ l of water. 5 ⁇ l of LigationHigh (TOYOBO # LGK-201) was added thereto, suspended, and reacted at 16 ° C.
  • a gel containing a conjugate of a double-stranded cDNA fragment having a length of about 500 to about 4000 bases and a Bst XI Adapter was cut out, and the Wizard (R) SV Gel and PCR Clean-Up System (promega # A9282) was used to purify the conjugate of double-stranded cDNA and Bst XI Adapter.
  • a cDNA library constructed using the pMX-SST vector was introduced into E. coli and amplified.
  • 5 ⁇ g of tRNA, 12.5 ⁇ l of 7.5 M sodium acetate, and 70 ⁇ l of ethanol were added and mixed by inversion, followed by centrifugation at 20,400 ⁇ g for 30 minutes, and the supernatant was discarded to obtain a precipitate.
  • 500 ⁇ l of 70% ethanol was added, centrifuged at 20,400 ⁇ g for 5 minutes, the supernatant was discarded, and the resulting precipitate was dissolved in 10 ⁇ l of water.
  • the resulting precipitate was dissolved in 10 ⁇ l of water.
  • virus packaging cell Plat-E (Gene Ther. 7 (12): 1063-6 (2000) Jun) 2 ⁇ 10 6 pieces are suspended in a 6cm dish containing 4ml of DMEM medium (Wako, # 044-29765), at 37 ° C and 5% CO 2 Cultured for 24 hours.
  • 100 ⁇ l of opti-MEM (GIBCO, # 31985070) and 9 ⁇ l of Fugene (Roche, # 1814443) were mixed and allowed to stand for 5 minutes at room temperature.
  • filtration supernatant 0.5 ml was added to a 10 cm dish containing 9.5 ml of RPMI-1640 (Kohjin Bio) medium containing 4 ⁇ 10 6 Ba / F3 cells.
  • SST5 'side-T3 5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3' (SEQ ID NO: 15)
  • the obtained sequence data is BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) and SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). Analyzed by using.
  • the SST-REX method was performed using cells as a material.
  • cDNAs from 87 transfectant Ba / F3 cells were sequenced and 40 different genes were used. I was able to get.
  • there were 15 types of duplicated genes in the first and second rounds and a total of 81 types of cDNA-derived genes could be obtained in the second round.
  • the transfectant Ba / F3 cell line used for gene analysis was confirmed to contain only one type of cDNA-derived gene, and was used in the subsequent experiments (hereinafter derived from the cDNA thus obtained). Cells containing the gene are referred to as “SST clonal cells”).
  • Example 2 Cloning of MANSC1 full-length gene and establishment of Ba / F3 cell line that expresses it
  • the MANSC1 gene included in the cDNA-derived gene list obtained in Example 1 includes the full-length gene. Cloning was performed to obtain SST clonal cells.
  • Design primers based on the information of NM_018050.2 on the nucleotide search site of NCBI (http://www.ncbi.nlm.nih.gov/nucleotide) using 30 ng of cDNA prepared by SST-REX of GCIY cells as a template PCR reaction was performed using PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A).
  • E. coli competent cells for heat shock were added to the total amount of the combined product, and left on ice for 30 minutes, followed by incubation at 42 ° C. for 90 seconds, and then 1 ml of LB medium was added and incubated at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 15,000 ⁇ g for 1 minute, the supernatant was removed, and the E. coli pellet was suspended with the remaining solution. Apply this whole amount to LB agar medium containing 50 ⁇ g / mL of ampicillin, incubate overnight at 37 ° C, and use the obtained colonies to perform PCR and sequence analysis in the same manner as the sequence analysis of Example 1 (4). Went. For clones in which a DNA fragment of the target length was confirmed, the PCR product was sequenced to confirm that the target sequence was inserted. PrimeSTAR MAX DNA polymerase was used as the polymerase for PCR during sequence analysis.
  • the colony in which the target sequence was inserted was inoculated into 3 ml of LB liquid medium, and cultured at 37 ° C. overnight.
  • the whole culture was centrifuged at 3,000 ⁇ g for 15 minutes, the supernatant was removed, and purified using QuickLyse Miniprep Kit (QIAGEN, # 27406) to obtain a plasmid containing the full length of the MANSC1 gene.
  • a retrovirus containing a vector was prepared in the same manner as in the packaging after packaging of the cDNA library shown in Example 1 (4) and thereafter.
  • a Ba / F3 cell line expressing the full-length MANSC1 gene was established and used for the subsequent experiments.
  • Example 3 Production of MANSC1 Monoclonal Antibody Mouse Balb / c is used as an immunized animal.
  • TiterMax Gold Alexis Biochemicals, ALX-510-002-L010
  • PBS PBS
  • 50 ⁇ l of the emulsified product was administered.
  • 5 ⁇ 10 6 SST clonal cells having the MANSC1 gene were administered as immunogen cells, and the immunogen cells were injected four times every two days.
  • the excised secondary lymphoid tissue was ground to obtain a cell population containing antibody-producing cells.
  • mice myeloma cell P3U1 P3-X63-Ag8.U1 was used.
  • Hybridomas include HAT (SIGMA, H0262), 5% BM-condimed (Roche, 663573), 15% FBS, 1% penicillin / streptomycin solution (GIBCO, 15140-122, penicillin-streptomycin liquid, hereinafter “P / Cultured for 10-14 days in RPMI1640 (Wako) selective medium containing “S”.
  • hybridomas were selected by flow cytometry shown in Example 4 that reacted with immunogen cells and did not react with SST clonal cells (negative control cells) that did not contain the antigen gene in the immunogen cells. Monocloning was performed by limiting dilution to obtain a hybridoma clone producing the anti-MANSC1 antibody ACT35-51_1A4B7D (FIG. 1).
  • the resulting hybridoma was maintained using RPMI-1640 medium containing the required amount of HT (SIGMA, HT media supplement (50X) Hybri-Max (Sigma-Aldrich H0137), 15% FBS, 1% P / S solution.
  • the isotype of the produced antibody was determined using an isostrip kit (Roche, 1493027), resulting in IgG2a / ⁇ .
  • the acquisition of the purified ACT35-51_1B4A7D antibody from the monocloned hybridoma was performed as follows.
  • the hybridoma was acclimated to a serum-free medium (Hybridoma-SFM: GIBCO, 12045-076) and expanded, and then cultured for a certain period to obtain a culture supernatant.
  • the IgG fraction contained in this culture supernatant was treated with Protein A Sepharose (GE Healthcare, 17-1279-03), MAPS-II binding buffer (BIO-RAD, 153-6161), MAPS-II elution buffer (BIO -RAD, 153-6162).
  • the eluted IgG was dialyzed against PBS to obtain a purified antibody fraction.
  • Example 4 Antibody screening using flow cytometry ACT35-51_1B4A7D antibody and various cells (Ba / F3 cell expressing target gene, Ba / F3 cell not expressing target gene, various cancer cells, etc.) The reactivity of was analyzed using flow cytometry.
  • PBS containing 0.5% BSA and 2 mM EDTA was used as the cell suspension buffer and the subsequent washing buffer.
  • Various cells to be reacted with the antibody were adjusted and dispensed in a 96-well plate (BD Falcon, 353911) so that the cell suspension contained 5 ⁇ 10 4 cells per well to 100 ⁇ l. .
  • the cells to be stained were cancer cell lines, they were detached from the culture plate using Cell Dissociation Buffer (GIBCO, 13151-014) and collected when they became 80% confluent.
  • GEBCO Cell Dissociation Buffer
  • antibody solution As an isotype control antibody of the antibody solution, a washing buffer containing 2 ⁇ g / ml each of mouse IgG1 (BioLegend, 400412), mouse IgG2a (BioLegend, 400224), mouse IgG2b (BioLegend, 400324) was used.
  • a gate was applied so as to select live cells from the measured values of forward scatter and side scatter. Measure the fluorescence intensity of PE based on the reactivity with the antibody against the selected live cells, and the reactivity intensity of the isotype control is used as a reference. Hybridoma cells that produced culture supernatants that were not reactive with the bacterium were selected as candidate clones (FIG. 1).
  • an antibody with a significant reactivity was selected based on the reaction intensity between the antibody and the isotype control antibody.
  • Example 5 Flow cytometry using cancer cells When cancer cells to be stained become 80% confluent, they are peeled off from the culture plate using Cell Dissociation Buffer (GIBCO, 13151-014) and collected. 1 ⁇ 10 5 cells were suspended in 100 ⁇ l each in 0.5% BSA, 2 mM EDTA / PBS (washing buffer shown in Example 4), and dispensed into a 96-well plate (BD Falcon, 353911). Thereafter, the reactivity between cancer cells and antibodies was analyzed using a flow cytometer in the same manner as in Example 4.
  • GCIY a gastric cancer cell line
  • ACT35-51_1B4A7D antibody a gastric cancer cell line
  • the ACT36-27_5D1 antibody reacted significantly with GCIY
  • the ACT35-51_1B4A7D antibody could not detect significant reactivity with GCIY (FIG. 2).
  • Example 6 Cell staining of cancer cells The reactivity between the ACT35-51_1B4A7D antibody and various cancer cells was analyzed by cell staining.
  • a black 96-well plate (BD Falcon, 353219) 1 ⁇ 10 4 cancer cells to be stained were suspended and seeded in 100 ⁇ l of medium, and cultured for 24 hours.
  • DMEM medium SIGMA
  • FBS FBS
  • P / S solution 1%
  • a buffer containing 25 mM HEPES (pH 7.4), 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO 4 , and 1.3 mM CaCl 2 was used as the washing buffer.
  • the washing buffer in which the hybridoma culture supernatant or purified antibody is dissolved at 2 ⁇ g / ml is removed from the cells obtained by removing the culture supernatant by centrifugation at 700 ⁇ g for 2 minutes. 50 ⁇ l of each was added.
  • mouse IgG1 BioLegend, 400412
  • mouse IgG2a BioLegend, 400224
  • mouse IgG2b BioLegend, 400324
  • the cell culture supernatant was removed by centrifugation, and the obtained cells were washed once with 100 ⁇ l of washing buffer in the same manner as described above. Thereafter, 50 ⁇ l of 4% paraformaldehyde / phosphate buffer (Wako, 161-20141) was added, and the cells were fixed by reacting at room temperature for 10 minutes. Then, it was washed twice with 100 ⁇ l of washing buffer. Next, 100 ⁇ l of a washing buffer containing 0.1% Triton X-100 was added and reacted at room temperature for 10 minutes to increase the permeability of the cell membrane, and then washed twice with 100 ⁇ l of washing buffer.
  • 4% paraformaldehyde / phosphate buffer (Wako, 161-20141) was added, and the cells were fixed by reacting at room temperature for 10 minutes. Then, it was washed twice with 100 ⁇ l of washing buffer.
  • 100 ⁇ l of a washing buffer containing 0.1% Triton X-100
  • staining and analysis were performed in the same manner as the method of staining only the cell surface.
  • the cells were examined for the presence or absence of antibody staining by measuring the fluorescence of PE using the nucleus stained with Hoechst33342 as the cell position reference.
  • MANSC1 When various cancer cell lines were stained with the ACT35-51_1B4A7D antibody, MANSC1 could not be detected under the staining conditions on the cell surface only (FIG. 3A). However, reactivity was observed between GCIY and U87MG by subjecting cancer cells to fixation and membrane permeabilization (FIG. 3B). From this, it was considered that in GCIY and U87MG, MANSC1 was expressed in an amount exceeding the detection limit, and the ACT35-51_1B4A7D antibody was reacting with it.
  • the ACT35-51_1B4A7D antibody was not reactive in GCIY and U87MG unless membrane permeabilization was performed, so the epitope to which this antibody reacts does not exist on the cell membrane surface, but is cleaved and secreted. It was speculated.
  • Example 7 Immunoprecipitation using MANSC1 transfectant culture supernatant Since it was suggested that the epitope of ACT35-51_1B4A7D antibody was cleaved and secreted outside the cell, the culture supernatant of MANSC1-expressing cells was used for immunoprecipitation. Since cancer cells are difficult to culture in a serum-free medium, the MANSC1 gene was transiently expressed in 293T cells, the medium was replaced with a serum-free medium, and immunoprecipitated using the concentrated sample.
  • 293T cells were seeded on 10 10 cm culture dishes, and gene transfer was performed using Fugene6 (Roche, # 1814443) when 80% confluent.
  • Fugene6 Fugene6
  • 600 ⁇ l of Opti-MEM (GIBCO, # 31985070) and 18 ⁇ l of Fugene6 were mixed per dish, left for 5 minutes at room temperature, 6 ⁇ g of MANSC1 DNA construct was added, and left for 15 minutes at room temperature.
  • the solution was added to the culture solution of 293T, washed 24 times later with 4 ml of DMEM without serum, and then 20 ml of FreeStyle 293 (GIBCO, # 12338-018) was added and cultured for 5 days.
  • FreeStyle 293 GIBCO, # 12338-018 was added and cultured for 5 days.
  • 293T cells into which only the vector was similarly introduced were also prepared.
  • the culture supernatant was collected, filtered through a 0.22 ⁇ m filter, and concentrated 100 times using Amicon Ultra (fractionated molecular weight 3000, Millipore, # UFC9 003 96).
  • the PVDF membrane was washed 3 times with PBS containing 0.05% Tween 20 (hereinafter referred to as PBS-T), and 2.5 ml of anti-rabbit IgG-POD (MBL, # 458) diluted 5000 times with 5% skim milk was added. And allowed to react at room temperature for 1 hour. After the reaction, the PVDF membrane was washed 5 times with PBS-T, developed with a chromogenic substrate (Millipore, Immobilon Western # WBKLS0500), and exposed to the film for 15 seconds.
  • PBS-T PBS containing 0.05% Tween 20
  • MBL anti-rabbit IgG-POD
  • MANSC1 signal was detected in the vicinity of 10 kDa when immunoprecipitated with ACT35-51_1B4A7D antibody, but no signal was detected when immunoprecipitated with isotype control (FIG. 4). ).
  • the culture supernatant of 293T into which only the vector was introduced no signal was detected in both the isotype control and those immunoprecipitated with the ACT35-51_1B4A7D antibody (FIG. 4).
  • Example 8 Effect of ACT35-51_1B4A7D monoclonal antibody on proliferation of cancer cells (MTT)
  • MTT cancer cells
  • the prostate cancer cell line AsPC1 was inactivated with RPMI1640 medium (WAKO) containing 10% FBS (Equitech, the same applies hereinafter) and 1% P / S, and other cancer cells.
  • RPMI1640 medium WAKO
  • FBS Fequitech, the same applies hereinafter
  • SIGMA DMEM medium
  • the medium for hybridoma producing the target antibody includes RPMI medium (Wako) containing the required amount of HT (Sigma-Aldrich, H0137, HT media supplement (50X) Hybri-Max), 15% FBS, 1% P / S solution. Was used.
  • Mouse IgG1 (BECKMAN COULTER, 731581), IgG2a (MBL, M076-3), IgG2b (MBL, M077-3), IgG3 (MBL, M078-3), 1 ml of hybridoma medium A mouse isotype control mixture dissolved in was used.
  • the culture supernatant containing the ACT35-51_1B4A7D antibody or mouse isotype control was added to the 96-well plate after incubation at 100 ⁇ l per well and incubated for 72 hours.
  • To each cell obtained by removing the culture supernatant by centrifugation add 100 ⁇ l of 5% WST-1 solution (vol./vol., Roche, 11, 644, 807, 001) prepared by dissolving in fresh medium, After incubating for 1 to 4 hours, the color development of WST-1 was measured with a microplate reader (BIO-RAD) every 1 hour.
  • WST-1 solution vol./vol., Roche, 11, 644, 807, 001
  • the graphed measurement results are shown in FIG. This data showed the color development of WST-1 after 3 hours by O.D. value (O.D450nm-O.D.630nm).
  • the ACT35-51_1B4A7D antibody suppressed GCIY cell proliferation to approximately 25% compared to the isotype control (FIG. 5B). That is, there was a growth suppression effect of about 75%.
  • the cell growth inhibitory effect of ACT35-51_1B4A7D antibody was remarkably observed in glioma-derived T98G cells, and its growth was suppressed to about 7% compared to the isotype control (FIG. 5C). That is, an about 93% growth inhibitory effect was observed.
  • Example 9 Effect of antibody by tail vein administration on mouse tumor bearing model The in vivo antitumor effect of ACT35-51_1B4A7D antibody was examined using a mouse tumor bearing model.
  • a cell suspension is prepared so that GCIY cells become 5 ⁇ 10 6 cells / 0.2 ml saline / mouse individual subcutaneously on the back of the neck of 6-week-old male SCID mice (purchased from CLEA Japan at 5 weeks of age). Transplanted. Measure the size of the tumor that engrafted 3 weeks after transplantation, and control the tumor-bearing mice, control group, positive control group, ACT35-51_1B4A7D antibody group so that the average tumor volume of each group is about 55 ⁇ 5 mm 3 The three groups were divided into 4 groups.
  • Sample administration to each group was performed on the tail vein of cancer-bearing mice from 3 weeks after cell transplantation.
  • Saline Otsuka raw diet
  • Taxotere 600 ⁇ g per mouse, sanofi-aventis
  • ACT35-51_1B4A7D antibody 10 mg / kg
  • body weight measurement and tumor measurement were performed immediately before each administration.
  • tumor volume 0.5 x major axis x minor axis x minor axis
  • the tumor volume data was compared with the control group to examine the antitumor effect over time.
  • the tumor increased with time, and at the end of the experiment 3 weeks after the start of administration, the tumor volume was about 25 times that at the start.
  • tumor growth suppression was observed from the second week after the start of administration, and in the third week after the end of the experiment, it remained at about 11 times the start of the experiment, about 59 times that of the control group. % Tumor volume suppression was observed.
  • An autopsy was performed 3 weeks after the start of administration of each sample.
  • the tumor-bearing mice were subjected to ether anesthesia lethality, and the tumors were removed subcutaneously from the back of the neck and their weights were measured.
  • the excised tumor weights were 1.33 g for the control group, 0.87 g for the positive control group, and 0.83 g for the ACT35-51_1B4A7D antibody group, respectively.
  • the excised tumor weight was reduced by about 38% compared to the control group, and the same tumor growth inhibitory effect as that of the positive control group was observed (FIG. 7).
  • the statistical test was performed by one-way analysis of variance (ANOVA), and when there was a difference at p ⁇ 0.05, a significant difference test was performed by Tukey's multiple comparison method. When the risk rate was p ⁇ 0.05, the control group was evaluated as having a significant difference. (*: p ⁇ 0.05, **: p ⁇ 0.01)
  • Example 10 Antibody variable region determination method
  • 2 ⁇ 10 6 ACT35-51_1B4A7D antibody-producing cell hybridoma cells were added to 1 ml of Trizol (invitrogen, # 15596-026). Suspended and allowed to stand for 5 minutes, 200 ⁇ l of chloroform was added, suspended for 15 seconds, and then centrifuged at 12,000 ⁇ g for 15 minutes to obtain a supernatant. This supernatant was mixed with 500 ⁇ l of isopropanol, and then centrifuged at 12,000 ⁇ g for 10 minutes.
  • the obtained pellet was washed with 80% ethanol to obtain 40 ⁇ g of total RNA.
  • the whole amount was dissolved in 20 ⁇ l of water.
  • double-stranded cDNA was prepared from total RNA using SuperScript TM Choice System (invitorgen, # 18090-019).
  • the obtained double-stranded cDNA was precipitated with ethanol, and the 5 ′ end and 3 ′ end of the double-stranded cDNA were bound using LigationHigh (TOYOBO # LGK-201), and PCR was performed using 1 ⁇ l of this as a template.
  • Primers designed for the heavy and light chain constant regions were used. Primer sequences are as follows.
  • Heavy chain 5 ′ side gtccacgaggtgctgcacaat (SEQ ID NO: 18) Heavy chain 3 ′ side gtcactggctcagggaaataacc (SEQ ID NO: 19) Light chain 5 ′ side aagatggatacagttggtgc (SEQ ID NO: 20) Light chain 3 ′ side tgtcaagagcttcaacagga (SEQ ID NO: 21)
  • the PCR product was electrophoresed on a 1.5% gel, and then excised and purified. Sequencing was performed using the purified DNA. For the light chain, the purified DNA was cloned and then sequenced.
  • the determined light chain variable region base sequence is SEQ ID NO: 9
  • the amino acid sequence is SEQ ID NO: 10
  • the heavy chain variable region base sequence is SEQ ID NO: 11
  • the amino acid sequence is SEQ ID NO: 12. Show.
  • amino acid sequences of these variable regions are numbered using sequence analysis (http://www.bioinf.org.uk/abysis/tools/analyze.cgi) on the site of UCR's “Andrew CR Martin's Bioinformatics Group”.
  • the CDR regions were identified according to the criteria described in “Table of CDR Definitions” (http://www.bioinf.org.uk/abs/#kabatnum).
  • the results of CDR prediction and the light chain and heavy chain signal sequences are shown in FIG.
  • the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 3-5, and the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 6-8.
  • Example 11 Epitope analysis of ACT35-51_1B4A7D antibody
  • Ba / F3 cells expressing MANSC1 peptides having several chain lengths were prepared and the reactivity with the antibody was evaluated. did.
  • MANSC1 extramembranous region 60aa (from N-terminal, the same applies hereinafter), 153aa, 186aa, 219aa, 250aa, 285aa, 385aa (extracellular full length) were used as peptides to be analyzed.
  • cDNA library subjected to the GCIY signal sequencing strap shown in Example 1 as a template, using the DNA of the following sequence as a primer, PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A) as a polymerase, seven kinds of genes was isolated.
  • pMX-SST was also digested with EcoRI and NotI and purified. Further, each was treated with LigationHigh (TOYOBO, # LGK-201), and then the same treatment as in Example 2 (after transformation to E. coli) was performed, and the transformed E. coli was applied to a 50 ⁇ g ampicillin-containing LB agarose plate. Was plated. PCR was performed from colonies obtained by overnight culture at 37 ° C. so as to contain the insert portion, and the pMX-SST vector containing the desired sequence was confirmed by sequencing. The following oligonucleotides were used as PCR primers for sequencing.
  • the ACT35-51_1B4A7D antibody does not react with clones expressing MANSC1 molecules up to 1-60, but reacts with clones expressing longer than 1 to 153 region. From these results, it has been clarified that an antibody epitope is contained between positions 61 to 153 from the N-terminus of the MANSC1 molecule.
  • MANSC1-Fc fusion protein pACT001 (pcDNA3.1) is a cassette vector in which the DNA encoding the extracellular domain of MANSC1 is amplified by PCR and then the Fc hinge part of human IgG is inserted.
  • MANSC1-Fc fusion protein expression vector was prepared by incorporating into the vector derived from ACTGen.
  • the DNA encoding the extracellular domain of MANSC1 uses cDNA derived from GCIY cells as a template, and PrimeSTAR Amplification was performed by PCR using MAX DNA polymerase (TaKaRa # R045A). The following primers were used.
  • the resulting expression vector was transferred to Fugene6 Transfection Reagent (Roche, # 11 988 387 001) was lipofected into 293T cells according to the capacity document, and MANSC1-Fc fusion protein was transiently produced in a serum-free medium (Free-style 293 expression medium).
  • MAPSII binding buffer BIO-RAD
  • purification was performed using Protein A Sepharose (GE Healthcare, # 17-1279-03). Then, the fraction eluted with L-Arginine (PH4.0) was dialyzed with PBS, and the obtained purified product was used for kinetic analysis.
  • the ACT35-51_1B4A7D antibody has a very low dissociation constant (KD) of 10 -10 to the MANSC1 protein, which clearly indicates that the ACT35-51_1B4A7D antibody has a high affinity for the MANSC1 protein. became.
  • Example 13 Analysis of anti-MANSC1 antibody 14 hybridoma clones producing anti-MANSC1 antibodies other than the aforementioned ACT35-51_1A4B7D were obtained by the method described in Example 3 (see Table 2). As for the monoclonal antibodies produced by these hybridoma clones, the reactivity with respect to the Ba / F3 cell line expressing MANSC1 protein was confirmed in the same manner as in the method described in Example 4. Some of the results obtained are shown in FIGS.
  • the monoclonal antibodies produced by these clones were subjected to an MTT assay using a stomach cancer cell line (GCIY) and a breast cancer cell line (ZR75-1) in the same manner as in the method described in Example 8.
  • GCIY stomach cancer cell line
  • ZR75-1 breast cancer cell line
  • ACT35-51_2C4E8C antibody and the ACT35-51_8H12A7H antibody only the MTT assay using a gastric cancer cell line (GCIY) was performed. The obtained results are shown in FIGS.
  • ACT35-51_2C4E8C antibody ACT35-51_9G12F12C antibody
  • ACT35-51_9G12F3E antibody ACT35-51_8H12A7H antibody
  • GCIY gastric cancer cell line
  • other monoclonal antibodies did not show growth inhibitory effects on the two cell lines tested (see FIGS. 30 to 34).
  • the heavy chains of the ACT35-51_9G12F12C antibody, the ACT35-51_9G12F3E antibody, and the ACT35-51_8H12A7H antibody have the same base sequence and amino acid sequence.
  • the light chains of the ACT35-51_9G12F12C antibody, the ACT35-51_9G12F3E antibody, and the ACT35-51_8H12A7H antibody were identical in amino acid sequence, but differed in nucleotide sequence.
  • the epitopes of monoclonal antibodies confirmed to have cancer cell growth-inhibiting effects are included in the amino acid sequence from positions 61 to 153 from the N-terminus of the MANSC1 protein. It was shown that In contrast, for monoclonal antibodies for which growth inhibitory effects on GCIY and ZR75-1 were not confirmed, these epitopes were located at positions 154 to 186, 187 to 219, or 220 to the N-terminal of the MANSC1 protein. It was shown to be included in the amino acid sequence at position 250.
  • an antibody that binds to an amino acid sequence contained in positions 61 to 153 from the N-terminus of the MANSC1 protein can be suitably used as an antibody having anticancer activity.
  • the amino acid sequence from position 33 to position 117 from the N-terminus of the MANSC1 protein is called a MANSC domain, which is a motif containing seven highly conserved cysteine sequences (motif at N terminus with seven cysteines).
  • the monoclonal antibody of the present invention Since the monoclonal antibody of the present invention has excellent anticancer activity, it can be used for treatment or prevention of cancer. In particular, it has a strong cell growth inhibitory effect on gastric cancer, glioma and breast cancer.
  • the monoclonal antibody of the present invention is extremely useful for medical treatment because it is considered to have an excellent effect on Skills gastric cancer, which is very malignant and has been difficult to treat.
  • the monoclonal antibody of the present invention can be applied to cancer diagnosis and cancer cell detection / selection.

Abstract

From a cDNA library derived from a cancer cell line, a cDNA encoding a protein that is expressed on cell surface or secreted by a cell is screened by using the SST-REX method. A monoclonal antibody against the protein encoded by the cDNA thus screened is constructed and the anticancer activity thereof is examined in vitro and in vivo. As a result, a monoclonal antibody capable of binding to MANSC1 protein and having an excellent anticancer activity is found out. Moreover, a region containing an epitope of this antibody in MANSC1 protein is identified and the structures of the light-chain and heavy-chain variable regions of this antibody are successfully determined.

Description

MANSC1蛋白質に結合し、抗癌活性を有する抗体Antibody that binds to MANSC1 protein and has anticancer activity
　本発明は、抗癌活性を有する抗体およびその用途に関する。 The present invention relates to an antibody having anticancer activity and use thereof.
　癌(腫瘍)は、わが国における死亡原因の第一位を占める疾患である。国立がんセンター内癌情報対策センターの統計によると、2006年に癌で死亡した人数はおよそ32万9千例あり、部位別に見ると、男性では肺（23％）、胃（17％）、肝臓（11％）、結腸（7％、大腸と併せると11％）、膵臓（6％）の順となっており、女性では、胃（13％）、肺（13％）、結腸（10％、大腸と併せると14％）、***（9％）、肝臓（8％）の順となっている。癌患者数は年々増加しており、有効性および安全性の高い薬剤や治療法の開発が強く望まれている。 Cancer (tumor) is a disease that occupies the top cause of death in Japan. According to statistics from the National Cancer Center's Cancer Information Center, there were approximately 329 thousand people who died from cancer in 2006. By region, the lungs (23%), stomach (17%), Liver (11%), colon (7%, 11% when combined with large intestine), pancreas (6%), followed by stomach (13%), lung (13%), colon (10%) When combined with the large intestine, 14%), breast (9%), liver (8%). The number of cancer patients is increasing year by year, and the development of highly effective and safe drugs and treatment methods is strongly desired.
　胃癌は、日本において罹患率、死亡率ともに非常に高い癌のひとつであるが、診断方法、および手術による外科的切除や化学療法を主とした治療方法の進歩により、現在では比較的治りやすい癌の1つともされている。しかしながら、スキルス胃癌に関しては、非常に悪性度の高い、治療が困難な胃癌の一つとされている。スキルス胃癌は、癌細胞が粘膜表面に現れずに胃壁全体あるいは半分～1/3以上にびまん性に浸潤し、肉眼的に明らかな腫瘤を形成せずに胃壁の肥厚や硬化をもたらし、病巣と周囲粘膜との境界が不明瞭であるという特徴を有する。スキルス胃癌は、通常の胃癌より発症年齢が低くて進行も早く、診断も困難である。診断がついた時点では、既に腹膜播種・転移を起こして6割が手術できない状態にあり、手術により切除された場合でも、5年生存率は、わずか15～20％である。 Gastric cancer is one of the cancers with very high morbidity and mortality in Japan, but it is now relatively easy to cure due to advances in diagnostic methods and treatment methods such as surgical excision and chemotherapy. It is also one of them. However, regarding Skills gastric cancer, it is regarded as one of very high-grade gastric cancer that is difficult to treat. Skills gastric cancer has cancer cells that do not appear on the mucosal surface and diffusely infiltrate the entire stomach wall or half to more than 1/3, resulting in thickening and hardening of the stomach wall without forming a macroscopically obvious tumor. It has the feature that the boundary with the surrounding mucosa is unclear. Skills gastric cancer has a lower age of onset, progresses faster, and is difficult to diagnose than normal gastric cancer. At the time of diagnosis, 60% of the patients were already inoperable due to peritoneal dissemination and metastasis, and even if resected by surgery, the 5-year survival rate is only 15-20%.
　近年、抗癌剤としての抗体の使用は、種々の病態（癌型）の治療におけるアプローチとして、その重要性が認められつつある。例えば、腫瘍特異的な抗原を標的とした抗体であれば、投与した抗体は腫瘍に集積することが推定されるため、補体依存性細胞傷害活性（CDC）や抗体依存的細胞性細胞傷害活性（ADCC）による、免疫システムを介した癌細胞への攻撃が期待できる。また、抗体に放射性核種や細胞毒性物質などの薬剤を結合しておくことにより、結合した薬剤を効率よく腫瘍部位に送達することが可能となる。これにより、他組織への薬剤到達量を減少させ、ひいては副作用の軽減を見込むことができる。腫瘍特異的抗原に細胞死を誘導する活性がある場合は、アゴニスティックな活性を持つ抗体を投与することで、また、腫瘍特異的抗原が細胞の増殖および生存に関与する場合は、中和活性を持つ抗体を投与することで、腫瘍特異的な抗体の集積と抗体の活性によって、腫瘍の増殖停止または退縮が期待できる。このような特性から、抗体は、抗癌剤として適用に好適であると考えられている。 In recent years, the use of antibodies as anticancer agents is gaining importance as an approach in the treatment of various disease states (cancer types). For example, if an antibody is targeted to a tumor-specific antigen, the administered antibody is presumed to accumulate in the tumor, so complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) can be expected to attack cancer cells via the immune system. Further, by binding a drug such as a radionuclide or a cytotoxic substance to the antibody, the bound drug can be efficiently delivered to the tumor site. As a result, the amount of drug reaching the other tissue can be reduced, and as a result, side effects can be reduced. If the tumor-specific antigen has activity to induce cell death, administer an antibody with agonistic activity, and if the tumor-specific antigen is involved in cell growth and survival, neutralize activity By administering an antibody having a tumor, tumor growth arrest or regression can be expected due to the accumulation of antibody specific to the tumor and the activity of the antibody. From such characteristics, the antibody is considered suitable for application as an anticancer agent.
　これまでに上市された抗体医薬としては、白血病・リンパ腫を対象として、CD20を標的としたrituximab（商品名rituxan）やiburitumomab　ozogamicin(商品名Zevailn)、CD33を標的としたgemutuzumab　ozogamitin（商品名Mylotarg）などが開発されている。また、上皮性固形癌を対象としたものとしては、乳癌では、Her2/neuを標的としたtrastuzumab（商品名Herceptin）やVEGFを標的としたbevacizumab（商品名Avastin）などが開発されている。このほか癌以外を対象疾患とするものとして、関節リウマチやキャッスルマン病に対して、ヒトIL-6受容体抗体であるtocilizumab（商品名Actemula）などが開発されている。 The antibody drugs marketed so far are leukemia and lymphoma targeting CD20 targeting rituximab (trade name rituxan) and iburitumomab ozogamicin (trade name Zevailn), and CD33 targeting gemutuzumab ozogamitin (trade name Mylotarg) Etc. are being developed. For breast cancer, trastuzumab (trade name Herceptin) targeting Her2 / neu and bevacizumab (trade name Avastin) targeting VEGF have been developed for breast cancer. In addition to cancer, other human diseases such as tocilizumab (trade name Actemula), which is a human IL-6 receptor antibody, have been developed for rheumatoid arthritis and Castleman's disease.
　しかしながら、2008年までに認可された抗体医薬は、米国でも20種類程度、日本でも10種類程度であり、特に固形癌に関してはまだ有効とされる抗体医薬は少ない。このため、さらなる有効な抗体医薬の開発が望まれている。 However, the number of antibody drugs approved by 2008 is about 20 in the United States and about 10 in Japan, and there are few antibody drugs that are still effective for solid cancer. For this reason, development of a more effective antibody drug is desired.
　ところで、細胞膜に存在し、細胞外ドメインのN末端側に、高度に保存された7つのシステイン配列を含むモチーフを有する蛋白質として、「Homo　sapiens　MANSC　domain　containing　1」（以下、「MANSC1」と称する）が知られている（MANSCは、motif　at　N　terminus　with　seven　cysteinesの略称）。このモチーフを有するMANSCドメインは、ESTに対するTBLASTNなどを用いた解析により、高等脊椎動物に限らず、軟体動物や脊索動物までの多細胞生物に高度に保存されていることが明らかとなっている。MANSCドメインは、肝細胞成長因子HGFの活性化因子阻害剤であるHAI-1や低密度リポ蛋白質受容体関連因子であるLRP-11などにも存在していることから、これら蛋白質の機能に基づき、組織の発生や再生、アルツハイマーなどの神経関連疾患、腫瘍の分化や転移などに関係している可能性が示唆されている（非特許文献１、非特許文献２、特許文献１）。しかしながら、これら文献においては、MANSC1分子自体の詳細な挙動および機能については開示されていない。 By the way, “Homo sapiens MANSC domain containing 1” (hereinafter referred to as “MANSC1”) is a protein that is present in the cell membrane and has a motif containing seven highly conserved cysteine sequences on the N-terminal side of the extracellular domain. (MANSC is an abbreviation for motif at N terminus with seven cysteines). The MANSC domain having this motif has been clarified to be highly conserved not only in higher vertebrates but also in multicellular organisms from molluscs and chordae by analysis using TBLASTN for EST. The MANSC domain is also present in HAI-1, an activator inhibitor of hepatocyte growth factor HGF, and LRP-11, a low-density lipoprotein receptor-related factor. It has been suggested that it may be related to the development and regeneration of tissues, neurological diseases such as Alzheimer's, tumor differentiation and metastasis (Non-patent Document 1, Non-patent Document 2, and Patent Document 1). However, these documents do not disclose the detailed behavior and function of the MANSC1 molecule itself.
　一方、MANSC1分子の挙動および機能に関しては、黒色腫において発現上昇している遺伝子の網羅的なリストの中の一つとして報告されている。（特許文献２の表15-21、配列番号947）。また、ヒトMANSC1のオルソログをコードする遺伝子の変異により、雌（-/-）マウスにおいて抑鬱様反応の低下がもたらされることが明らかになっている（特許文献３の明細書の段落892）。しかしながら、これらの文献のいずれにも、癌の発症へのMANSC1の寄与（因果関係）については何ら開示されていない。 On the other hand, the behavior and function of the MANSC1 molecule has been reported as one of the comprehensive list of genes whose expression is increased in melanoma. (Table 15-21 of Patent Document 2, SEQ ID NO: 947). It has also been clarified that mutations in the gene encoding the orthologue of human MANSC1 result in a decrease in depression-like response in female (− / −) mice (paragraph 892 of the specification of Patent Document 3). However, none of these documents disclose the contribution (causal relationship) of MANSC1 to the onset of cancer.
　従って、MANSC1に対する抗体が、抗癌活性を持ちうるかについては、いまだ明らかにされていないのが現状である。 Therefore, at present, it has not yet been clarified whether an antibody against MANSC1 can have anticancer activity.
国際公開第2007/66124号International Publication No. 2007/66124 特開2008-504034号公報JP 2008-504034 A 特開2009-527227号公報JP 2009-527227 A
　本発明はこのような状況に鑑みてなされたものであり、その目的は、優れた抗癌活性を有する新規抗体を提供することにある。さらなる本発明の目的は、このような抗体を有効成分とする抗癌剤を提供することにある。 The present invention has been made in view of such a situation, and an object thereof is to provide a novel antibody having excellent anticancer activity. A further object of the present invention is to provide an anticancer agent comprising such an antibody as an active ingredient.
　本発明者らは上記課題を解決すべく、まず、癌細胞株であるGCIY細胞由来のcDNAライブラリを作製し、SST-REX法により、その中から、細胞表面に発現あるいは細胞から分泌される蛋白質をコードするものを選抜した。次いで、選抜したcDNAがコードする蛋白質に対するモノクローナル抗体を作製して、各種癌細胞株に対する結合性、in　vitroおよびin　vivoにおける抗癌活性を検討した。その結果、得られたモノクローナル抗体が、MANSC1蛋白質に結合し、in　vitroおよびin　vivoにおいて優れた抗癌活性を有することを見出した。さらに、本発明者は、MANSC1蛋白質において、これら抗体のエピトープを含む領域を同定すると共に、この抗体の軽鎖および重鎖の可変領域の構造を決定することに成功し、本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventors first prepared a cDNA library derived from a cancer cell line, GCIY cell, and expressed it on the cell surface or secreted from the cell by the SST-REX method. The ones that code are selected. Subsequently, monoclonal antibodies against the protein encoded by the selected cDNA were prepared and examined for binding to various cancer cell lines and in vitro and in vivo anticancer activity. As a result, it was found that the obtained monoclonal antibody binds to the MANSC1 protein and has excellent anticancer activity in vitro and in vivo. Furthermore, the present inventor succeeded in identifying the region containing the epitope of these antibodies in MANSC1 protein and determining the structure of the variable region of the light chain and heavy chain of this antibody, thereby completing the present invention. It came.
　即ち、本発明は、MANSC1蛋白質に結合し、抗癌活性を有するモノクローナル抗体および該抗体を有効成分とする抗癌剤に関し、より詳しくは、
（１）　ヒト由来のMANSC1蛋白質に結合し、かつ、抗癌活性を有する抗体、
（２）　ヒト由来のMANSC1蛋白質の細胞外領域に結合する、（１）に記載の抗体、
（３）　癌が胃癌、グリオーマ、または乳癌である、（１）に記載の抗体、
（４）　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
　（ａ）配列番号：３～５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：６～８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：１０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：１２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（５）　下記（ａ）又は（ｂ）に記載の特徴を有する（１）に記載の抗体
　（ａ）配列番号：３３～３５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：３６～３８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：４０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：４２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（６）　下記（ａ）又は（ｂ）に記載の特徴を有する（１）に記載の抗体
　（ａ）配列番号：４３～４５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：４６～４８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：５０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：５２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（７）　下記（ａ）又は（ｂ）に記載の特徴を有する（１）に記載の抗体
　（ａ）配列番号：５３～５５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：５６～５８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：６０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：６２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（８）　下記（ａ）又は（ｂ）に記載の特徴を有する（１）に記載の抗体
　（ａ）配列番号：６３～６５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：６６～６８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：７０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：７２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（９）　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
　（ａ）配列番号：７３～７５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：７６～７８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：８０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：８２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（１０）　下記（ａ）又は（ｂ）に記載の特徴を有する（１）に記載の抗体
　（ａ）配列番号：８３～８５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：８６～８８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：９０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：９２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（１１）　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
　（ａ）配列番号：９３～９５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：９６～９８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
　（ｂ）配列番号：１０２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：１０４に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する、
（１２）　ヒト由来のMANSC1蛋白質の６１～１５３位のアミノ酸配列からなるペプチド配列に結合する、（１）に記載の抗体、
（１３）　ヒト由来のMANSC1蛋白質における、下記（ａ）～（ｈ）のいずれかに記載の抗体のエピト―プに結合する、（１）に記載の抗体
　（ａ）配列番号：１０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：１２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｂ）配列番号：４０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：４２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｃ）配列番号：５０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：５２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｄ）配列番号：６０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：６２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｅ）配列番号：７０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：７２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｆ）配列番号：８０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：８２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｇ）配列番号：９０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：９２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
　（ｈ）配列番号：１０２に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：１０４に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体、
（１４）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：３～５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：１０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：６～８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：１２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（１５）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：３３～３５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：４０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：３６～３８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：４２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（１６）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：４３～４５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：５０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：４６～４８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：５２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（１７）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：５３～５５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：６０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：５６～５８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：６２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（１８）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：６３～６５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：７０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：６６～６８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：７２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（１９）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：７３～７５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：８０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：７６～７８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：８２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド、
（２０）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：８３～８５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：９０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：８６～８８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：９２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（２１）　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
　（ａ）配列番号：９３～９５に記載のアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｂ）配列番号：１０２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の軽鎖またはその可変領域からなるペプチド
　（ｃ）配列番号：９６～９８に記載のアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド
　（ｄ）配列番号：１０４に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、（１）に記載の抗体の重鎖またはその可変領域からなるペプチド、
（２２）　（１）～（１３）のいずれかに記載の抗体または（１４）～（２１）のいずれかに記載のペプチドをコードするDNA、
（２３）　（１）～（１３）のいずれかに記載の抗体を産生する、または、（２２）に記載のDNAを含む、ハイブリドーマ、
（２４）　（１）～（１３）のいずれかに記載の抗体を有効成分とする、抗癌剤、
および
（２５）　癌が胃癌、グリオーマ、または乳癌である、（２４）に記載の抗癌剤、
を提供するものである。 That is, the present invention relates to a monoclonal antibody that binds to MANSC1 protein and has anticancer activity, and an anticancer agent comprising the antibody as an active ingredient, more specifically,
(1) an antibody that binds to a human-derived MANSC1 protein and has anticancer activity;
(2) The antibody according to (1), which binds to an extracellular region of a human-derived MANSC1 protein,
(3) The antibody according to (1), wherein the cancer is gastric cancer, glioma, or breast cancer,
(4) The antibody according to claim 1 having the characteristics described in the following (a) or (b): (a) the amino acid sequence according to SEQ ID NO: 3 to 5 or at least one of the amino acid sequences, In the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and at least one of the amino acid sequences set forth in SEQ ID NOs: 6 to 8 or the amino acid sequences, one or more (B) the amino acid sequence described in SEQ ID NO: 10 or the signal sequence is removed from the amino acid sequence, the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or inserted. A light chain variable region comprising the determined amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 12, the amino acid sequence from which the signal sequence has been removed, or at least one of these amino acid sequences, Retains a heavy chain variable region comprising an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted;
(5) The antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 33 to 35 or at least one of the amino acid sequences, In the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 36 to 38 or at least one of the amino acid sequences, one or more (B) the amino acid sequence set forth in SEQ ID NO: 40 or the signal sequence was removed from the amino acid sequence, the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, an amino acid sequence set forth in SEQ ID NO: 42, an amino acid sequence in which a signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(6) The antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 43 to 45 or at least one of the amino acid sequences, One or more of the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 46 to 48 or at least one of the amino acid sequences (B) the amino acid sequence shown in SEQ ID NO: 50 or the signal sequence was removed from the amino acid sequence, the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 52, an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(7) The antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 53 to 55 or at least one of the amino acid sequences, One or more of the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 56 to 58 or at least one of the amino acid sequences (B) the amino acid sequence set forth in SEQ ID NO: 60 or the signal sequence was removed from the amino acid sequence, the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 62, an amino acid sequence in which a signal sequence is removed from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(8) The antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 63 to 65 or at least one of the amino acid sequences, In the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and at least one of the amino acid sequences set forth in SEQ ID NOs: 66 to 68 or the amino acid sequences, one or more (B) the amino acid sequence described in SEQ ID NO: 70 or the signal sequence was removed from the amino acid sequence, the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or Is a light chain variable region comprising an inserted amino acid sequence, an amino acid sequence set forth in SEQ ID NO: 72, an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or at least one of these amino acid sequences, 1 or Retains a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(9) The antibody according to claim 1 having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 73 to 75 or at least one of the amino acid sequences, A light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 76 to 78 or at least one of the amino acid sequences. (B) the amino acid sequence described in SEQ ID NO: 80 or the signal sequence was removed from the amino acid sequence, the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or Or at least one of the light chain variable region comprising the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 82, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Or a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(10) The antibody according to (1) having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 83 to 85 or at least one of the amino acid sequences, A light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 86 to 88 or at least one of the amino acid sequences. (B) the amino acid sequence set forth in SEQ ID NO: 90 or the signal sequence was removed from the amino acid sequence, which has the heavy chain variable region comprising the amino acid sequence substituted, deleted, added and / or inserted. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or Or at least one of the light chain variable region comprising the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 92, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Or a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(11) The antibody according to claim 1 having the characteristics described in (a) or (b) below: (a) the amino acid sequence according to SEQ ID NO: 93 to 95 or at least one of the amino acid sequences, One or more of the light chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 96 to 98 or at least one of the amino acid sequences (B) the amino acid sequence of SEQ ID NO: 102 or the signal sequence was removed from the amino acid sequence. In the amino acid sequence or at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added, and In the light chain variable region comprising the inserted amino acid sequence and / or the amino acid sequence set forth in SEQ ID NO: 104, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences, Or a heavy chain variable region comprising an amino acid sequence in which a plurality of amino acids are substituted, deleted, added and / or inserted,
(12) The antibody according to (1), which binds to a peptide sequence consisting of amino acid sequences at positions 61 to 153 of a human-derived MANSC1 protein,
(13) The antibody according to (1), which binds to the epitope of the antibody according to any one of the following (a) to (h) in a human-derived MANSC1 protein (a) according to SEQ ID NO: 10 An antibody having a light chain variable region comprising the amino acid sequence and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and SEQ ID NO: (C) a light chain variable region comprising the amino acid sequence described in SEQ ID NO: 50 and a heavy chain comprising the amino acid sequence described in SEQ ID NO: 52 (D) an antibody retaining a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 60 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 62. (e) a sequence Number: 70 An antibody having a light chain variable region comprising a non-acid sequence and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 72 (f) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 80 and the sequence An antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 90 and a heavy comprising the amino acid sequence of SEQ ID NO: 92 An antibody retaining a chain variable region (h) an antibody retaining a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 102 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104;
(14) The peptide according to any one of the following (a) to (d): (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 3 to 5 or its variable A peptide comprising the region (b) a peptide comprising the light chain of the antibody according to claim 1 or a variable region thereof comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence from which the signal sequence has been removed. c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 6 to 8, and (d) the amino acid sequence according to SEQ ID NO: 12, or from the amino acid sequence A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(15) The peptide according to any one of the following (a) to (d): (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 33 to 35 or a variable thereof A peptide comprising the region (b) a peptide comprising the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or the light chain of the antibody or the variable region thereof according to (1) c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence set forth in SEQ ID NO: 36 to 38; (d) the amino acid sequence set forth in SEQ ID NO: 42 or from the amino acid sequence; A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(16) The peptide according to any one of (a) to (d) below: (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NOs: 43 to 45 or a variable thereof A peptide comprising the region (b) a peptide comprising the amino acid sequence of SEQ ID NO: 50 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or the light chain of the antibody or the variable region thereof according to (1) c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof comprising the amino acid sequence set forth in SEQ ID NO: 46-48 (d) the amino acid sequence set forth in SEQ ID NO: 52 or the amino acid sequence A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(17) The peptide according to any one of (a) to (d) below: (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 53 to 55 or a variable thereof A peptide comprising the region (b) a peptide comprising the amino acid sequence of SEQ ID NO: 60 or the light chain of the antibody or a variable region thereof according to (1), comprising the amino acid sequence of SEQ ID NO: 60 or a signal sequence removed from the amino acid sequence ( c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence set forth in SEQ ID NO: 56 to 58 (d) from the amino acid sequence set forth in SEQ ID NO: 62 or from the amino acid sequence A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(18) The peptide according to any one of (a) to (d) below: (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 63 to 65 or a variable thereof A peptide comprising the region (b) a peptide comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence or the light chain of the antibody or a variable region thereof according to (1) c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof comprising the amino acid sequence set forth in SEQ ID NO: 66 to 68 (d) the amino acid sequence set forth in SEQ ID NO: 72 or the amino acid sequence A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(19) The peptide according to any one of the following (a) to (d): (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 73 to 75 or a variable thereof A peptide comprising the region (b) a peptide comprising the amino acid sequence of SEQ ID NO: 80 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or the light chain of the antibody or the variable region thereof according to (1) c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof comprising the amino acid sequence set forth in SEQ ID NO: 76 to 78 (d) the amino acid sequence set forth in SEQ ID NO: 82 or from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising an amino acid sequence from which a signal sequence has been removed;
(20) The peptide according to any one of the following (a) to (d): (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 83 to 85 or a variable thereof (B) a peptide comprising the antibody light chain or the variable region thereof according to (1), comprising the amino acid sequence represented by SEQ ID NO: 90 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence (b) c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence set forth in SEQ ID NO: 86 to 88. (d) the amino acid sequence set forth in SEQ ID NO: 92 or the amino acid sequence. A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(21) The peptide according to any one of (a) to (d) below: (a) the light chain of the antibody according to (1) comprising the amino acid sequence according to SEQ ID NO: 93 to 95 or a variable thereof (B) a peptide comprising the amino acid sequence of SEQ ID NO: 102 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or a peptide comprising the variable region thereof (1) c) a peptide comprising the heavy chain of the antibody according to (1) or the variable region thereof, comprising the amino acid sequence set forth in SEQ ID NO: 96 to 98; (d) the amino acid sequence set forth in SEQ ID NO: 104 or from the amino acid sequence; A peptide comprising the heavy chain of the antibody according to (1) or a variable region thereof, comprising an amino acid sequence from which the signal sequence has been removed;
(22) DNA encoding the antibody according to any one of (1) to (13) or the peptide according to any one of (14) to (21),
(23) A hybridoma that produces the antibody according to any one of (1) to (13) or contains the DNA according to (22),
(24) An anticancer agent comprising the antibody according to any one of (1) to (13) as an active ingredient,
And (25) the anticancer agent according to (24), wherein the cancer is gastric cancer, glioma, or breast cancer,
Is to provide.
　本発明により、ヒト由来のMANSC1蛋白質に結合し、in　vitroおよびin　vivoにおいて優れた抗癌活性を有する抗体が提供された。本発明の抗体を用いれば、癌の治療や予防が可能となる。本発明の抗体は、特に、胃癌細胞、グリオーマ、または乳癌細胞の増殖抑制に効果的である。 According to the present invention, an antibody that binds to a human-derived MANSC1 protein and has excellent anticancer activity in vitro and in vivo is provided. Use of the antibody of the present invention makes it possible to treat or prevent cancer. The antibody of the present invention is particularly effective in suppressing the growth of gastric cancer cells, gliomas, or breast cancer cells.
ACT35-51_1B4A7D抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（Ａ）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（Ｂ）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_1B4A7D antibody and Ba / F3 cell which expresses a MANSC1 gene. Analysis of antibody responses to transfectant Ba / F3 cells (A) expressing the full-length MANSC1 gene (A) and control Ba / F3 cells (B) not expressing the MANSC1 gene using a flow cytometer did. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_1B4A7D抗体と、胃癌細胞株（GCIY）との反応性をフローサイトメーターで解析した結果を示す図である。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は陰性対照として用いたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。It is a figure which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and a gastric cancer cell line (GCIY) with the flow cytometer. The filled histogram portion of each flow cytometer data shows its reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) used as a negative control. ACT35-51_1B4A7D抗体と、各種培養癌細胞表面との反応性を細胞染色で解析した結果を示す顕微鏡写真である。癌細胞株として、膀胱癌細胞株（T24）、前立腺癌細胞株（PC3、Du145）、膵臓癌細胞株（BxPC3、AsPC1）、グリオーマ細胞株（U251、U87MG、T98G）、胃癌細胞株（MKN1、GCIY）を用いた。図左は、Hoechst33342を用いた核染色像、図中は、抗体による染色像、図右は、Hoechst33342を用いた核染色像と抗体による染色像とを重ねた像を示す。It is a microscope picture which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and the surface of various cultured cancer cells by cell staining. Cancer cell lines include bladder cancer cell lines (T24), prostate cancer cell lines (PC3, Du145), pancreatic cancer cell lines (BxPC3, AsPC1), glioma cell lines (U251, U87MG, T98G), gastric cancer cell lines (MKN1, GCIY) was used. The left figure shows a nuclear stained image using Hoechst33342, the figure shows an antibody-stained image, and the right figure shows an image obtained by superimposing a nuclear stained image using Hoechst33342 and an antibody-stained image. ACT35-51_1B4A7D抗体と、細胞固定および膜透過処理を行った各種培養癌細胞との反応性を細胞染色で解析した結果を示す顕微鏡写真である。癌細胞株として、膀胱癌細胞株（T24）、前立腺癌細胞株（PC3、Du145）、膵臓癌細胞株（BxPC3、AsPC1）、グリオーマ細胞株（U251、U87MG、T98G）、胃癌細胞株（MKN1、GCIY）を用いた。図左は、Hoechst33342を用いた核染色像、図中は、抗体による染色像、図右は、Hoechst33342を用いた核染色像と抗体による染色像とを重ねた像を示す。It is a microscope picture which shows the result of having analyzed the reactivity of ACT35-51_1B4A7D antibody and various cultured cancer cells which performed the cell fixation and the membrane permeation process by cell staining. Cancer cell lines include bladder cancer cell lines (T24), prostate cancer cell lines (PC3, Du145), pancreatic cancer cell lines (BxPC3, AsPC1), glioma cell lines (U251, U87MG, T98G), gastric cancer cell lines (MKN1, GCIY) was used. The left figure shows a nuclear stained image using Hoechst33342, the figure shows an antibody-stained image, and the right figure shows an image obtained by superimposing a nuclear stained image using Hoechst33342 and an antibody-stained image. MANSC1遺伝子を発現している293T細胞の培養上清に対して、ACT35-51_1B4A7D抗体を用いて免疫沈降を行った結果を示す電気泳動写真である。陰性対照細胞として、ベクターのみを導入した293T細胞（モック）を用いた。また、陰性対照抗体としてマウスIgG2aを用いた。It is an electrophoresis photograph showing the result of immunoprecipitation using the ACT35-51_1B4A7D antibody on the culture supernatant of 293T cells expressing the MANSC1 gene. As negative control cells, 293T cells (mock) into which only the vector was introduced were used. Moreover, mouse IgG2a was used as a negative control antibody. ACT35-51_1B4A7Dモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。縦軸はWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。対象癌細胞株として、膀胱癌細胞株（T24）、前立腺癌細胞株（PC3、Du145）を用いた。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1. Bladder cancer cell lines (T24) and prostate cancer cell lines (PC3, Du145) were used as the target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_1B4A7Dモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。縦軸はWST-1添加3時間後のO.D.(O.D.450nm-O.D.630 nm)値を示す。対象癌細胞株として、膵臓癌細胞株（BxPC3、AsPC1）、胃癌細胞株（GCIY）を用いた。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after addition of WST-1. Pancreatic cancer cell lines (BxPC3, AsPC1) and gastric cancer cell lines (GCIY) were used as target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_1B4A7Dモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。縦軸はWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。対象癌細胞株として、グリオーマ細胞株（U251、U87MG、T98G）を用いた。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_1B4A7D monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1. Glioma cell lines (U251, U87MG, T98G) were used as the target cancer cell lines. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_1B4A7D抗体を投与したマウス担癌モデルにおける腫瘍体積推移を示す図である。対照として生理食塩水を、陽性対照としてTaxotereを用いた。It is a figure which shows the tumor volume transition in the mouse | mouth cancer bearing model which administered ACT35-51_1B4A7D antibody. Saline was used as a control and Taxotere was used as a positive control. ACT35-51_1B4A7D抗体の投与３週間後のマウス担癌モデルにおける摘出腫瘍重量を示す図である。対照として生理食塩水を、陽性対照としてTaxotereを用いた。It is a figure which shows the excised tumor weight in the mouse | mouth cancer bearing model 3 weeks after administration of ACT35-51_1B4A7D antibody. Saline was used as a control and Taxotere was used as a positive control. ACT35-51_1B4A7D抗体を投与したマウス担癌モデルにおける体重推移を示す図である。対照として生理食塩水を、陽性対照としてTaxotereを用いた。It is a figure which shows the body weight transition in the mouse | mouth cancer bearing model which administered ACT35-51_1B4A7D antibody. Saline was used as a control and Taxotere was used as a positive control. ACT35-51_1B4A7D抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_1B4A7D antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. ACT35-51_1B4A7D抗体と、様々な長さのMANSC1遺伝子を発現するトランスフェクタントBa/F3細胞との反応性を示す図である。抗原であるMANSC1分子のN末端より60アミノ酸、153アミノ酸、186アミノ酸、219アミノ酸、250アミノ酸、280アミノ酸、385アミノ酸に対応する遺伝子を発現するBa/F3細胞に対する、ACT35-51_1B4A7D抗体および対照としてのMPL抗体の反応性を、フローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、各MANSC1分子に対する抗体との反応を、白のヒストグラム部分は対照として用いたマウスIgG2a（ベックマン・コールター、#731589）の反応を示す。It is a figure which shows the reactivity of the ACT35-51_1B4A7D antibody and the transfectant Ba / F3 cell which expresses MANSC1 gene of various length. ACT35-51_1B4A7D antibody against Ba / F3 cells expressing genes corresponding to 60, 153, 186, 219, 250, 280, and 385 amino acids from the N-terminus of the antigen MANSC1 molecule and as a control The reactivity of the MPL antibody was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the antibody against each MANSC1 molecule, and the white histogram portion shows the response of mouse IgG2a (Beckman Coulter, # 731589) used as a control. ACT35-51_1B4A7D抗体とMANSC1-Fc融合タンパク質との相互作用をProteOnによりモニターした結果を示すグラフである。It is a graph which shows the result of having monitored interaction with ACT35-51_1B4A7D antibody and MANSC1-Fc fusion protein by ProteOn. ACT35-51_3C3F3抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_3C3F3 antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_5D6C11抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_5D6C11 antibody and the Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_5E2H6抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_5E2H6 antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_7D9C8抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_7D9C8 antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_8G11B7抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2b（ベックマン・コールター、#731597）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_8G11B7 antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2b (Beckman Coulter, # 731597) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_2C4E8C抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_2C4E8C antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_9G12F12C抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_9G12F12C antibody and Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_9G12F3E抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2a（ベックマン・コールター、#731589）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_9G12F3E antibody and the Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2a (Beckman Coulter, # 731589) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_8H12A7H抗体と、MANSC1遺伝子を発現するBa/F3細胞との反応性を示す図である。免疫原細胞であるMANSC1全長遺伝子を発現するトランスフェクタントBa/F3細胞（vs ACT00035-0051）と、MANSC1遺伝子を発現していない対照Ba/F3細胞（vs Ba/F3）に対する各抗体の反応をフローサイトメーターで解析した。各フローサイトメーターデータの塗りつぶしヒストグラム部分は、それそれのサンプル抗体との反応を、白のヒストグラム部分は、陰性対照としたマウスIgG2b（ベックマン・コールター、#731597）との反応を示す。陽性対照として、抗MPL抗体を用いた。It is a figure which shows the reactivity of ACT35-51_8H12A7H antibody and the Ba / F3 cell which expresses a MANSC1 gene. Response of each antibody to transfectant Ba / F3 cells (vs ACT00035-0051) expressing the full-length MANSC1 gene, which is an immunogen, and control Ba / F3 cells (vs Ba / F3) not expressing the MANSC1 gene Was analyzed with a flow cytometer. The filled histogram portion of each flow cytometer data shows the reaction with the sample antibody, and the white histogram portion shows the reaction with mouse IgG2b (Beckman Coulter, # 731597) as a negative control. An anti-MPL antibody was used as a positive control. ACT35-51_3C3F3モノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_3C3F3 monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_5D6C11モノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_5D6C11 monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_5E2H6モノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_5E2H6 monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_7D9C8モノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_7D9C8 monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_8G11B7モノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2b（MBL、#M077-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_8G11B7 monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2b (MBL, # M077-3) was used as a negative control antibody. ACT35-51_2C4E8Cモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。縦軸はWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。対象癌細胞株として、胃癌細胞株（GCIY）を用いた。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_2C4E8C monoclonal antibody has on the proliferation of cancer cells. The vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1. A gastric cancer cell line (GCIY) was used as the target cancer cell line. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_9G12F12Cモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of the ACT35-51_9G12F12C monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_9G12F3Eモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2a（MBL、#M076-3）を用いた。It is a figure which shows the result analyzed with the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_9G12F3E monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2a (MBL, # M076-3) was used as a negative control antibody. ACT35-51_8H12A7Hモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。縦軸はWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。対象癌細胞株として、胃癌細胞株（GCIY)を用いた。陰性対照抗体として、マウスIgG2b（MBL、#M077-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_8H12A7H monoclonal antibody. The vertical axis represents the O.D. (O.D.450 nm-O.D.630 nm) value 3 hours after the addition of WST-1. A gastric cancer cell line (GCIY) was used as the target cancer cell line. Mouse IgG2b (MBL, # M077-3) was used as a negative control antibody. ACT35-51_2F9Bモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG3（MBL、#M078-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_2F9B monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG3 (MBL, # M078-3) was used as a negative control antibody. ACT35-51_5C4Fモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgM（MBL、#M079-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_5C4F monoclonal antibody has on the proliferation of cancer cells. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgM (MBL, # M079-3) was used as a negative control antibody. ACT35-51_6F6Cモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG1（MBL、#M075-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which the ACT35-51_6F6C monoclonal antibody has on the proliferation of cancer cells. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG1 (MBL, # M075-3) was used as a negative control antibody. ACT35-51_8F7Cモノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgG2b（MBL、#M077-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_8F7C monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgG2b (MBL, # M077-3) was used as a negative control antibody. ACT35-51_1A7H7モノクローナル抗体の癌細胞の増殖に与える影響について、MTTアッセイで解析した結果を示す図である。対象癌細胞株として、胃癌細胞株（GCIY）、乳癌細胞株（ZR75-1）を用いた。縦軸は、胃癌細胞株（GCIY）においてはWST-1添加3時間後のO.D.(O.D.450nm-O.D.630nm)値を示し、乳癌細胞株（ZR75-1）においてはWST-1添加4時間後のO.D.(O.D.450nm-O.D.630nm)値を示す。陰性対照抗体として、マウスIgM（MBL、#M079-3）を用いた。It is a figure which shows the result analyzed by the MTT assay about the influence which it has on the proliferation of the cancer cell of ACT35-51_1A7H7 monoclonal antibody. Gastric cancer cell lines (GCIY) and breast cancer cell lines (ZR75-1) were used as the target cancer cell lines. The vertical axis shows the OD (OD450nm-OD630nm) value 3 hours after WST-1 addition in the gastric cancer cell line (GCIY), and 4 hours after WST-1 addition in the breast cancer cell line (ZR75-1). OD (OD450nm-OD630nm) value is shown. Mouse IgM (MBL, # M079-3) was used as a negative control antibody. ACT35-51_3C3F3抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_3C3F3 antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. ACT35-51_5D6C11抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_5D6C11 antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by solid lines. ACT35-51_5E2H6抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_5E2H6 antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. ACT35-51_7D9C8抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_7D9C8 antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by solid lines. ACT35-51_8G11B7抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_8G11B7 antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. ACT35-51_2C4E8C抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_2C4E8C antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by a solid line. ACT35-51_9G12F12C抗体、ACT35-51_9G12F3E抗体、及びACT35-51_8H12A7H抗体の可変領域のアミノ酸配列とCDR予測を示した図である。CDR予測の結果を破線で、軽鎖および重鎖のシグナル配列を実線で示す。It is the figure which showed the amino acid sequence and CDR prediction of the variable region of ACT35-51_9G12F12C antibody, ACT35-51_9G12F3E antibody, and ACT35-51_8H12A7H antibody. The result of CDR prediction is indicated by a broken line, and the light chain and heavy chain signal sequences are indicated by solid lines.
　本発明は、ヒト由来のMANSC1蛋白質に結合し、抗癌活性を有する抗体を提供する。本発明における「抗体」は、免疫グロブリンのすべてのクラスおよびサブクラスを含む。「抗体」には、ポリクローナル抗体、モノクローナル抗体が含まれ、また、抗体の機能的断片の形態も含む意である。「ポリクローナル抗体」は、異なるエピトープに対する異なる抗体を含む抗体調製物である。また、「モノクローナル抗体」とは、実質的に均一な抗体の集団から得られる抗体（抗体断片を含む）を意味する。ポリクローナル抗体とは対照的に、モノクローナル抗体は、抗原上の単一の決定基を認識するものである。本発明の抗体は、好ましくはモノクローナル抗体である。本発明の抗体は、自然環境の成分から分離され、および／または回収された（即ち、単離された）抗体である。 The present invention provides an antibody that binds to a human-derived MANSC1 protein and has anticancer activity. The “antibody” in the present invention includes all classes and subclasses of immunoglobulins. “Antibody” includes polyclonal antibodies and monoclonal antibodies, and also includes forms of functional fragments of antibodies. “Polyclonal antibodies” are antibody preparations comprising different antibodies directed against different epitopes. The “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population. In contrast to polyclonal antibodies, monoclonal antibodies are those that recognize a single determinant on an antigen. The antibody of the present invention is preferably a monoclonal antibody. The antibodies of the invention are antibodies that have been separated and / or recovered (ie, isolated) from components of the natural environment.
　本発明の抗体が結合する「ヒト由来のMANSC1蛋白質（NCBI　Reference　Sequence：NM_018050.2）」は、細胞膜に存在し、細胞外ドメインのN末端側に、高度に保存された7つのシステイン配列を含むモチーフを有する蛋白質である。ヒト由来のMANSC1蛋白質は、431アミノ酸配列からなる蛋白質であり、そのうち、N末端から26アミノ酸部分をシグナル配列、27番目から385番目までを細胞外領域、386番目から408番目までを膜貫通領域、409番目以降を膜内領域とする膜貫通型蛋白質であると推定される。典型的なヒト由来のMANSC1蛋白質のアミノ酸配列を配列番号：２に、MANSC1遺伝子の塩基配列を配列番号：１に示す。ヒト由来のMANSC1蛋白質は、このような典型的なアミノ酸配列を有するもの以外に、天然においてアミノ酸が変異したものも存在しうる。従って、本発明における「ヒト由来のMANSC1蛋白質」は、好ましくは、配列番号：２に記載のアミノ酸配列からなる蛋白質であるが、それ以外に、配列番号：２で表されるアミノ酸配列において、1もしくは複数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列からなるものも含まれる。アミノ酸配列の置換、欠失、挿入もしくは付加は、一般的には、10アミノ酸以内（例えば、5アミノ酸以内、3アミノ酸以内、1アミノ酸）である。 The “human-derived MANSC1 protein (NCBI Reference Sequence: NM_018050.2)” to which the antibody of the present invention binds is present in the cell membrane and contains seven highly conserved cysteine sequences on the N-terminal side of the extracellular domain. A protein having a motif. The human-derived MANSC1 protein is a protein consisting of a 431 amino acid sequence, of which the 26 amino acid portion from the N-terminal is a signal sequence, the 27th to 385th extracellular region, the 386th to 408th is a transmembrane region, It is presumed to be a transmembrane protein having the 409th and subsequent regions in the intramembrane region. The amino acid sequence of a typical human-derived MANSC1 protein is shown in SEQ ID NO: 2, and the nucleotide sequence of the MANSC1 gene is shown in SEQ ID NO: 1. A human-derived MANSC1 protein may naturally have a mutated amino acid in addition to such a typical amino acid sequence. Therefore, the “human-derived MANSC1 protein” in the present invention is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, but in addition to that, in the amino acid sequence represented by SEQ ID NO: 2, Or what consists of an amino acid sequence by which several amino acid was substituted, deleted, inserted, or added is also contained. The substitution, deletion, insertion or addition of the amino acid sequence is generally within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 1 amino acid).
　本発明において「抗癌活性」とは、in　vitroおよび/またはin　vivoにおいて、癌細胞の増殖を抑制する活性を意味する。抗癌活性は、例えば、実施例８に記載のMTTアッセイあるいは実施例９に記載の担癌モデルを用いた解析により評価することができる。本発明の抗体の好ましい態様は、実施例８に記載のMTTアッセイを行った場合に、抗体添加72時間後において、胃癌細胞株（例えば、GCIY）の増殖を、対照と比較して、50％以上（例えば、60％以上、70%以上）抑制する抗体である。本発明の抗体の他の好ましい態様は、実施例８に記載のMTTアッセイを行った場合に、抗体添加72時間後において、グリオーマ細胞株（例えば、T98G）の増殖を、対照と比較して、50％以上（例えば、60％以上、70%以上、80％以上、90％以上）抑制する抗体である。本発明の抗体の他の好ましい態様は、実施例９に記載の担癌モデルを用いた解析により、抗体投与3週間後において、腫瘍体積を、対照と比較して、30％以上（例えば、35％以上、40％以上、45％以上、50％以上、55％以上）減少させる抗体である。本発明の抗体の他の好ましい態様は、実施例９に記載の担癌モデルを用いた解析により、抗体投与3週間後において、摘出腫瘍重量を、対照と比較して、20％以上（例えば、25％以上、30％以上、35％以上）減少させる抗体である。抗癌剤として用いる場合、これら抗体は、さらに、投与対象の体重を減少させないという特性を持つことが好ましい。本発明の抗体は、上記の活性を複数併せ持つことが特に好ましい。 In the present invention, “anticancer activity” means an activity of suppressing the growth of cancer cells in vitro and / or in vivo. The anticancer activity can be evaluated, for example, by analysis using the MTT assay described in Example 8 or the cancer-bearing model described in Example 9. A preferred embodiment of the antibody of the present invention shows that when the MTT assay described in Example 8 is performed, the growth of a gastric cancer cell line (eg, GCIY) is 50% compared to the control after 72 hours of antibody addition. An antibody that suppresses the above (for example, 60% or more, 70% or more). Another preferred embodiment of the antibody of the present invention is that when the MTT assay described in Example 8 is performed, the proliferation of a glioma cell line (for example, T98G) is 72 hours after the addition of the antibody compared to the control. It is an antibody that suppresses 50% or more (for example, 60% or more, 70% or more, 80% or more, 90% or more). In another preferred embodiment of the antibody of the present invention, the analysis using the cancer-bearing model described in Example 9 shows that the tumor volume is 30% or more (for example, 35 % Or more, 40% or more, 45% or more, 50% or more, 55% or more). In another preferred embodiment of the antibody of the present invention, an analysis using the cancer-bearing model described in Example 9 shows that the weight of the excised tumor is 20% or more (for example, 25% or more, 30% or more, 35% or more). When used as an anticancer agent, these antibodies preferably further have the property of not reducing the body weight of the administration subject. It is particularly preferred that the antibody of the present invention has a plurality of the above activities.
　本発明の抗体の他の好ましい態様は、ヒト由来のMANSC1蛋白質に対して高い親和性を示す抗体であり、精製MANSC1タンパク質を用いる実施例１２に記載の表面プラズモン共鳴（surface plasmon Resonance、SPR）法によって得られる解離定数の値が、好ましくは１０－９以下の抗体であり、より好ましくは５×１０－１０以下の抗体である。 Another preferred embodiment of the antibody of the present invention is an antibody having a high affinity for human-derived MANSC1 protein, and the surface plasmon resonance (SPR) method described in Example 12 using purified MANSC1 protein. The value of the dissociation constant obtained by is preferably 10 −9 or less, more preferably 5 × 10 −10 or less.
　本発明の抗体の他の好ましい態様は、軽鎖CDR1～CDR3（配列番号：３～配列番号：５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：６～配列番号：８に記載のアミノ酸配列）を含む重鎖可変領域を保持する抗体、
軽鎖CDR1～CDR3（配列番号：３３～配列番号：３５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：３６～配列番号：３８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体、
軽鎖CDR1～CDR3（配列番号：４３～配列番号：４５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：４６～配列番号：４８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体、
軽鎖CDR1～CDR3（配列番号：５３～配列番号：５５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：５６～配列番号：５８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体、
軽鎖CDR1～CDR3（配列番号：６３～配列番号：６５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：６６～配列番号：６８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体、
軽鎖CDR1～CDR3（配列番号：７３～配列番号：７５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：７６～配列番号：７８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体、
軽鎖CDR1～CDR3（配列番号：８３～配列番号：８５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：８６～配列番号：８８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体、
軽鎖CDR1～CDR3（配列番号：９３～配列番号：９５に記載のアミノ酸配列）を含む軽鎖可変領域と、重鎖CDR1～CDR3（配列番号：９６～配列番号：９８に記載のアミノ酸配列）を含む重鎖可変領域とを保持する抗体である。 In another preferred embodiment of the antibody of the present invention, a light chain variable region comprising light chains CDR1 to CDR3 (amino acid sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 5) and heavy chain CDR1 to CDR3 (SEQ ID NO: 6). An antibody having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8),
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 33 to SEQ ID NO: 35) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 36 to SEQ ID NO: 38) An antibody having a heavy chain variable region comprising,
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 43 to SEQ ID NO: 45) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 46 to SEQ ID NO: 48) An antibody having a heavy chain variable region comprising,
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 53 to SEQ ID NO: 55) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 56 to SEQ ID NO: 58) An antibody having a heavy chain variable region comprising,
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 63 to SEQ ID NO: 65) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 66 to SEQ ID NO: 68) An antibody having a heavy chain variable region comprising,
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 73 to SEQ ID NO: 75) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 76 to SEQ ID NO: 78) An antibody having a heavy chain variable region comprising,
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 83 to SEQ ID NO: 85) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 86 to SEQ ID NO: 88) An antibody having a heavy chain variable region comprising,
Light chain variable region including light chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 93 to SEQ ID NO: 95) and heavy chain CDR1 to CDR3 (amino acid sequence described in SEQ ID NO: 96 to SEQ ID NO: 98) And a heavy chain variable region containing the antibody.
　例えば、軽鎖可変領域が配列番号：１０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：１２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：４０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：４２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：５０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：５２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：６０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：６２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：７０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：７２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：８０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：８２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：９０に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：９２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体、
軽鎖可変領域が配列番号：１０２に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなり、重鎖可変領域が配列番号：１０４に記載のアミノ酸配列（または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列）からなる抗体が挙げられる。 For example, the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 10 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 12 (or An amino acid sequence in which a signal sequence is removed from the amino acid sequence),
The light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 40 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 42 (or the amino acid). An amino acid sequence in which the signal sequence is removed from the sequence),
The light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 50 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 52 (or the amino acid). An amino acid sequence in which the signal sequence is removed from the sequence),
The light chain variable region is composed of the amino acid sequence set forth in SEQ ID NO: 60 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 62 (or the amino acid). An amino acid sequence in which the signal sequence is removed from the sequence),
The light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 70 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 72 (or the amino acid). An amino acid sequence in which the signal sequence is removed from the sequence),
The light chain variable region is composed of the amino acid sequence set forth in SEQ ID NO: 80 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 82 (or the amino acid). An amino acid sequence in which the signal sequence is removed from the sequence),
The light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 90 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 92 (or the amino acid). An amino acid sequence in which the signal sequence is removed from the sequence),
The light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 102 (or an amino acid sequence obtained by removing the signal sequence from the amino acid sequence), and the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 104 (or the amino acid). An antibody consisting of an amino acid sequence in which the signal sequence is removed from the sequence).
　一旦、上記軽鎖可変領域と重鎖可変領域とからなる抗体が得られた場合、当業者であれば、その抗体が認識するヒト由来のMANSC1蛋白質上のペプチド領域（エピトープ）を特定して、その領域に結合し、かつ、抗癌活性を示す種々の抗体を作製することができる。抗体のエピトープは、ヒト由来のMANSC1蛋白質のアミノ酸配列から得られたオーバーラップする合成オリゴペプチドへの結合を調べるなどの周知の方法によって決定することができる（例えば、Ed　Harlow　and　D.Lane,　Using　Antibodies,　a　Laboratory　Manual,　Cold　Spring　Harbor　Laboratory　Press,、米国特許4708871号）。ファージディスプレイによるペプチドライブラリーをエピトープマッピングに用いることもできる。二つの抗体が同一または立体的に重なり合ったエピトープと結合するかどうかは、競合アッセイ法により決定することができる。本発明の抗体が認識するMANSC1蛋白質上のペプチド領域は、好ましくは、MANSC1蛋白質の細胞外領域である。本発明の抗体が認識するMANSC1蛋白質の細胞外領域は、好ましくは、MANSC1蛋白質のアミノ酸配列の61位から153位の範囲内の領域である。 Once an antibody comprising the light chain variable region and the heavy chain variable region is obtained, those skilled in the art specify a peptide region (epitope) on the human-derived MANSC1 protein recognized by the antibody, Various antibodies that bind to the region and show anticancer activity can be produced. The epitope of the antibody can be determined by a known method such as examining the binding to an overlapping synthetic oligopeptide obtained from the amino acid sequence of MANSC1 protein derived from human (for example, Ed Harlow and D. Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, US Pat. No. 4,088,871). A peptide library by phage display can also be used for epitope mapping. Whether two antibodies bind to the same or sterically overlapping epitopes can be determined by competition assays. The peptide region on the MANSC1 protein recognized by the antibody of the present invention is preferably an extracellular region of the MANSC1 protein. The extracellular region of the MANSC1 protein recognized by the antibody of the present invention is preferably a region within the range of positions 61 to 153 of the amino acid sequence of the MANSC1 protein.
　本発明の抗体には、マウス抗体、キメラ抗体、ヒト化抗体、ヒト抗体、および、これら抗体の機能的断片が含まれる。本発明の抗体を医薬としてヒトに投与する場合は、副作用低減の観点から、キメラ抗体、ヒト化抗体、あるいはヒト抗体が望ましい。 The antibodies of the present invention include mouse antibodies, chimeric antibodies, humanized antibodies, human antibodies, and functional fragments of these antibodies. When the antibody of the present invention is administered to a human as a pharmaceutical, a chimeric antibody, a humanized antibody, or a human antibody is desirable from the viewpoint of reducing side effects.
　本発明において「キメラ抗体」とは、ある種の抗体の可変領域とそれとは異種の抗体の定常領域とを連結した抗体である。キメラ抗体は、例えば、抗原をマウスに免疫し、そのマウスモノクローナル抗体の遺伝子から抗原と結合する抗体可変部（可変領域）を切り出して、ヒト骨髄由来の抗体定常部（定常領域）遺伝子と結合し、これを発現ベクターに組み込んで宿主に導入して産生させることにより取得することができる（例えば、特開平8-280387号公報、米国特許第4816397号公報、米国特許第4816567号公報、米国特許第5807715号公報）。また、本発明において「ヒト化抗体」とは、非ヒト由来の抗体の抗原結合部位（CDR）の遺伝子配列をヒト抗体遺伝子に移植（CDRグラフティング）した抗体であり、その作製方法は、公知である（例えば、EP239400、EP125023、WO90/07861、WO96/02576参照）。本発明において、「ヒト抗体」とは、すべての領域がヒト由来の抗体である。ヒト抗体の作製においては、ヒトB細胞より活性のある抗体の産生をスクリーニングする方法、ファージディスプレイ法、免疫することで、ヒト抗体のレパートリーを生産することが可能なトランスジェニック動物（例えばマウス）を利用すること等が可能である。ヒト抗体の作製手法は、公知である（例えば、Nature,　362：255-258(1993)、Intern.　Rev.　Immunol,　13：65-93(1995)、J.　Mol.　Biol,　222：581-597(1991)、Nature　Genetics,　15：146-156(1997)、Proc.　Natl.　Acad.　Sci.　USA,　97:722-727(2000)、特開平10-146194号公報、特開平10-155492号公報、特許2938569号公報、特開平11-206387号公報、特表平8-509612号公報、特表平11-505107号公報）。 In the present invention, a “chimeric antibody” is an antibody in which a variable region of a certain antibody is linked to a constant region of a heterogeneous antibody. A chimeric antibody, for example, immunizes a mouse with an antigen, cuts out an antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, and binds to a human bone marrow-derived antibody constant region (constant region) gene. Can be obtained by incorporating it into an expression vector and introducing it into a host for production (for example, Japanese Patent Application Laid-Open No. 8-280387, US Pat. No. 4816397, US Pat. No. 4,816,567, US Pat. No. 5807715). In the present invention, the “humanized antibody” is an antibody obtained by transplanting the gene sequence of the antigen-binding site (CDR) of a non-human-derived antibody to a human antibody gene (CDR grafting), and its production method is publicly known. (See, for example, EP239400, EP125023, WO90 / 07861, WO96 / 02576). In the present invention, a “human antibody” is an antibody derived from all regions. In the production of human antibodies, a method for screening production of active antibodies from human B cells, a phage display method, and a transgenic animal (for example, a mouse) capable of producing a repertoire of human antibodies by immunization. It can be used. Methods for producing human antibodies are known (for example, Nature, 362: 255-258 (1993), Intern. Rev. Immunol, 13: 65-93 (1995), J. Mol. Biol, 222: 581-597). (1991), Nature Genetics, 15: 146-156 (1997), Proc. Natl. Acad. Sci. USA, 97: 722-727 (2000), JP 10-146194, JP 10-155492 No. 2938569, JP-A-11-206387, JP-A-8-509612, JP-A-11-505107).
　本発明において抗体の「機能的断片」とは、抗体の一部分（部分断片）であって、ヒト由来のMANSC1蛋白質を特異的に認識するものを意味する。具体的には、Fab、Fab’、F(ab’）2、可変領域断片（Fv）、ジスルフィド結合Fv、一本鎖Fv（scFv）、sc（Fv）2、ダイアボディー、多特異性抗体、およびこれらの重合体などが挙げられる。 In the present invention, the “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a human-derived MANSC1 protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.
　ここで「Fab」とは、１つの軽鎖および重鎖の一部からなる免疫グロブリンの一価の抗原結合断片を意味する。抗体のパパイン消化によって、また、組換え方法によって得ることができる。「Fab'」は、抗体のヒンジ領域の１つまたはそれより多いシステインを含めて、重鎖CH1ドメインのカルボキシ末端でのわずかの残基の付加によって、Fabとは異なる。「F（ab’）2」とは、両方の軽鎖と両方の重鎖の部分からなる免疫グロブリンの二価の抗原結合断片を意味する。 Here, “Fab” means a monovalent antigen-binding fragment of an immunoglobulin composed of one light chain and part of a heavy chain. It can be obtained by papain digestion of antibodies and by recombinant methods. “Fab ′” differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the hinge region of the antibody. "F (ab ') 2" means a divalent antigen-binding fragment of an immunoglobulin that consists of both light chains and parts of both heavy chains.
　「可変領域断片（Fv）」は、完全な抗原認識および結合部位を有する最少の抗体断片である。Fvは、重鎖可変領域および軽鎖可変領域が非共有結合により強く連結されたダイマーである。「一本鎖Fv（scFv）」は、抗体の重鎖可変領域および軽鎖可変領域を含み、これらの領域は、単一のポリペプチド鎖に存在する。「sc（Fv）2」は、2つの重鎖可変領域および２つの軽鎖可変領域をリンカー等で結合して一本鎖にしたものである。「ダイアボディー」とは、二つの抗原結合部位を有する小さな抗体断片であり、この断片は、同一ポリペプチド鎖の中に軽鎖可変領域に結合した重鎖可変領域を含み、各領域は別の鎖の相補的領域とペアを形成している。「多特異性抗体」は、少なくとも２つの異なる抗原に対して結合特異性を有するモノクローナル抗体である。例えば、二つの重鎖が異なる特異性を持つ二つの免疫グロブリン重鎖／軽鎖対の同時発現により調製することができる。 “Variable region fragment (Fv)” is the smallest antibody fragment with complete antigen recognition and binding sites. Fv is a dimer in which a heavy chain variable region and a light chain variable region are strongly linked by a non-covalent bond. “Single-chain Fv (scFv)” comprises the heavy and light chain variable regions of an antibody, and these regions are present in a single polypeptide chain. “Sc (Fv) 2” is a chain formed by joining two heavy chain variable regions and two light chain variable regions with a linker or the like. A “diabody” is a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable region bound to a light chain variable region in the same polypeptide chain, each region comprising a separate It forms a pair with the complementary region of the strand. A “multispecific antibody” is a monoclonal antibody that has binding specificities for at least two different antigens. For example, it can be prepared by co-expression of two immunoglobulin heavy / light chain pairs where the two heavy chains have different specificities.
　本発明においては、本発明において同定されたCDRを含む抗体の軽鎖若しくは重鎖またはそれらの可変領域からなるペプチドを提供する。好ましいペプチドは、配列番号：３から５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
　配列番号：３３から３５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
配列番号：４３から４５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
配列番号：５３から５５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
配列番号：６３から６５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
配列番号：７３から７５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
配列番号：８３から８５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチド、
配列番号：９３から９５に記載のアミノ酸配列を含む本発明の抗体の軽鎖若しくはその可変領域からなるペプチドであり、特に好ましくは、配列番号：１０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：４０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：５０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：６０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：７０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：８０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：９０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：１０２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチドである。 The present invention provides a peptide consisting of the light chain or heavy chain of an antibody comprising the CDR identified in the present invention, or a variable region thereof. A preferred peptide is a peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 3 to 5 or a variable region thereof,
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence of SEQ ID NOs: 33 to 35 or a variable region thereof,
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 43 to 45 or a variable region thereof,
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence of SEQ ID NOs: 53 to 55 or a variable region thereof,
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 63 to 65 or a variable region thereof;
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence of SEQ ID NOs: 73 to 75 or a variable region thereof,
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 83 to 85 or a variable region thereof,
A peptide comprising the light chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 93 to 95 or a variable region thereof, particularly preferably the amino acid sequence set forth in SEQ ID NO: 10 or a signal sequence derived from the amino acid sequence. A peptide comprising an amino acid sequence from which
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 70 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 80 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 90 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 102 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence.
　他の好ましいペプチドは、配列番号：６から８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：３６から３８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：４６から４８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：５６から５８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：６６から６８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：７６から７８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：８６から８８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチド、
配列番号：９６から９８に記載のアミノ酸配列を含む本発明の抗体の重鎖若しくはその可変領域からなるペプチドであり、特に好ましくは、配列番号：１２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：４２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：５２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：６２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：７２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：８２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：９２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチド、
配列番号：１０４に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含むペプチドである。 Another preferred peptide is a peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 8, or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 36 to 38 or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 46 to 48 or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 56 to 58 or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 66 to 68 or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 76 to 78 or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NOs: 86 to 88 or a variable region thereof,
A peptide comprising the heavy chain of the antibody of the present invention comprising the amino acid sequence set forth in SEQ ID NO: 96 to 98 or a variable region thereof, particularly preferably the amino acid sequence set forth in SEQ ID NO: 12 or a signal sequence derived from the amino acid sequence. A peptide comprising an amino acid sequence from which
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 42 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 52 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 62 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 72 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 82 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 92 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence;
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 104 or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence.
　また、これらペプチドを、例えば、リンカー等により連結することで、機能的な抗体を作製することが可能である。 Also, functional antibodies can be produced by linking these peptides with, for example, a linker.
　本発明の抗体には、望ましい活性（抗原への結合活性、抗癌活性、および／または他の生物学的特性）を減少させることなく、そのアミノ酸配列が修飾された抗体が含まれる。本発明の抗体のアミノ酸配列変異体は、本発明の抗体鎖をコードするDNAへの変異導入によって、またはペプチド合成によって作製することができる。そのような修飾には、例えば、本発明の抗体のアミノ酸配列内の残基の置換、欠失、付加および／または挿入を含む。抗体のアミノ酸配列が改変される部位は、改変される前の抗体と同等の活性を有する限り、抗体の重鎖または軽鎖の定常領域であってもよく、また、可変領域（フレームワーク領域およびCDR）であってもよい。CDR以外のアミノ酸の改変は、抗原との結合親和性への影響が相対的に少ないと考えられるが、現在では、CDRのアミノ酸を改変して、抗原へのアフィニティーが高められた抗体をスクリーニングする手法が公知である（PNAS,　102：8466-8471(2005)、Protein　Engineering,　Design　&　Selection,　21：485-493(2008)、国際公開第2002/051870号、J.　Biol.　Chem.,　280：24880-24887(2005)、Protein　Engineering,　Design　&　Selection,　21：345-351(2008)）。 The antibody of the present invention includes an antibody whose amino acid sequence has been modified without reducing the desired activity (antigen binding activity, anticancer activity, and / or other biological properties). An amino acid sequence variant of the antibody of the present invention can be prepared by introducing a mutation into DNA encoding the antibody chain of the present invention or by peptide synthesis. Such modifications include, for example, residue substitutions, deletions, additions and / or insertions within the amino acid sequences of the antibodies of the invention. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR). Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen. Currently, however, the amino acid of the CDR is modified to screen for antibodies with increased affinity for the antigen. Methods are known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002/051870, J. Biol. Chem., 280: 24880-24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).
　改変されるアミノ酸数は、好ましくは、10アミノ酸以内、より好ましくは5アミノ酸以内、最も好ましくは3アミノ酸以内（例えば、2アミノ酸以内、1アミノ酸）である。アミノ酸の改変は、好ましくは、保存的な置換である。本発明において「保存的な置換」とは、化学的に同様な側鎖を有する他のアミノ酸残基で置換することを意味する。化学的に同様なアミノ酸側鎖を有するアミノ酸残基のグループは、本発明の属する技術分野でよく知られている。例えば、酸性アミノ酸（アスパラギン酸およびグルタミン酸）、塩基性アミノ酸（リシン・アルギニン・ヒスチジン）、中性アミノ酸においては、炭化水素鎖を持つアミノ酸（グリシン・アラニン・バリン・ロイシン・イソロイシン・プロリン）、ヒドロキシ基を持つアミノ酸（セリン・トレオニン）、硫黄を含むアミノ酸（システイン・メチオニン）、アミド基を持つアミノ酸（アスパラギン・グルタミン）、イミノ基を持つアミノ酸（プロリン）、芳香族基を持つアミノ酸（フェニルアラニン・チロシン・トリプトファン）で分類することができる。また、「同等の活性を有する」とは、抗原への結合活性または抗癌活性が対象抗体（代表的には、ACT35-51_1B4A7D抗体）と同等（例えば、70％以上、好ましくは80%以上、より好ましくは90%以上）であることを意味する。抗原への結合活性は、例えば、抗原を発現するBa/F3細胞を作製し、抗体サンプルとの反応性をフローサイトメーターで解析することにより評価することができる（実施例４、１１）。また、抗癌活性は、上記した通り、例えば、実施例８に記載のMTTアッセイあるいは実施例９に記載の担癌モデルを用いた解析により評価することができる。 The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid). The amino acid modification is preferably a conservative substitution. In the present invention, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan). Further, “having equivalent activity” means that the antigen binding activity or anticancer activity is equivalent to the target antibody (typically, ACT35-51_1B4A7D antibody) (eg, 70% or more, preferably 80% or more, More preferably 90% or more). The binding activity to the antigen can be evaluated, for example, by preparing Ba / F3 cells expressing the antigen and analyzing the reactivity with the antibody sample with a flow cytometer (Examples 4 and 11). Further, as described above, the anticancer activity can be evaluated by, for example, analysis using the MTT assay described in Example 8 or the cancer-bearing model described in Example 9.
　また、本発明の抗体の改変は、例えば、グリコシル化部位の数または位置を変化させるなどの抗体の翻訳後プロセスの改変であってもよい。これにより、例えば、抗体のADCC活性を向上させることができる。抗体のグリコシル化とは、典型的には、N-結合またはO-結合である。抗体のグリコシル化は、抗体を発現するために用いる宿主細胞に大きく依存する。グリコシル化パターンの改変は、糖生産に関わる特定の酵素の導入または欠失などの公知の方法で行うことができる（特開2008-113663、米国特許第5047335号、米国特許第5510261号、米国特許第5278299号、国際公開第99/54342号）。さらに、本発明においては、抗体の安定性を増加させる等の目的で脱アミド化されるアミノ酸若しくは脱アミド化されるアミノ酸に隣接するアミノ酸を他のアミノ酸に置換することにより脱アミド化を抑制してもよい。また、グルタミン酸を他のアミノ酸へ置換して、抗体の安定性を増加させることもできる。本発明は、こうして安定化された抗体をも提供するものである。 Further, the modification of the antibody of the present invention may be modification of a post-translational process of the antibody, for example, changing the number or position of glycosylation sites. Thereby, for example, the ADCC activity of the antibody can be improved. Antibody glycosylation is typically N-linked or O-linked. Antibody glycosylation is highly dependent on the host cell used to express the antibody. The glycosylation pattern can be modified by a known method such as introduction or deletion of a specific enzyme involved in sugar production (JP 2008-113663, US Pat. No. 5,473,335, US Pat. No. 5,510,261, US Pat. No. 5278299, International Publication No. 99/54342). Furthermore, in the present invention, deamidation is suppressed by substituting an amino acid adjacent to the amino acid deamidated or deamidated with another amino acid for the purpose of increasing the stability of the antibody. May be. Alternatively, glutamic acid can be substituted with other amino acids to increase antibody stability. The present invention also provides the antibody thus stabilized.
　本発明の抗体は、ポリクローナル抗体であれば、抗原（ヒト由来のMANSC1蛋白質、その部分ペプチド、またはこれらを発現する細胞など）で免疫動物を免疫し、その抗血清から、従来の手段（例えば、塩析、遠心分離、透析、カラムクロマトグラフィーなど）によって、精製して取得することができる。また、モノクローナル抗体は、ハイブリドーマ法や組換えDNA法によって作製することができる。 If the antibody of the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (such as a human-derived MANSC1 protein, a partial peptide thereof, or a cell expressing these), and the conventional means (for example, Salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.
　ハイブリドーマ法としては、代表的には、コーラーおよびミルスタインの方法（Kohler　&　Milstein,　Nature,　256：495(1975)）が挙げられる。この方法における細胞融合工程に使用される抗体産生細胞は、抗原（ヒト由来のMANSC1蛋白質、その部分ペプチド、またはこれらを発現する細胞など）で免疫された動物（例えば、マウス、ラット、ハムスター、ウサギ、サル、ヤギ）の脾臓細胞、リンパ節細胞、末梢血白血球などである。免疫されていない動物から予め単離された上記の細胞またはリンパ球などに対して、抗原を培地中で作用させることによって得られた抗体産生細胞も使用することが可能である。ミエローマ細胞としては公知の種々の細胞株を使用することが可能である。抗体産生細胞およびミエローマ細胞は、それらが融合可能であれば、異なる動物種起源のものでもよいが、好ましくは、同一の動物種起源のものである。ハイブリドーマは、例えば、抗原で免疫されたマウスから得られた脾臓細胞と、マウスミエローマ細胞との間の細胞融合により産生され、その後のスクリーニングにより、ヒト由来のMANSC1蛋白質に特異的なモノクローナル抗体を産生するハイブリドーマを得ることができる。ヒト由来のMANSC1蛋白質に対するモノクローナル抗体は、ハイブリドーマを培養することにより、また、ハイブリドーマを投与した哺乳動物の腹水から、取得することができる。 The hybridoma method typically includes the Kohler and Milstein method (Kohler & Milstein, Nature, 256: 495 (1975)). The antibody-producing cells used in the cell fusion step in this method are animals (eg, mice, rats, hamsters, rabbits) immunized with antigens (human-derived MANSC1 protein, partial peptides thereof, or cells expressing these). Monkeys, goats) spleen cells, lymph node cells, peripheral blood leukocytes and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal in a medium. As the myeloma cells, various known cell lines can be used. The antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with antigen and mouse myeloma cells, and subsequent screening produces monoclonal antibodies specific for human-derived MANSC1 protein. Hybridomas can be obtained. Monoclonal antibodies against human-derived MANSC1 protein can be obtained by culturing hybridomas or from ascites of mammals to which hybridomas have been administered.
　組換えDNA法は、上記本発明の抗体またはペプチドをコードするDNAをハイブリドーマやB細胞等からクローニングし、適当なベクターに組み込んで、これを宿主細胞（例えば哺乳類細胞株、大腸菌、酵母細胞、昆虫細胞、植物細胞など）に導入し、本発明の抗体を組換え抗体として産生させる手法である（例えば、P.J.Delves,　Antibody　Production:　Essential　Techniques,　1997　WILEY、P.Shepherd　and　C.　Dean　Monoclonal　Antibodies,　2000　OXFORD　UNIVERSITY　PRESS、Vandamme　A.M.　et　al.,　Eur.　J.　Biochem.　192:767-775(1990)）。本発明の抗体をコードするDNAの発現においては、重鎖または軽鎖をコードするDNAを別々に発現ベクターに組み込んで宿主細胞を形質転換してもよく、重鎖および軽鎖をコードするDNAを単一の発現ベクターに組み込んで宿主細胞を形質転換してもよい（WO94/11523号公報参照）。本発明の抗体は、上記宿主細胞を培養し、宿主細胞内または培養液から分離・精製し、実質的に純粋で均一な形態で取得することができる。抗体の分離・精製は、通常のポリペプチドの精製で使用されている方法を使用することができる。トランスジェニック動物作製技術を用いて、抗体遺伝子が組み込まれたトランスジェニック動物（ウシ、ヤギ、ヒツジまたはブタなど）を作製すれば、そのトランスジェニック動物のミルクから、抗体遺伝子に由来するモノクローナル抗体を大量に取得することも可能である。 In the recombinant DNA method, a DNA encoding the antibody or peptide of the present invention is cloned from a hybridoma, a B cell or the like and incorporated into an appropriate vector, which is then introduced into a host cell (eg, a mammalian cell line, E. coli, yeast cell, insect). Cells, plant cells, etc.) and a method for producing the antibody of the present invention as a recombinant antibody (for example, PJDelves, Antibody Production: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem. 192: 767-775 (1990)). In the expression of the DNA encoding the antibody of the present invention, DNA encoding the heavy chain or the light chain may be separately incorporated into an expression vector to transform the host cell. Host cells may be transformed into a single expression vector (see WO94 / 11523). The antibody of the present invention can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. For the separation and purification of the antibody, the methods used in the usual purification of polypeptides can be used. If transgenic animals (such as cows, goats, sheep or pigs) in which an antibody gene is incorporated are produced using transgenic animal production technology, a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.
　本発明は、上記本発明の抗体またはペプチドをコードするDNA、該DNAを含むベクター、該DNAを保持する宿主細胞、および該宿主細胞を培養し、抗体を回収することを含む抗体の生産方法をも提供するものである。 The present invention provides a DNA encoding the antibody or peptide of the present invention, a vector containing the DNA, a host cell holding the DNA, and a method for producing the antibody comprising culturing the host cell and recovering the antibody Is also provided.
　本発明の抗体は、抗癌活性を有することから、癌の治療または予防に利用することができる。従って、本発明は、本発明の抗体を有効成分とする抗癌剤、および、本発明の抗体の治療上または予防上の有効量を、ヒトを含む哺乳類に投与する工程を含んでなる、癌の治療または予防の方法をも提供するものである。本発明の治療または予防の方法は、ヒト以外にも、例えば、イヌ、ネコ、ウシ、ウマ、ヒツジ、ブタ、ヤギ、ウサギなどを含各種哺乳動物に応用することが可能である。 Since the antibody of the present invention has anticancer activity, it can be used for treatment or prevention of cancer. Therefore, the present invention is a cancer treatment comprising the steps of administering an anticancer agent comprising the antibody of the present invention as an active ingredient and a therapeutically or prophylactically effective amount of the antibody of the present invention to mammals including humans. It also provides a preventive method. The treatment or prevention method of the present invention can be applied to various mammals including dogs, cats, cows, horses, sheep, pigs, goats, rabbits and the like, in addition to humans.
　本実施例において、本発明の抗体は、癌の中でも、特に胃癌細胞、グリオーマ細胞、および乳癌細胞の増殖を強く抑制したことから、胃癌（例えば、スキルス胃癌）やグリオーマや乳癌の治療または予防に特に効果的である。 In this example, the antibodies of the present invention strongly suppressed the growth of gastric cancer cells, glioma cells, and breast cancer cells, among other cancers. It is particularly effective.
　本発明の抗体を有効成分とする抗癌剤は、本発明の抗体と任意の成分、例えば生理食塩水、葡萄糖水溶液またはリン酸塩緩衝液などを含有する組成物の形態で使用することができる。本発明の抗癌剤は、必要に応じて液体または凍結乾燥した形態で製形化しても良く、任意に薬学的に許容される担体もしくは媒体、例えば、安定化剤、防腐剤、等張化剤などを含有させることもできる。 The anticancer agent comprising the antibody of the present invention as an active ingredient can be used in the form of a composition containing the antibody of the present invention and an optional component such as physiological saline, sucrose aqueous solution or phosphate buffer. The anticancer agent of the present invention may be formed into a liquid or lyophilized form as necessary, and optionally a pharmaceutically acceptable carrier or medium, such as a stabilizer, preservative, isotonic agent, etc. Can also be included.
　薬学的に許容される担体としては、凍結乾燥した製剤の場合、マンニトール、ラクトース、サッカロース、ヒトアルブミンなどを例として挙げることができ、液状製剤の場合には、生理食塩水、注射用水、リン酸塩緩衝液、水酸化アルミニウムなどを例として挙げることができるが、これらに限定されるものではない。 Examples of the pharmaceutically acceptable carrier include mannitol, lactose, saccharose, human albumin and the like in the case of a lyophilized preparation. In the case of a liquid preparation, physiological saline, water for injection, phosphoric acid, etc. Examples thereof include, but are not limited to, a salt buffer and aluminum hydroxide.
　抗癌剤の投与方法は、投与対象の年齢、体重、性別、健康状態などにより異なるが、経口投与、非経口投与（例えば、静脈投与、動脈投与、局所投与）のいずれかの投与経路で投与することができる。好ましい投与方法は、非経口投与である。抗癌剤の投与量は、患者の年齢、体重、性別、健康状態、癌の進行の程度および投与する抗癌剤の成分により変動しうるが、一般的に静脈内投与の場合、成人には体重1kg当たり1日0.1～1000 mg、好ましくは１～100 mgである。 The administration method of the anticancer agent varies depending on the age, weight, sex, health status, etc. of the administration subject, but it should be administered by any of the administration routes of oral administration or parenteral administration (eg, intravenous administration, arterial administration, local administration). Can do. A preferred method of administration is parenteral administration. The dose of an anticancer drug may vary depending on the patient's age, weight, sex, health status, degree of cancer progression, and the components of the anticancer drug to be administered. The daily dose is 0.1 to 1000 mg, preferably 1 to 100 mg.
　本発明の抗体は、癌の治療や予防のみならず、癌の診断への応用も考えられる。本発明の抗体を癌の診断に用いる場合あるいは癌の治療における腫瘍部位の検出に用いる場合、本発明の抗体は、標識したものであってもよい。標識としては、例えば、放射性物質、蛍光色素、化学発光物質、酵素、補酵素を用いることが可能であり、具体的には、ラジオアイソトープ、フルオレセイン、ローダミン、ダンシルクロリド、ルシフェラーゼ、ペルオキシダーゼ、アルカリフォスファターゼ、リゾチーム、ビオチン／アビジンなどが挙げられる。本発明の抗体を診断剤として調剤するには、合目的な任意の手段を採用して任意の剤型でこれを得ることができる。例えば、精製した抗体についてその抗体価を測定し、適当にPBS（Phosphate　buffer　saline,生理食塩を含むリン酸緩衝液）等で希釈した後、0.1％アジ化ナトリウム等を防腐剤として加えることができる。また、例えば、ラテックス等に本発明の抗体を吸着させたものについて抗体価を求め、適当に希釈し、防腐剤を添加して用いることもできる。 The antibody of the present invention can be applied not only to cancer treatment and prevention but also to cancer diagnosis. When the antibody of the present invention is used for diagnosis of cancer or used for detection of a tumor site in cancer treatment, the antibody of the present invention may be labeled. As the label, for example, a radioactive substance, a fluorescent dye, a chemiluminescent substance, an enzyme, and a coenzyme can be used. Specifically, radioisotope, fluorescein, rhodamine, dansyl chloride, luciferase, peroxidase, alkaline phosphatase, Examples include lysozyme and biotin / avidin. In order to prepare the antibody of the present invention as a diagnostic agent, it can be obtained in any dosage form by employing any suitable means. For example, the antibody titer of a purified antibody can be measured and appropriately diluted with PBS (Phosphate buffer saline, phosphate buffer containing physiological saline) or the like, and then 0.1% sodium azide or the like can be added as a preservative. . In addition, for example, the antibody titer of a substance obtained by adsorbing the antibody of the present invention on latex or the like can be obtained, diluted appropriately, and added with a preservative.
　また、本発明において、MANSC1蛋白質に対する抗体が抗癌活性を有することが判明したことから、MANSC1蛋白質またはその部分ペプチドを癌ワクチンとして、ヒトを含む哺乳動物に投与することも可能である(例えば、特開2007-277251、特開2006-052216を参照のこと）。本発明は、このような癌ワクチン用途に用いられる、MANSC1蛋白質またはその部分ペプチドを含む癌ワクチン組成物をも提供するものである。製剤化する場合には、上記本発明の抗癌剤と同様に、薬学的に許容される担体もしくは媒体、例えば、安定化剤、防腐剤、等張化剤などを含有させることができる。 In the present invention, since it has been found that an antibody against MANSC1 protein has anticancer activity, MANSC1 protein or a partial peptide thereof can be administered as a cancer vaccine to mammals including humans (for example, (See JP 2007-277251, JP 2006-052216). The present invention also provides a cancer vaccine composition containing MANSC1 protein or a partial peptide thereof used for such cancer vaccine applications. In the case of formulating, a pharmaceutically acceptable carrier or medium such as a stabilizer, preservative, isotonic agent and the like can be contained in the same manner as the anticancer agent of the present invention.
　以下、本発明を実施例により、さらに詳細に説明するが、本発明はこれら実施例に制限されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
　（実施例１）　SST-REXの実施
　スキルス胃癌株化細胞GCIY細胞細胞表面に発現している膜あるいは分泌遺伝子情報を網羅的に得るためにSST-REXを実施した。 (Example 1) Implementation of SST-REX SST-REX was performed in order to obtain comprehensive information on membrane or secretory genes expressed on the cell surface of Skills gastric cancer cell line GCIY cells.
　（１）cDNAの作製
　GCIY細胞2×107個をTrizol(invitrogen、#15596-026)1mlに懸濁して5分放置し、クロロフォルムを200μl添加して15秒間懸濁後、12,000×gで15分間遠心した。この遠心後の上清と500μlのイソプロパノールを混ぜ合わせた後、12,000×gで10分間遠心した。得られたペレットを80％エタノールで洗浄し、全RNA　200μg以上を得て、以後の実験に供した。 (1) Preparation of cDNA 7 GCIY cells (2 × 10 7 cells) are suspended in 1 ml of Trizol (invitrogen, # 15596-026) and left for 5 minutes. After adding 200 μl of chloroform and suspending for 15 seconds, 15 × 12,000 × g Centrifuge for minutes. The supernatant after centrifugation and 500 μl of isopropanol were mixed, and then centrifuged at 12,000 × g for 10 minutes. The obtained pellet was washed with 80% ethanol to obtain 200 μg or more of total RNA, and used for the subsequent experiments.
　得られた全RNAすべてを100μlの水に溶かし、FastTrack2.0　mRNA　Isolation　kit(invitrogen、＃K1593-02)を用いて、mRNA　3μgを得た。SuperScriptTM　Choice　System（invitorgen、＃18090-019）を用いて、得られたmRNAから2本鎖cDNAの作製を行った。 All of the obtained total RNA was dissolved in 100 μl of water, and 3 μg of mRNA was obtained using FastTrack 2.0 mRNA Isolation kit (invitrogen, # K1593-02). Using the SuperScript ™ Choice System (invitorgen, # 18090-019), double-stranded cDNA was prepared from the obtained mRNA.
　なお、GCIY細胞は、BorrmanIV型胃癌および、その腹膜播種性転移に罹患した女性の淡血性黄色透明手術時の腹水から樹立された胃癌細胞株であり、多剤耐性遺伝子(mdr-1)の発現、ならびに、CEA、CA19-9、およびαFPの分泌が見られる低分化型腺癌細胞である。 GCIY cells are a gastric cancer cell line established from Borrman IV gastric cancer and peritoneal disseminated metastasis of ascites from a woman who suffered from peritoneal disseminated metastasis, and expressed multidrug resistance gene (mdr-1). , And poorly differentiated adenocarcinoma cells in which secretion of CEA, CA19-9, and αFP is observed.
　（２）pMX-SSTベクターへのcDNA配列の組み込み(キメラ化)
　レトロウイルスベクターpMX-SSTに得られたcDNAを組み込むためにpMX-SSTベクター（Nature　Biotechnology　17：487-490(1999)）5μgを制限酵素BstXIを用いて、100μlの反応系で45℃で4時間切断処理した。反応液すべてを1％アガロースゲルにて電気泳動し、ベクター部位に相当する約5000塩基の長さのDNA断片を切り出した。さらにWizard(R)　SV　Gel　and　PCR　Clean-Up　System（promega、＃A9282）を用いて、約5000塩基の長さのDNA断片を精製した。このようにして得られたDNA断片をpMX-SSTベクターをBstXIで制限酵素処理したものとし、これを1μl当たり50ナノグラム含む水溶液となるよう調製した。 (2) Integration of cDNA sequence into pMX-SST vector (chimerization)
In order to incorporate the obtained cDNA into the retroviral vector pMX-SST, 5 μg of pMX-SST vector (Nature Biotechnology 17: 487-490 (1999)) was added at 4 ° C. in a 100 μl reaction system at 45 ° C. with the restriction enzyme Bst XI. Time cut processing. All the reaction solutions were electrophoresed on a 1% agarose gel, and a DNA fragment having a length of about 5000 bases corresponding to the vector site was excised. Further Wizard (R) SV Gel and PCR Clean-Up System (promega, # A9282) was used to purify the DNA fragment having a length of about 5000 bases. The DNA fragment thus obtained was treated with a restriction enzyme treatment of pMX-SST vector with Bst XI, and prepared to be an aqueous solution containing 50 nanogram per 1 μl.
　先に調製した2本鎖cDNAは、平滑末端であり、BstXIで制限酵素処理したpMX-SSTと直接結合させることはできない。そこで、2本鎖cDNAの両端にBstXIの制限酵素切断後のDNA配列を持たせるための作業を行った。BstXI　Adapter（invitorgen、＃N408-18）9μgを10μlの水に溶かしたBstXI　Adapter水溶液に2本鎖cDNAを溶かした。これにLigationHigh(TOYOBO#LGK-201)を5μl添加し、懸濁して、16℃で16時間反応させて、BstXI　Adapterと2本鎖cDNAとを結合させた。その後、SuperScriptTM　Choice　System（invitorgen、＃18090-019）に添付のサイズ分画カラムを用いて、鎖長が約400塩基以下のDNA断片を除去した。その後、得られた容量の10分の1量の3M酢酸ナトリウムと2.5倍量のエタノールを添加し、転倒混和した後、20,400×ｇで30分遠心した。遠心後の上清を除去して得た沈殿を15μlの水に溶かし、1.5％のアガロースゲルにて電気泳動した。その後、約500塩基から約4000塩基の長さを持った2本鎖cDNA断片とBstXI　Adapterとの結合体を含んだゲルを切り出し、Wizard(R)　SV　Gel　and　PCR　Clean-Up　System（promega　＃A9282）を用いて、2本鎖cDNAとBstXI　Adapterとの結合体を精製した。 The previously prepared double-stranded cDNA has blunt ends and cannot be directly ligated with pMX-SST treated with Bst XI. Therefore, an operation was carried out to provide both ends of the double-stranded cDNA with a DNA sequence after cleavage of the Bst XI restriction enzyme. Double-stranded cDNA was dissolved in an aqueous solution of Bst XI Adapter in which 9 μg of Bst XI Adapter (invitorgen, # N408-18) was dissolved in 10 μl of water. 5 μl of LigationHigh (TOYOBO # LGK-201) was added thereto, suspended, and reacted at 16 ° C. for 16 hours to bind the Bst XI Adapter and the double-stranded cDNA. Thereafter, using a size fractionation column attached to SuperScript ™ Choice System (invitorgen, # 18090-019), a DNA fragment having a chain length of about 400 bases or less was removed. Thereafter, 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added and mixed by inversion, followed by centrifugation at 20,400 × g for 30 minutes. The precipitate obtained by removing the supernatant after centrifugation was dissolved in 15 μl of water and electrophoresed on a 1.5% agarose gel. Thereafter, a gel containing a conjugate of a double-stranded cDNA fragment having a length of about 500 to about 4000 bases and a Bst XI Adapter was cut out, and the Wizard (R) SV Gel and PCR Clean-Up System (promega # A9282) was used to purify the conjugate of double-stranded cDNA and Bst XI Adapter.
　BstXIで制限酵素処理したpMX-SSTベクター50ng、取得した2本鎖cDNAとBstXI　Adapterとの結合体の全量、およびT4　DNA　ligaseを、20μlの反応系にて室温で3時間処理し、pMX-SSTベクターをBstXIで制限酵素処理したものと上記結合体とを結合させた。反応液の組成は能書にしたがって調整した。 Bst XI restriction enzyme treated with pMX-SST vector 50 ng, obtained double-stranded cDNA and conjugates of the total amount of the Bst XI Adapter, and T4 DNA ligase, and 3 hours at room temperature 20μl of the reaction system, pMX -SST vector treated with restriction enzyme with Bst XI was combined with the above conjugate. The composition of the reaction solution was adjusted according to the booklet.
　（３）cDNAライブラリの増幅
　pMX-SSTベクターを用いて構築したcDNAライブラリを大腸菌に導入して増幅を行った。cDNAライブラリに、5μgのtRNA、12.5μlの7.5M酢酸ナトリウム、および70μlのエタノールを加え、転倒混和した後、20,400×gで30分遠心し、上清を捨て沈殿を得た。得られた沈殿に500μlの70％エタノールを加え、20,400×gで5分遠心し、上清を捨て得られた沈殿を10μlの水に溶かした。cDNAを大腸菌内で増幅させるために、そのうちの2μlを、コンピテントセル（invitrogen、＃18920－015）23μlと混ぜ、1.8ｋＶの条件でエレクトロポレーションを行い、1mlのSOC培地に全量を懸濁した。この作業を2回行い、大腸菌を懸濁したSOC培地を37℃で90分間、振とう培養した。その後、この培養溶液全量を、培地１ml当たりアンピシリン100μgを含むLB培地500mlに投入し、37℃で16時間、振とう培養した。 (3) Amplification of cDNA Library A cDNA library constructed using the pMX-SST vector was introduced into E. coli and amplified. To the cDNA library, 5 μg of tRNA, 12.5 μl of 7.5 M sodium acetate, and 70 μl of ethanol were added and mixed by inversion, followed by centrifugation at 20,400 × g for 30 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting precipitate, 500 μl of 70% ethanol was added, centrifuged at 20,400 × g for 5 minutes, the supernatant was discarded, and the resulting precipitate was dissolved in 10 μl of water. In order to amplify the cDNA in E. coli, 2 μl of this was mixed with 23 μl of competent cells (invitrogen, # 18920-015), electroporated under conditions of 1.8 kV, and the entire volume was suspended in 1 ml of SOC medium. did. This operation was performed twice, and the SOC medium in which Escherichia coli was suspended was cultured at 37 ° C. for 90 minutes with shaking. Thereafter, the entire amount of the culture solution was put into 500 ml of LB medium containing 100 μg of ampicillin per ml of the medium, and cultured with shaking at 37 ° C. for 16 hours.
　cDNAライブラリの大腸菌への導入数、およびpMX-SSTベクターと結合したcDNAの鎖長を確認するために、培養液5μlを取り出し、50μg/mlのアンピシリンを含むLB寒天培地にプレーティングした。 In order to confirm the number of cDNA libraries introduced into E. coli and the length of the cDNA bound to the pMX-SST vector, 5 μl of the culture solution was taken out and plated on an LB agar medium containing 50 μg / ml ampicillin.
　その結果、5μlをプレーティングしたLB寒天培地に150個のコロニーの生育が見られた。これにより培養液500ml中に1.5×107個の独立したcDNAライブラリがあると考えられた。また、コロニーのうち任意の16個についてプラスミドを抽出し、制限酵素BstXIで制限酵素処理し、処理物を1％アガロースゲルにて電気泳動を行い、pMX-SSTベクター上のcDNAの長さを計測した。その結果、平均値は約1000塩基であった。 As a result, 150 colonies grew on the LB agar medium plated with 5 μl. Thus, it was considered that there were 1.5 × 10 7 independent cDNA libraries in 500 ml of the culture solution. In addition, a plasmid was extracted from any 16 colonies, treated with the restriction enzyme Bst XI, electrophoresed on a 1% agarose gel, and the length of the cDNA on the pMX-SST vector was determined. Measured. As a result, the average value was about 1000 bases.
　残りの培養液から集菌し、10　NucleoBond（R）　AX　500　columns（日本ジェネティクス、＃740574）を用いてプラスミドを精製し、増幅されたcDNAライブラリ系を確立した。 Harvested from the remaining culture, 10 NucleoBond (R) AX 500 columns ( Japan Genetics, # 740574) plasmid was purified using established a amplified cDNA library system.
　（４）cDNAライブラリのパッケージングおよびSST-REX法の実施
　cDNAライブラリ由来遺伝子が組み込まれたpMX-SSTレトロウイルスベクターRNAを含むレトロウイルスを産生させるため、ウイルスパッケイジング細胞Plat-E（Gene　Ther.　7(12):1063-6(2000)Jun）2×106個を、4mlのDMEM培地（Wako、＃044-29765）を含む6cmディッシュに懸濁し、37℃で5％CO2の条件で24時間培養した。一方、100μlのopti-MEM（GIBCO、#31985070）と9μlのFugene（Roche、#1814443）を混ぜ、5分室温で放置後、3μgのcDNAライブラリを添加し、15分室温で放置した。cDNAライブラリを含む溶液を、培養後のPlat-E細胞に滴下し、24時間後に上清を入れ替えて同一条件で培養を続けた。さらに24時間後の上清を0.45μmのフィルターを通してろ過した。 (4) Packaging of cDNA library and implementation of SST-REX method In order to produce retrovirus containing pMX-SST retrovirus vector RNA in which a cDNA library-derived gene is incorporated, virus packaging cell Plat-E (Gene Ther. 7 (12): 1063-6 (2000) Jun) 2 × 10 6 pieces are suspended in a 6cm dish containing 4ml of DMEM medium (Wako, # 044-29765), at 37 ° C and 5% CO 2 Cultured for 24 hours. On the other hand, 100 μl of opti-MEM (GIBCO, # 31985070) and 9 μl of Fugene (Roche, # 1814443) were mixed and allowed to stand for 5 minutes at room temperature. Then, 3 μg of cDNA library was added, and the mixture was allowed to stand for 15 minutes at room temperature. The solution containing the cDNA library was dropped onto the cultured Plat-E cells, and the supernatant was replaced 24 hours later and the culture was continued under the same conditions. Further, the supernatant after 24 hours was filtered through a 0.45 μm filter.
　この取得したろ過上清0.5mlを、4×106個のBa/F3細胞を含むRPMI-1640(コージンバイオ)培地9.5mlが入れられた10cm　dish中に加えた。 0.5 ml of the obtained filtration supernatant was added to a 10 cm dish containing 9.5 ml of RPMI-1640 (Kohjin Bio) medium containing 4 × 10 6 Ba / F3 cells.
　さらに10μlのポリブレン（CHEMICON、＃TR-1003-G）と10ngのIL-3を添加し、24時間培養した。その後、細胞を3回RPMI-1640培地で洗浄し、新しいRPMI-1640培地200mlに懸濁して96ウェルプレート20枚に均等分量になるようにまき、Ba/F3細胞の自律増殖能に基づくセレクションおよびクローニングを試みた。10日後から20日後までに増殖が見られた細胞をSST-REXに基づいて選抜されたものとし、該細胞が各ウェルいっぱいに増殖するまでさらに培養を続けた。 Furthermore, 10 μl of polybrene (CHEMICON, # TR-1003-G) and 10 ng of IL-3 were added and cultured for 24 hours. After that, the cells are washed 3 times with RPMI-1640 medium, suspended in 200 ml of fresh RPMI-1640 medium, and evenly spread onto 20 96-well plates, and selection based on the autonomous growth ability of Ba / F3 cells and Attempted cloning. Cells that grew after 10 to 20 days were selected based on SST-REX, and the culture was further continued until the cells grew to full wells.
　（５）SST-REXで得られた遺伝子産物の解析
　各ウェルから得られた細胞の半分量は拡大培養して、細胞ストックとした。さらに、細胞ストックからの細胞を培養して、組み込まれたcDNA由来のペプチド分子を細胞外に発現するトランスフェクタントBa/F3細胞を、抗体作製のための免疫源細胞として、また、スクリーニング対象の細胞として用いた。各ウェルから得られた細胞の残り半分からはゲノムを抽出してシークエンスを行い、導入されたcDNA由来の遺伝子を解析した。シークエンスにおいては、得られたゲノムに対して、LA　taq　DNA　polymerase（Takara、＃RR002）またはPrimeSTAR　MAX　DNA　polymerase(TaKaRa、#R045A)を用いて、PCRを行った。PCRプライマーには、以下の配列を用いた。
 
SST3'側－T7　5'-TAATACGACTCACTATAGGGCGCGCAGCTGTAAACGGTAG-3'（配列番号：１３）
SST5'側－T3　5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3'（配列番号：１４）
 
　PCR産物をWizard(R)　SV　Gel　and　PCR　Clean-Up　System（promega、＃A9282）などを用いて精製した。その後、精製したPCR産物について、BigDye　Terminator　v3.1　Cycle　sequencing（ABI、＃4337456）およびDNAシークエンサーABI3100XLを用いて、シークエンスを行った。シークエンスのプライマーには以下のものを用いた。
 
SST5'側－T3　5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3'（配列番号：１５）
 
　得られたシークエンスデータは、BLAST検索（http://www.ncbi.nlm.nih.gov/BLAST/）とSignalP　3.0　Server（http://www.cbs.dtu.dk/services/SignalP/）を利用して解析した。 (5) Analysis of gene product obtained by SST-REX Half the amount of cells obtained from each well was expanded and used as a cell stock. In addition, transfectant Ba / F3 cells that express the peptide molecules derived from the incorporated cDNA outside the cells are cultured as cells that are the source of antibodies and screened. Used as cells. From the remaining half of the cells obtained from each well, the genome was extracted and sequenced, and the introduced cDNA-derived gene was analyzed. In the sequence, PCR was performed on the obtained genome using LA taq DNA polymerase (Takara, # RR002) or PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A). The following sequences were used as PCR primers.

SST3 'side-T7 5'-TAATACGACTCACTATAGGGCGCGCAGCTGTAAACGGTAG-3' (SEQ ID NO: 13)
SST5 'side-T3 5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3' (SEQ ID NO: 14)

The PCR product Wizard (R) SV Gel and PCR Clean-Up System (promega, # A9282) was purified by using a. Thereafter, the purified PCR product was sequenced using BigDye Terminator v3.1 Cycle sequencing (ABI, # 4337456) and DNA sequencer ABI3100XL. The following primers were used as sequence primers.

SST5 'side-T3 5'-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3' (SEQ ID NO: 15)

The obtained sequence data is BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) and SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). Analyzed by using.
　上記の通り、細胞を材料として、SST-REX法を実施した結果、1回目の実施では、87個のトランスフェクタントBa/F3細胞からのcDNA由来の遺伝子のシークエンスを行い、異なる遺伝子40種類を取得できた。2回目の実施では176個のトランスフェクタントBa/F3細胞からのcDNA由来の遺伝子のシークエンスを行い、異なる遺伝子56種類を取得できた。なお、1回目と2回目で重複した遺伝子が15種類あり、2度の実施で合計81種類のcDNA由来の遺伝子を取得することができた。遺伝子解析に供したトランスフェクタントBa/F3細胞系には、cDNA由来の遺伝子1種類のみ含まれていることを確認し、以降の実験に供した（以後このようにして得られたcDNA由来遺伝子を含む細胞を「SSTクローン細胞」と称する）。 As described above, the SST-REX method was performed using cells as a material. As a result, in the first implementation, cDNAs from 87 transfectant Ba / F3 cells were sequenced and 40 different genes were used. I was able to get. In the second implementation, we sequenced cDNA-derived genes from 176 transfectant Ba / F3 cells and obtained 56 different genes. In addition, there were 15 types of duplicated genes in the first and second rounds, and a total of 81 types of cDNA-derived genes could be obtained in the second round. The transfectant Ba / F3 cell line used for gene analysis was confirmed to contain only one type of cDNA-derived gene, and was used in the subsequent experiments (hereinafter derived from the cDNA thus obtained). Cells containing the gene are referred to as “SST clonal cells”).
　（実施例２）　MANSC1全長遺伝子のクローニングと、それを発現するBa/F3細胞株の樹立
　さらに、実施例1で得られたcDNA由来遺伝子リスト中に含まれた、MANSC1遺伝子について、遺伝子全長を含むSSTクローン細胞を得るためにクローニングを行った。 (Example 2) Cloning of MANSC1 full-length gene and establishment of Ba / F3 cell line that expresses it In addition, the MANSC1 gene included in the cDNA-derived gene list obtained in Example 1 includes the full-length gene. Cloning was performed to obtain SST clonal cells.
　GCIY細胞のSST-REXで作製したcDNAのうち30ngをテンプレートとし、NCBIのヌクレオチド検索サイト（http://www.ncbi.nlm.nih.gov/nucleotide）におけるNM_018050.2の情報に基づく設計プライマーとPrimeSTAR　MAX　DNA　polymerase（TaKaRa、＃R045A）を用い、PCR反応を行った。
 
フォワードプライマー:ccggaattcatccttgacctttgaagacc（配列番号：１６）
リバースプライマー:ttttccttttgcggccgcgatgtccacatagatcccat（配列番号：１７）
 
　得られたPCR産物を1％アガロースゲルで電気泳動し、目的の長さのDNA断片をゲルから抽出した。抽出したDNA断片をEcoRI（TaKaRa社製　＃1040A）とNotI（TaKaRa、＃1166A）で制限酵素処理した。同時に、pMX-SSTベクターについても、EcoRIとNotIで制限酵素処理した。制限酵素処理したDNA断片100ngとpMX-SSTベクター40ngとをLigationHigh(TOYOBO、#LGK-201)を用いて、2時間かけて結合させた。 Design primers based on the information of NM_018050.2 on the nucleotide search site of NCBI (http://www.ncbi.nlm.nih.gov/nucleotide) using 30 ng of cDNA prepared by SST-REX of GCIY cells as a template PCR reaction was performed using PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A).

Forward primer: cgggaattcatccttgacctttgaagacc (SEQ ID NO: 16)
Reverse primer: ttttccttttgcggccgcgatgtccacatagatcccat (SEQ ID NO: 17)

The obtained PCR product was electrophoresed on a 1% agarose gel, and a DNA fragment of the desired length was extracted from the gel. The extracted DNA fragments were treated with restriction enzymes with EcoRI (TaKaRa # 1040A) and NotI (TaKaRa, # 1166A). At the same time, the pMX-SST vector was treated with restriction enzymes with EcoRI and NotI. 100 ng of the restriction enzyme-treated DNA fragment and 40 ng of the pMX-SST vector were ligated over 2 hours using LigationHigh (TOYOBO, # LGK-201).
　結合させたもの全量に、熱ショック用の大腸菌コンピテントセルを100μl加え、氷上に30分放置した後、42℃で90秒インキュベートし、その後1mlのLB培地を加え37℃で1時間インキュベートした。その後、15,000×gで1分の遠心を行い、上清を除去し、残存液で大腸菌ペレットを懸濁した。この全量をアンピシリン50μg/mL含むLB寒天培地に塗りこみ、37℃で1晩インキュベートし、得られたコロニーを用いて、実施例1(4)のシークエンス解析と同様の方法で、PCRおよびシークエンス解析を行った。目的の長さのDNA断片が確認できたクローンについては、PCR産物をシークエンスして、目的の配列が挿入されていることを確認した。なお、シークエンス解析の際のPCR用ポリメラーゼにはPrimeSTAR　MAX　DNA　polymeraseを用いた。 100 μl of E. coli competent cells for heat shock were added to the total amount of the combined product, and left on ice for 30 minutes, followed by incubation at 42 ° C. for 90 seconds, and then 1 ml of LB medium was added and incubated at 37 ° C. for 1 hour. Thereafter, centrifugation was performed at 15,000 × g for 1 minute, the supernatant was removed, and the E. coli pellet was suspended with the remaining solution. Apply this whole amount to LB agar medium containing 50 μg / mL of ampicillin, incubate overnight at 37 ° C, and use the obtained colonies to perform PCR and sequence analysis in the same manner as the sequence analysis of Example 1 (4). Went. For clones in which a DNA fragment of the target length was confirmed, the PCR product was sequenced to confirm that the target sequence was inserted. PrimeSTAR MAX DNA polymerase was used as the polymerase for PCR during sequence analysis.
　その後、目的の配列が挿入されているコロニーをLB液体培地3mlに植菌し、37℃で1晩の培養を行った。培養物全量に対して、3,000×g、15分の遠心を行い、上清を除去し、QuickLyse　Miniprep　Kit(QIAGEN、＃27406)を用いて精製し、MANSC1遺伝子全長を含むプラスミドを得た。 Thereafter, the colony in which the target sequence was inserted was inoculated into 3 ml of LB liquid medium, and cultured at 37 ° C. overnight. The whole culture was centrifuged at 3,000 × g for 15 minutes, the supernatant was removed, and purified using QuickLyse Miniprep Kit (QIAGEN, # 27406) to obtain a plasmid containing the full length of the MANSC1 gene.
　その後、得られたプラスミドを用いて、実施例1(4)以降に示すcDNAライブラリのパッケージング以降と同様の操作にて、ベクターを含むレトロウイルスの作製を行った。次いで、全長MANSC1遺伝子を発現するBa/F3細胞株を樹立し、以降の実験に供した。 Thereafter, using the obtained plasmid, a retrovirus containing a vector was prepared in the same manner as in the packaging after packaging of the cDNA library shown in Example 1 (4) and thereafter. Next, a Ba / F3 cell line expressing the full-length MANSC1 gene was established and used for the subsequent experiments.
　（実施例３）　MANSC1モノクローナル抗体の作製
　免疫動物はマウスBalb/cを使用し、まず、免疫賦活剤として、TiterMax　Gold(Alexis　Biochemicals、ALX-510-002-L010)を等量のPBSと混和して乳化したもの50μlを投与した。翌日、MANSC1遺伝子を有するSSTクローン細胞を免疫原細胞として5×106個投与し、さらに免疫原細胞を2日おきに4回注入した。最初の免疫から約2週間後、摘出した二次リンパ組織をすりつぶし、抗体産生細胞を含む細胞集団を得た。それらの細胞と融合パートナー細胞を混合し、ポリエチレングリコール(MERCK、1.09727.0100)を用いた細胞融合によりハイブリドーマ]を作製した。融合パートナー細胞としては、マウスミエローマ細胞P3U1(P3-X63-Ag8.U1)を用いた。 Example 3 Production of MANSC1 Monoclonal Antibody Mouse Balb / c is used as an immunized animal. First, TiterMax Gold (Alexis Biochemicals, ALX-510-002-L010) is mixed with an equal amount of PBS as an immunostimulator. 50 μl of the emulsified product was administered. On the next day, 5 × 10 6 SST clonal cells having the MANSC1 gene were administered as immunogen cells, and the immunogen cells were injected four times every two days. About 2 weeks after the first immunization, the excised secondary lymphoid tissue was ground to obtain a cell population containing antibody-producing cells. These cells and fusion partner cells were mixed and a hybridoma] was prepared by cell fusion using polyethylene glycol (MERCK, 1.09727.0100). As a fusion partner cell, mouse myeloma cell P3U1 (P3-X63-Ag8.U1) was used.
　ハイブリドーマは、HAT（SIGMA、H0262）、5%　BM-condimed(Roche、663573)、15％FBS、1%ペニシリン/ストレプトイマイシン溶液（GIBCO、15140-122、Penicillin-streptomycin　liquid、、以降「P/S」と略す）を含むRPMI1640(Wako)選択培地で10～14日間培養した。次に、実施例4に示すフローサイトメトリーにより免疫原細胞に反応し、免疫源細胞に抗原遺伝子を含まないSSTクローン細胞（陰性対照細胞）に反応しないハイブリドーマを選択した。限界希釈によりモノクローン化し、抗MANSC1抗体ACT35-51_1A4B7Dを産生するハイブリドーマクローンを得た（図1）。 Hybridomas include HAT (SIGMA, H0262), 5% BM-condimed (Roche, 663573), 15% FBS, 1% penicillin / streptomycin solution (GIBCO, 15140-122, penicillin-streptomycin liquid, hereinafter “P / Cultured for 10-14 days in RPMI1640 (Wako) selective medium containing “S”. Next, hybridomas were selected by flow cytometry shown in Example 4 that reacted with immunogen cells and did not react with SST clonal cells (negative control cells) that did not contain the antigen gene in the immunogen cells. Monocloning was performed by limiting dilution to obtain a hybridoma clone producing the anti-MANSC1 antibody ACT35-51_1A4B7D (FIG. 1).
　得られたハイブリドーマは、必要量HT(SIGMA、HT　media　supplement(50X)Hybri-Max　(Sigma-Aldrich　H0137)、15%　FBS、1%　P/S溶液を含むRPMI-1640培地を用いて、維持した。産生抗体のアイソタイプを、アイソストリップキット(Roche、1493027)を用いて決定した結果、IgG2a/κであった。 The resulting hybridoma was maintained using RPMI-1640 medium containing the required amount of HT (SIGMA, HT media supplement (50X) Hybri-Max (Sigma-Aldrich H0137), 15% FBS, 1% P / S solution. The isotype of the produced antibody was determined using an isostrip kit (Roche, 1493027), resulting in IgG2a / κ.
　モノクローン化されたハイブリドーマからの精製されたACT35-51_1B4A7D抗体の取得は、次のように行った。ハイブリドーマを無血清培地(Hybridoma-SFM:GIBCO、12045-076)に馴化して拡大培養後、一定期間培養して培養上清を得た。次いで、この培養上清に含まれるIgG画分をProtein　A　セファロース(GEヘルスケア、17-1279-03)、MAPS-II結合バッファー(BIO-RAD、153-6161)、MAPS-II溶出バッファー(BIO-RAD、153-6162)を用いて精製した。溶出されたIgGをPBSで透析し、精製抗体画分を得た。 The acquisition of the purified ACT35-51_1B4A7D antibody from the monocloned hybridoma was performed as follows. The hybridoma was acclimated to a serum-free medium (Hybridoma-SFM: GIBCO, 12045-076) and expanded, and then cultured for a certain period to obtain a culture supernatant. Next, the IgG fraction contained in this culture supernatant was treated with Protein A Sepharose (GE Healthcare, 17-1279-03), MAPS-II binding buffer (BIO-RAD, 153-6161), MAPS-II elution buffer (BIO -RAD, 153-6162). The eluted IgG was dialyzed against PBS to obtain a purified antibody fraction.
　（実施例４）　フローサイトメトリーを用いた抗体スクリーニング
　ACT35-51_1B4A7D抗体と、各種細胞（目的遺伝子を発現するBa/F3細胞、目的遺伝子を発現していないBa/F3細胞、各種癌細胞など）との反応性を、フローサイトメトリーを用いて解析した。 (Example 4) Antibody screening using flow cytometry ACT35-51_1B4A7D antibody and various cells (Ba / F3 cell expressing target gene, Ba / F3 cell not expressing target gene, various cancer cells, etc.) The reactivity of was analyzed using flow cytometry.
　本実施例においては、細胞懸濁バッファーおよび以降の洗浄バッファーには、0.5%　BSAと2mM　EDTAを含有するPBSを用いた。抗体と反応させる各種細胞(対象細胞)を、96穴プレート(BD　Falcon、353911)に、細胞懸濁液が1ウェルあたり5×104個の細胞を含み100μlに成るよう調整し、分注した。 In this example, PBS containing 0.5% BSA and 2 mM EDTA was used as the cell suspension buffer and the subsequent washing buffer. Various cells to be reacted with the antibody (target cells) were adjusted and dispensed in a 96-well plate (BD Falcon, 353911) so that the cell suspension contained 5 × 10 4 cells per well to 100 μl. .
　また、染色対象の細胞が癌細胞株の場合は、80%コンフルエントになった時点でCell　Dissociation　Buffer(GIBCO、13151-014)を用いて培養プレートから剥がして回収した。 In addition, when the cells to be stained were cancer cell lines, they were detached from the culture plate using Cell Dissociation Buffer (GIBCO, 13151-014) and collected when they became 80% confluent.
　細胞懸濁液の各サンプルに、ハイブリドーマ培養上清あるいは2μg/mlの精製抗体（以降、「抗体溶液」と称する）50μlを添加し、抗体と細胞とを反応させた。抗体溶液のアイソタイプ対照抗体としては、mouse　IgG1(BioLegend、400412)、mouse　IgG2a(BioLegend、400224)、mouse　IgG2b(BioLegend、400324)をそれぞれ2μg/mlずつ含む洗浄バッファーを用いた。ハイブリドーマの培養上清と対象細胞を室温で30分反応後、700×gで2分間の遠心を行って培養上清を除去し、さらに洗浄バッファーを100μl加え、再度700×gで2分間の遠心を行って上清を除去し、細胞を洗浄した。 To each sample of the cell suspension, 50 μl of hybridoma culture supernatant or 2 μg / ml purified antibody (hereinafter referred to as “antibody solution”) was added to react the antibody with the cells. As an isotype control antibody of the antibody solution, a washing buffer containing 2 μg / ml each of mouse IgG1 (BioLegend, 400412), mouse IgG2a (BioLegend, 400224), mouse IgG2b (BioLegend, 400324) was used. After reacting the hybridoma culture supernatant and target cells for 30 minutes at room temperature, centrifuge at 700 xg for 2 minutes to remove the culture supernatant, add 100 μl of washing buffer, and again centrifuge at 700 xg for 2 minutes. To remove the supernatant and wash the cells.
　次に、洗浄後の細胞ペレットに、検出用2次抗体として、Goat　anti-mouse　IgG,　F(ab')2-PE(Beckman　Coulter、IM0855)を洗浄バッファーで200倍に希釈したものを50μl添加し、暗所において室温で30分反応させた。反応後、700×gで2分間の遠心を行って上清を除去し、さらに洗浄バッファーを100μl加え、再度700×gで2分間の遠心を行って上清を除去し、細胞を洗浄した。その後、適当量の洗浄バッファーで細胞を懸濁し、フローサイトメーター(Beckman　Coulter、FC500MPL)により、抗体と細胞の反応性について解析した。 Next, 50 μl of Goat anti-mouse IgG, F (ab ′) 2-PE (Beckman Coulter, IM0855) diluted 200-fold with a washing buffer is added to the washed cell pellet as a secondary antibody for detection. And allowed to react for 30 minutes at room temperature in the dark. After the reaction, the supernatant was removed by centrifugation at 700 × g for 2 minutes, 100 μl of washing buffer was further added, and the supernatant was removed again by centrifugation at 700 × g for 2 minutes to wash the cells. Thereafter, the cells were suspended in an appropriate amount of washing buffer, and the reactivity between the antibody and the cells was analyzed by a flow cytometer (Beckman Coulter, FC500MPL).
　反応性の測定では、前方散乱と側方散乱の測定値より生細胞を選択するようにゲートをかけた。選択された生細胞に対して抗体との反応性に基づくPEの蛍光強度を測定し、アイソタイプ対照の反応強度を基準として、免疫原細胞に対して有意に反応性が認められるが、陰性対照細胞に対して反応性が認められない培養上清を産生するハイブリドーマ細胞を候補クローンとして選択した（図１）。 In the measurement of reactivity, a gate was applied so as to select live cells from the measured values of forward scatter and side scatter. Measure the fluorescence intensity of PE based on the reactivity with the antibody against the selected live cells, and the reactivity intensity of the isotype control is used as a reference. Hybridoma cells that produced culture supernatants that were not reactive with the bacterium were selected as candidate clones (FIG. 1).
　抗体と各種細胞との反応性解析では、該抗体とアイソタイプ対照抗体との反応強度を基準として、有意に反応性が認められる抗体を選択した。 In the reactivity analysis between the antibody and various cells, an antibody with a significant reactivity was selected based on the reaction intensity between the antibody and the isotype control antibody.
　（実施例５）　癌細胞を用いたフローサイトメトリー
　染色対象とする癌細胞が80%コンフルエントになった時点でCell　Dissociation　Buffer(GIBCO、13151-014)を用いて培養プレートから剥がして回収し、回収した細胞1x105個を0.5%　BSA、2mM　EDTA/PBS(実施例4に示す洗浄バッファー)に100μlずつ懸濁し、96ウェルプレート(BD　Falcon、353911)に分注した。以降実施例4と同様の方法で、癌細胞と抗体との反応性をフローサイトメーターを用いて解析した。 (Example 5) Flow cytometry using cancer cells When cancer cells to be stained become 80% confluent, they are peeled off from the culture plate using Cell Dissociation Buffer (GIBCO, 13151-014) and collected. 1 × 10 5 cells were suspended in 100 μl each in 0.5% BSA, 2 mM EDTA / PBS (washing buffer shown in Example 4), and dispensed into a 96-well plate (BD Falcon, 353911). Thereafter, the reactivity between cancer cells and antibodies was analyzed using a flow cytometer in the same manner as in Example 4.
　ACT35-51_1B4A7D抗体との反応性を解析する癌細胞としては、胃癌細胞株であるGCIYを用いた。その結果、ACT36-27_5D1抗体はGCIYに対して有意に反応したが、ACT35-51_1B4A7D抗体においては、GCIYに対する有意な反応性を検出することはできなかった（図２）。 GCIY, a gastric cancer cell line, was used as a cancer cell for analyzing the reactivity with the ACT35-51_1B4A7D antibody. As a result, the ACT36-27_5D1 antibody reacted significantly with GCIY, but the ACT35-51_1B4A7D antibody could not detect significant reactivity with GCIY (FIG. 2).
　（実施例６）　癌細胞の細胞染色
　ACT35-51_1B4A7D抗体と各種癌細胞との反応性を、細胞染色により解析した。黒色96ウェルプレート(BD　Falcon、353219)に、染色の対象とした癌細胞1x104個を100μlの培地に懸濁して播種し、24時間培養した。癌各種癌細胞の培地には、非働化処理した10％FBS（Equitech）および1%P/S溶液を含むDMEM培地(SIGMA)を用いた。本実施例では、洗浄バッファーとして、25mM　HEPES(pH7.4)、120mM　NaCl、4.8mM　KCl、1.2mM　MgSO4、1.3mM　CaCl2を含有するバッファーを用いた。 (Example 6) Cell staining of cancer cells The reactivity between the ACT35-51_1B4A7D antibody and various cancer cells was analyzed by cell staining. In a black 96-well plate (BD Falcon, 353219), 1 × 10 4 cancer cells to be stained were suspended and seeded in 100 μl of medium, and cultured for 24 hours. As a medium for various cancer cells, DMEM medium (SIGMA) containing 10% FBS (Equitech) and 1% P / S solution which had been inactivated was used. In this example, a buffer containing 25 mM HEPES (pH 7.4), 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO 4 , and 1.3 mM CaCl 2 was used as the washing buffer.
　細胞表面のみを染色する場合、ハイブリドーマ培養上清もしくは精製抗体を2μg/mlで溶解させた洗浄バッファーを、700×gで2分間の遠心により培養上清を除去して得た細胞に対して、それぞれ50μl添加した。ACT35-51_1B4A7D抗体に対する陰性対照としては、mouse　IgG1(BioLegend、400412)、mouse　IgG2a(BioLegend、400224)、mouse　IgG2b(BioLegend、400324)を2μg/mlの濃度で洗浄バッファーに溶解させ、50μl添加した溶液を用いた。各抗体を室温で30分反応後、700×gで2分間の遠心を行って上清を除去し、さらに洗浄バッファーを100μl加え、再度700×gで2分間の遠心を行って上清を除去して、細胞を洗浄した。 When staining only the cell surface, the washing buffer in which the hybridoma culture supernatant or purified antibody is dissolved at 2 μg / ml is removed from the cells obtained by removing the culture supernatant by centrifugation at 700 × g for 2 minutes. 50 μl of each was added. As a negative control for ACT35-51_1B4A7D antibody, mouse IgG1 (BioLegend, 400412), mouse IgG2a (BioLegend, 400224), mouse IgG2b (BioLegend, 400324) were dissolved in a washing buffer at a concentration of 2 μg / ml, and 50 μl was added. Was used. After reacting each antibody at room temperature for 30 minutes, centrifuge at 700 xg for 2 minutes to remove the supernatant, add 100 μl of washing buffer, and centrifuge again at 700 xg for 2 minutes to remove the supernatant. The cells were then washed.
　洗浄後、Goat　anti-mouse　IgG,F(ab’)2-PE(Beckman　Coulter、IM0855)を洗浄バッファーで200倍希釈し、さらに10mg/mlのHoechst33342(Invitrogen、H1399)を2,000倍希釈したものを「検出用2次抗体・核染色試薬」として、細胞に50μl添加し、室温で30分暗所にて反応させた。その後、上記と同様の洗浄を2回行い、100μlの洗浄バッファーを添加した後、In　Cell　Analyzer　1000(GEヘルスケア)を用いて細胞染色を観察した。 After washing, Goat anti-mouse IgG, F (ab ') 2-PE (Beckman Coulter, IM0855) diluted 200-fold with washing buffer, and 10 mg / ml Hoechst33342 (Invitrogen, H1399) diluted 2,000-fold As a “secondary antibody for detection / nuclear staining reagent”, 50 μl was added to the cells and reacted at room temperature for 30 minutes in the dark. Thereafter, the same washing as described above was performed twice, 100 μl of washing buffer was added, and then cell staining was observed using In Cell Analyzer 1000 (GE Healthcare).
　細胞表面と細胞内部を染色する場合は、遠心にて細胞培養液上清を除去し、得られた細胞を100μlの洗浄バッファーで1回上記と同様に洗浄した。その後、4%パラホルムアルデヒド・リン酸緩衝液(Wako、161-20141)を50μl添加し、室温で10分反応させて細胞を固定した。その後、100μlの洗浄バッファーで2回洗浄した。次に、0.1%　Triton　X-100を含有する洗浄バッファーを100μl添加し、室温で10分反応させて、細胞膜の透過性を高め、その後100μlの洗浄バッファーで2回洗浄した。それ以降は、細胞表面のみを染色する方法と同様にして、染色および解析を行った。細胞の観察は、Hoechst33342で染色される核を細胞の位置の基準とし、PEの蛍光を測定することで抗体による染色の有無を検討した。 When staining the cell surface and the cell interior, the cell culture supernatant was removed by centrifugation, and the obtained cells were washed once with 100 μl of washing buffer in the same manner as described above. Thereafter, 50 μl of 4% paraformaldehyde / phosphate buffer (Wako, 161-20141) was added, and the cells were fixed by reacting at room temperature for 10 minutes. Then, it was washed twice with 100 μl of washing buffer. Next, 100 μl of a washing buffer containing 0.1% Triton X-100 was added and reacted at room temperature for 10 minutes to increase the permeability of the cell membrane, and then washed twice with 100 μl of washing buffer. Thereafter, staining and analysis were performed in the same manner as the method of staining only the cell surface. The cells were examined for the presence or absence of antibody staining by measuring the fluorescence of PE using the nucleus stained with Hoechst33342 as the cell position reference.
　各種癌細胞株をACT35-51_1B4A7D抗体で細胞染色したところ、細胞表面のみの染色条件ではMANSC1を検出できなかった（図３Ａ）。しかし、癌細胞を固定・膜透過処理を行うことで、GCIYとU87MGで反応性が見られた（図３Ｂ）。このことから、GCIYとU87MGにおいては、MANSC1が検出限界以上の量で発現しており、ACT35-51_1B4A7D抗体がそれに反応していると考えられた。ACT35-51_1B4A7D抗体は、GCIYとU87MGにおいて、膜透過処理を行わないと反応性が見られなかったことから、本抗体が反応するエピトープは細胞膜表面上には存在せず、切断および分泌されていると推測された。 When various cancer cell lines were stained with the ACT35-51_1B4A7D antibody, MANSC1 could not be detected under the staining conditions on the cell surface only (FIG. 3A). However, reactivity was observed between GCIY and U87MG by subjecting cancer cells to fixation and membrane permeabilization (FIG. 3B). From this, it was considered that in GCIY and U87MG, MANSC1 was expressed in an amount exceeding the detection limit, and the ACT35-51_1B4A7D antibody was reacting with it. The ACT35-51_1B4A7D antibody was not reactive in GCIY and U87MG unless membrane permeabilization was performed, so the epitope to which this antibody reacts does not exist on the cell membrane surface, but is cleaved and secreted. It was speculated.
　（実施例７）　MANSC1トランスフェクタント培養上清を用いた免疫沈降
　ACT35-51_1B4A7D抗体のエピトープは細胞外へ切断および分泌されている可能性が示唆されたことから、MANSC1発現細胞の培養上清を用いて免疫沈降を行った。癌細胞は無血清培地での培養が困難であるため、MANSC1遺伝子を293T細胞に一過性に発現させ、培地を無血清培地に置換後、濃縮したサンプルを用いて免疫沈降を行った。 (Example 7) Immunoprecipitation using MANSC1 transfectant culture supernatant Since it was suggested that the epitope of ACT35-51_1B4A7D antibody was cleaved and secreted outside the cell, the culture supernatant of MANSC1-expressing cells Was used for immunoprecipitation. Since cancer cells are difficult to culture in a serum-free medium, the MANSC1 gene was transiently expressed in 293T cells, the medium was replaced with a serum-free medium, and immunoprecipitated using the concentrated sample.
　293T細胞を10cm培養ディッシュ10枚に播種し、80%コンフルエントになった段階でFugene6（Roche、#1814443）を用いて遺伝子導入を行った。遺伝子導入はディッシュ1枚あたり、Opti-MEM（GIBCO、#31985070）600μlとFugene6を18μl混合し、5分間室温で放置後、6μgのMANSC1のDNAコンストラクトを添加し、15分室温で放置した。その溶液を293Tの培養液に添加し、24時間後に血清を含まない4mlのDMEMで3回洗浄後、FreeStyle　293（GIBCO、#12338-018）を20ml添加して5日間培養を行った。対照として、ベクターのみを同様に遺伝子導入した293T細胞も作製した。 293T cells were seeded on 10 10 cm culture dishes, and gene transfer was performed using Fugene6 (Roche, # 1814443) when 80% confluent. For gene transfer, 600 μl of Opti-MEM (GIBCO, # 31985070) and 18 μl of Fugene6 were mixed per dish, left for 5 minutes at room temperature, 6 μg of MANSC1 DNA construct was added, and left for 15 minutes at room temperature. The solution was added to the culture solution of 293T, washed 24 times later with 4 ml of DMEM without serum, and then 20 ml of FreeStyle 293 (GIBCO, # 12338-018) was added and cultured for 5 days. As a control, 293T cells into which only the vector was similarly introduced were also prepared.
　5日の培養後、培養上清を回収し、0.22μmのフィルターろ過を行った後、Amicon　Ultra(分画分子量3000、ミリポア、#UFC9　003　96)を用いて100倍濃縮した。 After 5 days of culture, the culture supernatant was collected, filtered through a 0.22 μm filter, and concentrated 100 times using Amicon Ultra (fractionated molecular weight 3000, Millipore, # UFC9 003 96).
　濃縮した培養上清200μlにPBSを800μl加え、そこにProtein　Aセファロース(GEヘルスケア、17-1279-03)を20μl加え、ローテーター（TAITEC、#RT-5）を用い4℃で30分攪拌して反応させることで、Protein　Aセファロースに非特異的に結合する蛋白質を除去した。反応後、15000×g、4℃、5分間で遠心し培養上清を回収して、再度Protein　Aセファロースを20μl加え、同様の手法で非特異的に反応する蛋白質を除去した。 Add 800 μl of PBS to 200 μl of concentrated culture supernatant, add 20 μl of Protein A Sepharose (GE Healthcare, 17-1279-03), and stir at 4 ° C for 30 minutes using a rotator (TAITEC, # RT-5) The protein that non-specifically binds to Protein A Sepharose was removed. After the reaction, the mixture was centrifuged at 15000 × g and 4 ° C. for 5 minutes to recover the culture supernatant, and 20 μl of Protein A Sepharose was added again to remove non-specifically reacting proteins by the same method.
　Protein　Aセファロースに非特異的に反応する蛋白質を除去した培養上清に、ACT35-51_1B4A7D抗体、もしくはmouse　IgG2a抗体を5μg加え、4℃で60分攪拌して反応させた。抗体の反応後、Protein　Aセファロースを20μl加え、4℃で30分攪拌して反応させて免疫沈降した。免疫沈降後、15000×g、4℃、5分間で遠心し、上清を除去してProtein　Aセファロースを回収した。回収したProtein　AセファロースにPBSを500μlくわえ、4℃で5分攪拌して洗浄した。洗浄後、15000×g、4℃、5分間で遠心し、PBSを除去してProtein　Aセファロースを回収した。この洗浄操作を3回繰り返した。 5 μg of ACT35-51_1B4A7D antibody or mouse IgG2a antibody was added to the culture supernatant from which the protein non-specifically reacting with Protein A Sepharose was removed, and the mixture was reacted by stirring at 4 ° C. for 60 minutes. After the reaction of the antibody, 20 μl of Protein A Sepharose was added, and the mixture was stirred at 4 ° C. for 30 minutes to react and immunoprecipitate. After immunoprecipitation, centrifugation was performed at 15000 × g, 4 ° C. for 5 minutes, and the supernatant was removed to recover Protein A Sepharose. The collected protein A sepharose was mixed with 500 μl of PBS and washed by stirring at 4 ° C. for 5 minutes. After washing, the mixture was centrifuged at 15000 × g, 4 ° C. for 5 minutes, and PBS was removed to recover Protein A Sepharose. This washing operation was repeated three times.
　洗浄後、Protein　Aセファロースに20μlのSDS-PAGEサンプルバッファーを添加し、100℃で5分間煮沸した。サンプルバッファーを12.5%のSDS-PAGEに負荷し、30mAの電流をかけて電気泳動を行った。電気泳動後、PVDF膜に160mAの電流をかけて1時間転写した。転写後のPVDF膜を5%スキムミルクを室温で1時間反応させてブロッキングを行った。次に抗MANSC1ポリクローナル抗体(AVIVA　systems、#ARP34359_P050)を5%スキムミルクで3000倍希釈したもの2.5mlを、室温で1時間反応させた。反応後PVDF膜を0.05%Tween20を含むPBS(以下、PBS-Tと称する)で3回洗浄後、抗ウサギIgG-POD(MBL、#458)を5%スキムミルクで5000倍希釈したもの2.5mlを、室温で1時間反応させた。反応後PVDF膜をPBS-Tで5回洗浄し、発色基質(ミリポア、Immobilon　Western　#WBKLS0500)を用いて発色させ、フィルムに15秒間感光させた。 After washing, 20 μl of SDS-PAGE sample buffer was added to Protein A Sepharose and boiled at 100 ° C. for 5 minutes. Sample buffer was loaded on 12.5% SDS-PAGE, and electrophoresis was performed with a current of 30 mA. After electrophoresis, the PVDF membrane was transferred for 1 hour with a current of 160 mA. The PVDF membrane after transfer was blocked by reacting 5% skim milk at room temperature for 1 hour. Next, 2.5 ml of anti-MANSC1 polyclonal antibody (AVIVA systems, # ARP34359_P050) diluted 3000 times with 5% skim milk was reacted at room temperature for 1 hour. After the reaction, the PVDF membrane was washed 3 times with PBS containing 0.05% Tween 20 (hereinafter referred to as PBS-T), and 2.5 ml of anti-rabbit IgG-POD (MBL, # 458) diluted 5000 times with 5% skim milk was added. And allowed to react at room temperature for 1 hour. After the reaction, the PVDF membrane was washed 5 times with PBS-T, developed with a chromogenic substrate (Millipore, Immobilon Western # WBKLS0500), and exposed to the film for 15 seconds.
　MANSC1を導入した293Tの培養上清では、ACT35-51_1B4A7D抗体で免疫沈降したものは10kDa近傍にMANSC1のシグナルが検出されたが、アイソタイプ対照で免疫沈降したものはシグナルが検出されなかった（図４）。また、ベクターのみを導入した293Tの培養上清ではアイソタイプ対照、ACT35-51_1B4A7D抗体で免疫沈降したもの共にシグナルは検出されなかった（図４）。これらの結果からも、MANSC1の一部は切断され、培養上清中に分泌する可能性が示唆された。 In the culture supernatant of 293T introduced with MANSC1, MANSC1 signal was detected in the vicinity of 10 kDa when immunoprecipitated with ACT35-51_1B4A7D antibody, but no signal was detected when immunoprecipitated with isotype control (FIG. 4). ). In addition, in the culture supernatant of 293T into which only the vector was introduced, no signal was detected in both the isotype control and those immunoprecipitated with the ACT35-51_1B4A7D antibody (FIG. 4). These results also suggested that part of MANSC1 may be cleaved and secreted into the culture supernatant.
　（実施例８）　ACT35-51_1B4A7Dモノクローナル抗体の癌細胞の増殖に対する効果（MTT）
　ACT35-51_1B4A7D抗体の癌細胞の増殖に与える影響について、MTTアッセイを用いて解析した。各種癌細胞の培地には、前立腺癌細胞株AsPC1については非働化処理した10％FBS（Equitech、以降同じ）と1％P/Sを含むRPMI1640培地(WAKO)を、それ以外の癌細胞については非働化処理した10％FBSおよび1％P/S溶液を含むDMEM培地(SIGMA)を用いた。対象抗体を産生するハイブリドーマ用培地には、必要量のHT(Sigma-Aldrich、H0137、HT　media　supplement(50X)　Hybri-Max)、15％FBS、1％P/S溶液を含むRPMI培地(Wako)を用いた。 (Example 8) Effect of ACT35-51_1B4A7D monoclonal antibody on proliferation of cancer cells (MTT)
The effect of ACT35-51_1B4A7D antibody on cancer cell proliferation was analyzed using MTT assay. For various cancer cells, the prostate cancer cell line AsPC1 was inactivated with RPMI1640 medium (WAKO) containing 10% FBS (Equitech, the same applies hereinafter) and 1% P / S, and other cancer cells. DMEM medium (SIGMA) containing inactivated 10% FBS and 1% P / S solution was used. The medium for hybridoma producing the target antibody includes RPMI medium (Wako) containing the required amount of HT (Sigma-Aldrich, H0137, HT media supplement (50X) Hybri-Max), 15% FBS, 1% P / S solution. Was used.
　また、対照として、Mouse　IgG1(BECKMAN　COULTER、731581)、IgG2a(MBL、M076-3)、IgG2b(MBL、M077-3)、IgG3(MBL、M078-3)を各1μlずつ、1mlのハイブリドーマ用培地に溶かしたマウスアイソタイプ対照混合液を用いた。 As controls, 1 μl each of Mouse IgG1 (BECKMAN COULTER, 731581), IgG2a (MBL, M076-3), IgG2b (MBL, M077-3), IgG3 (MBL, M078-3), 1 ml of hybridoma medium A mouse isotype control mixture dissolved in was used.
　各実験は、抗体培養上清1試験あたり3ウェルずつ行った。各種癌細胞を、１ウェルあたり、100μl培地中で2×103個ずつ、96ウェルプレート(IWAKI)に播種し、5％CO2条件にて37℃で24時間インキュベートした。 Each experiment was performed in 3 wells per antibody culture supernatant test. Various cancer cells were seeded in a 96-well plate (IWAKI) at 2 × 10 3 cells in 100 μl medium per well and incubated at 37 ° C. for 24 hours under 5% CO 2 condition.
　インキュベート後の96ウェルプレートに、ACT35-51_1B4A7D抗体を含む培養上清あるいはマウスアイソタイプ対照を1ウェルあたり100μlずつ添加し、72時間インキュベートした。遠心により培養上清を除去して得たそれぞれの細胞に対して、新鮮な培地に溶かして調整した5%　WST-1溶液(vol./vol.、Roche、11　644　807　001)100μlを加え、1～4時間インキュベートし、1時間おきにマイクロプレートリーダー(BIO-RAD)にて、WST-1の発色を測定した。 The culture supernatant containing the ACT35-51_1B4A7D antibody or mouse isotype control was added to the 96-well plate after incubation at 100 μl per well and incubated for 72 hours. To each cell obtained by removing the culture supernatant by centrifugation, add 100 μl of 5% WST-1 solution (vol./vol., Roche, 11, 644, 807, 001) prepared by dissolving in fresh medium, After incubating for 1 to 4 hours, the color development of WST-1 was measured with a microplate reader (BIO-RAD) every 1 hour.
　グラフ化した測定結果を図５に示す。このデータは3時間後のWST-1の発色をO.D.値(O.D450nm-O.D.630nm)で示した。MTTアッセイにおいて、ACT35-51_1B4A7D抗体は、GCIY細胞の増殖を、アイソタイプ対照に比べ、約25％にまで抑制した（図５Ｂ）。即ち、約75％の増殖抑制効果があった。さらに、ACT35-51_1B4A7D抗体の細胞増殖抑制効果は、グリオーマ由来のT98G細胞において顕著に現れ、アイソタイプ対照に比べ、その増殖を約7％にまで抑制した（図５Ｃ）。即ち、約93％の増殖抑制効果が認められた。 The graphed measurement results are shown in FIG. This data showed the color development of WST-1 after 3 hours by O.D. value (O.D450nm-O.D.630nm). In the MTT assay, the ACT35-51_1B4A7D antibody suppressed GCIY cell proliferation to approximately 25% compared to the isotype control (FIG. 5B). That is, there was a growth suppression effect of about 75%. Furthermore, the cell growth inhibitory effect of ACT35-51_1B4A7D antibody was remarkably observed in glioma-derived T98G cells, and its growth was suppressed to about 7% compared to the isotype control (FIG. 5C). That is, an about 93% growth inhibitory effect was observed.
　（実施例９）　マウス担癌モデルに対する尾静脈投与による抗体の効果
　ACT35-51_1B4A7D抗体のin　vivoにおける抗腫瘍効果についてマウス担癌モデルを用いて検討した。 (Example 9) Effect of antibody by tail vein administration on mouse tumor bearing model The in vivo antitumor effect of ACT35-51_1B4A7D antibody was examined using a mouse tumor bearing model.
　6週齢雄のSCIDマウス（日本クレア株式会社より5週齢で購入）の頚背部皮下に、GCIY細胞が5×10６個／0.2ｍl　saline／マウス個体となるように、細胞懸濁液を移植した。移植後3週間目に生着した腫瘍の大きさを計測し、各群の平均腫瘍体積がおよそ55±5mm3となるように、担癌マウスを対照群、陽性対照群、ACT35-51_1B4A7D抗体群の3群に分け、1群を4匹とした。 A cell suspension is prepared so that GCIY cells become 5 × 10 6 cells / 0.2 ml saline / mouse individual subcutaneously on the back of the neck of 6-week-old male SCID mice (purchased from CLEA Japan at 5 weeks of age). Transplanted. Measure the size of the tumor that engrafted 3 weeks after transplantation, and control the tumor-bearing mice, control group, positive control group, ACT35-51_1B4A7D antibody group so that the average tumor volume of each group is about 55 ± 5 mm 3 The three groups were divided into 4 groups.
　各群それぞれへのサンプル投与は、細胞移植後3週間目から担癌マウスの尾静脈に対して行った。対照群に生理食塩水（大塚生食注）を、陽性対照群にTaxotere（マウス個体あたり600μg、サノフィ・アベンティス）を、抗体投与群にACT35-51_1B4A7D抗体（10mg/kg）を、それぞれ週に1回（3週間連続で合計3回）尾静脈より投与した。また毎回の投与直前には体重計測および腫瘍計測を行った。体重計測は動物天秤を使用し、腫瘍計測はデジマチックキャリパーにて長径および短径を計測し、式「腫瘍体積(mm3)＝0.5×長径×短径×短径」により腫瘍体積を求めた。 Sample administration to each group was performed on the tail vein of cancer-bearing mice from 3 weeks after cell transplantation. Saline (Otsuka raw diet) for the control group, Taxotere (600 μg per mouse, sanofi-aventis) for the positive control group, and ACT35-51_1B4A7D antibody (10 mg / kg) for the antibody administration group once a week It was administered via the tail vein (3 times in total for 3 consecutive weeks). In addition, body weight measurement and tumor measurement were performed immediately before each administration. An animal balance was used for body weight measurement, and for tumor measurement, the major axis and minor axis were measured with a digimatic caliper, and the tumor volume was determined by the formula "tumor volume (mm 3 ) = 0.5 x major axis x minor axis x minor axis" .
　腫瘍体積データを対照群と比較して、経時的な抗腫瘍効果を検討した。その結果、対照群では経時的に腫瘍は増大し、投与開始3週間後の実験終了時では、開始時の約25倍の腫瘍体積であった。これに対して、Taxotereを投与した群では投与開始後2週間目から腫瘍増殖抑制が観察され、実験終了となる3週間目では、実験開始時の約11倍程度に留まり、対照群に対する約59％の腫瘍体積抑制が観察された。また、ACT35-51_1B4A7D抗体を投与した群では投与開始後1週間目より、対照群に対して腫瘍増殖抑制傾向が観察され、実験終了時では、腫瘍体積は投与開始時の約16倍程度に留まり、対照群の腫瘍体積を約44％抑制した（図６）。 The tumor volume data was compared with the control group to examine the antitumor effect over time. As a result, in the control group, the tumor increased with time, and at the end of the experiment 3 weeks after the start of administration, the tumor volume was about 25 times that at the start. In contrast, in the group administered Taxotere, tumor growth suppression was observed from the second week after the start of administration, and in the third week after the end of the experiment, it remained at about 11 times the start of the experiment, about 59 times that of the control group. % Tumor volume suppression was observed. In addition, in the group administered ACT35-51_1B4A7D antibody, a tendency to suppress tumor growth was observed with respect to the control group from the first week after the start of administration, and at the end of the experiment, the tumor volume remained about 16 times that at the start of administration. The tumor volume of the control group was suppressed by about 44% (FIG. 6).
　各サンプルの投与開始から3週間後、剖検を行った。担癌マウスをエーテル麻酔致死後、頚背部皮下より腫瘍を摘出してその重量を計測し、各群の腫瘍重量を比較した。摘出腫瘍重量はそれぞれ、対照群1.33ｇ、陽性対照群0.87g、ACT35-51_1B4A7D抗体群0.83gであった。ACT35-51_1B4A7D抗体を投与した群では、対照群と比較して、摘出腫瘍重量が約38％程度減少しており、陽性対照群と同程度の腫瘍増殖抑制効果が観察された（図７）。 An autopsy was performed 3 weeks after the start of administration of each sample. The tumor-bearing mice were subjected to ether anesthesia lethality, and the tumors were removed subcutaneously from the back of the neck and their weights were measured. The excised tumor weights were 1.33 g for the control group, 0.87 g for the positive control group, and 0.83 g for the ACT35-51_1B4A7D antibody group, respectively. In the group to which the ACT35-51_1B4A7D antibody was administered, the excised tumor weight was reduced by about 38% compared to the control group, and the same tumor growth inhibitory effect as that of the positive control group was observed (FIG. 7).
　体重推移に関しては、陽性対照群では投与開始2週間後から体重減少が顕著に認められたが、ACT35-51_1B4A7D抗体群では体重の減少は認められなかった。剖検時体重から腫瘍重量を差し引いた算出体重においても、陽性対照群の体重減少は明らかであるが、ACT35-51_1B4A7D抗体群では体重の増加が観察された（図８）。 Regarding the body weight transition, a significant decrease in body weight was observed in the positive control group from 2 weeks after the start of administration, but no decrease in body weight was observed in the ACT35-51_1B4A7D antibody group. Also in the calculated body weight obtained by subtracting the tumor weight from the body weight at the time of necropsy, a decrease in the body weight of the positive control group was obvious, but an increase in the body weight was observed in the ACT35-51_1B4A7D antibody group (FIG. 8).
　なお、統計学的検定は一元配置分散分析(ANOVA)を行い、p<0.05で差のある場合にTukeyの多重比較法による有意差検定を行った。対照群に対して、危険率がp<0.05の場合に有意差ありと評価した。(*:p<0.05,**:p<0.01) The statistical test was performed by one-way analysis of variance (ANOVA), and when there was a difference at p <0.05, a significant difference test was performed by Tukey's multiple comparison method. When the risk rate was p <0.05, the control group was evaluated as having a significant difference. (*: p <0.05, **: p <0.01)
　（実施例１０）　抗体可変領域決定方法
　ACT35-51_1A4B7D抗体の可変領域の遺伝子配列を明らかにするため、ACT35-51_1B4A7D抗体産生細胞ハイブリドーマ細胞2×106をTrizol(invitrogen、#15596-026)1mlに懸濁し5分放置し、クロロフォルムを200μl添加して、15秒間懸濁後、12,000×gで15分間遠心し、上清を得た。この上清と500μlのイソプロパノールを混合した後、12,000×gで10分間遠心した。得られたペレットを80％エタノールで洗浄し、全RNA　40μgを得た。その全量を20μlの水で溶かした。そのうち、全RNA　5μg分の溶液を使用して、SuperScriptTM　Choice　System（invitorgen、＃18090-019）を用いて、全RNAから2本鎖cDNAを作製した。得られた2本鎖cDNAをエタノール沈殿後、LigationHigh(TOYOBO#LGK-201)を用いて2本鎖cDNAの5’末端と3’末端を結合させ、そのうち1μlを鋳型としてPCRを行った。プライマーとしては、重鎖と軽鎖の定常領域に対して設計したものを使用した。プライマーの配列は、次の通りである。
 
重鎖5'側gtccacgaggtgctgcacaat（配列番号：１８）
重鎖3'側gtcactggctcagggaaataacc（配列番号：１９）
軽鎖5'側aagatggatacagttggtgc（配列番号：２０）
軽鎖3'側tgtcaagagcttcaacagga（配列番号：２１）
 
　PCR産物を1.5％ゲルにて電気泳動を行った後、切り出して精製を行った。精製したDNAを用いてシークエンスを行った。軽鎖については、精製したDNAをクローニングした後、シークエンスを行った。決定された軽鎖の可変領域の塩基配列を配列番号：９に、アミノ酸配列を配列番号：１０に、重鎖の可変領域の塩基配列を配列番号：１１に、アミノ酸配列を配列番号：１２に示す。 (Example 10) Antibody variable region determination method In order to clarify the gene sequence of the variable region of ACT35-51_1A4B7D antibody, 2 × 10 6 ACT35-51_1B4A7D antibody-producing cell hybridoma cells were added to 1 ml of Trizol (invitrogen, # 15596-026). Suspended and allowed to stand for 5 minutes, 200 μl of chloroform was added, suspended for 15 seconds, and then centrifuged at 12,000 × g for 15 minutes to obtain a supernatant. This supernatant was mixed with 500 μl of isopropanol, and then centrifuged at 12,000 × g for 10 minutes. The obtained pellet was washed with 80% ethanol to obtain 40 μg of total RNA. The whole amount was dissolved in 20 μl of water. Among them, using a solution corresponding to 5 μg of total RNA, double-stranded cDNA was prepared from total RNA using SuperScript ™ Choice System (invitorgen, # 18090-019). The obtained double-stranded cDNA was precipitated with ethanol, and the 5 ′ end and 3 ′ end of the double-stranded cDNA were bound using LigationHigh (TOYOBO # LGK-201), and PCR was performed using 1 μl of this as a template. Primers designed for the heavy and light chain constant regions were used. Primer sequences are as follows.

Heavy chain 5 ′ side gtccacgaggtgctgcacaat (SEQ ID NO: 18)
Heavy chain 3 ′ side gtcactggctcagggaaataacc (SEQ ID NO: 19)
Light chain 5 ′ side aagatggatacagttggtgc (SEQ ID NO: 20)
Light chain 3 ′ side tgtcaagagcttcaacagga (SEQ ID NO: 21)

The PCR product was electrophoresed on a 1.5% gel, and then excised and purified. Sequencing was performed using the purified DNA. For the light chain, the purified DNA was cloned and then sequenced. The determined light chain variable region base sequence is SEQ ID NO: 9, the amino acid sequence is SEQ ID NO: 10, the heavy chain variable region base sequence is SEQ ID NO: 11, and the amino acid sequence is SEQ ID NO: 12. Show.
　また、これら可変領域のアミノ酸配列について、UCLの「Andrew　C.R.　Martin's　Bioinformatics　Group」のサイトにおける配列分析（http://www.bioinf.org.uk/abysis/tools/analyze.cgi）を利用してナンバリングし、「Table　of　CDR　Definitions」に記載の基準（http://www.bioinf.org.uk/abs/#kabatnum）に従ってCDR領域を同定した。CDR予測の結果と軽鎖および重鎖のシグナル配列を図９に示す。また、軽鎖のCDR1、CDR2、CDR3のアミノ酸配列を配列番号:３～５に、重鎖のCDR1、CDR2、CDR3のアミノ酸配列を配列番号:６～８に示す。 In addition, the amino acid sequences of these variable regions are numbered using sequence analysis (http://www.bioinf.org.uk/abysis/tools/analyze.cgi) on the site of UCR's “Andrew CR Martin's Bioinformatics Group”. The CDR regions were identified according to the criteria described in “Table of CDR Definitions” (http://www.bioinf.org.uk/abs/#kabatnum). The results of CDR prediction and the light chain and heavy chain signal sequences are shown in FIG. The amino acid sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 3-5, and the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 6-8.
　（実施例１１）　ACT35-51_1B4A7D抗体のエピトープ解析
　ACT35-51_１B4A7D抗体のエピトープを特定するために、数種の鎖長のMANSC1ペプチドを発現するBa/F3細胞を作製し、抗体との反応性を評価した。 (Example 11) Epitope analysis of ACT35-51_1B4A7D antibody In order to identify the epitope of ACT35-51_1B4A7D antibody, Ba / F3 cells expressing MANSC1 peptides having several chain lengths were prepared and the reactivity with the antibody was evaluated. did.
　MANSC1、膜外領域60aa（N末端より。以下、同様）、153aa、186aa、219aa、250aa、285aa、385aa(細胞外全長)を解析対象のペプチドとした。実施例1に示すGCIYシグナルシークエンストラップ法を実施したcDNAライブラリを鋳型として、下記配列のDNAをプライマーとして使用し、PrimeSTAR　MAX　DNA　polymerase(TaKaRa、#R045A)をポリメラーゼとして使用して、７種類の遺伝子の単離を行った。
フォワードプライマー(2種類の遺伝子共通):（配列番号：１６）
リバースプライマー(Rに付加した数値は、増幅産物がコードするペプチドの鎖長を意味する)
 
R60:（配列番号：２２）ttttccttttgcggccgcttgagttgaagtatatacgg
R153:（配列番号：２３）ttttccttttgcggccgctaggggagtgactgcttgtg
R186:（配列番号：２４）ttttccttttgcggccgcaaatagtttctccaagtgat
R219:（配列番号：２５）ttttccttttgcggccgccagcagatgcgctatttctt
R250:（配列番号：２６）ttttccttttgcggccgcggtgggtagaagggtggcgg
R285:（配列番号：２７）ttttccttttgcggccgctgtagaaatgagggtcgtgg
R385:（配列番号：２８）ttttccttttgcggccgcaagccatttttcaaatggaa
 
　得られた各PCR産物を1％アガロースゲルにて電気泳動を行った後、切り出し精製を行い、EcoRIとNotIで制限酵素処理を行った。pMX-SSTもEcoRIとNotIで制限酵素処理を行い切り出し精製した。さらにそれぞれをLigationHigh(TOYOBO、#LGK-201)にて処理し、その後、実施例2（大腸菌にトランスホーメーション以降）と同様の処理を行い、50μgアンピシリン含有LBアガロースプレートに、トランスホーメーションした大腸菌をプレーティングした。37℃で１晩培養して得られたコロニーからインサート部分を含有するようにPCRを行い、希望する配列を含んだpMX-SSTベクターであるかにつき、シークエンスにて確認した。シークエンス用PCRプライマーとしては、次のオリゴヌクレオチドを用いた。
 
SST3’側　5'-ggcgcgcagctgtaaacggtag-3'（配列番号：２９）SST5’側　5'-cgggggtggaccatcctcta-3'（配列番号：３０）
 
　その後、実施例1(4)（ウイルスパッケイジング以降）と同様の方法で、各種鎖長のMANSC1遺伝子配列を含むBa/F3細胞を作製した。さらに、実施例4と同様の手法で、各種鎖長のMANSC1分子を発現するBa/F3細胞とACT35-51_１B4A7D抗体との反応性をフローサイトメーターにて解析した（図１０）。その結果、ACT35-51_1B4A7D抗体は、MANSC1分子の1-60まで発現させたクローンに対しては反応性を示さず、1から153の領域よりも長いものを発現するクローンに対して反応性を示したことから、MANSC1分子のN末端から61位から153位の間に抗体のエピトープが含まれることが明らかとなった。 MANSC1, extramembranous region 60aa (from N-terminal, the same applies hereinafter), 153aa, 186aa, 219aa, 250aa, 285aa, 385aa (extracellular full length) were used as peptides to be analyzed. Using the cDNA library subjected to the GCIY signal sequencing strap shown in Example 1 as a template, using the DNA of the following sequence as a primer, PrimeSTAR MAX DNA polymerase (TaKaRa, # R045A) as a polymerase, seven kinds of genes Was isolated.
Forward primer (common to two genes): (SEQ ID NO: 16)
Reverse primer (The number added to R means the chain length of the peptide encoded by the amplification product)

R60: (SEQ ID NO: 22) ttttccttttgcggccgcttgagttgaagtatatacgg
R153: (SEQ ID NO: 23) ttttccttttgcggccgctaggggagtgactgcttgtg
R186: (SEQ ID NO: 24) ttttccttttgcggccgcaaatagtttctccaagtgat
R219: (SEQ ID NO: 25) ttttccttttgcggccgccagcagatgcgctatttctt
R250: (SEQ ID NO: 26) ttttccttttgcggccgcggtgggtagaagggtggcgg
R285: (SEQ ID NO: 27) ttttccttttgcggccgctgtagaaatgagggtcgtgg
R385: (SEQ ID NO: 28) ttttccttttgcggccgcaagccatttttcaaatggaa

Each PCR product obtained was electrophoresed on a 1% agarose gel, cut out and purified, and then treated with restriction enzymes with EcoRI and NotI. pMX-SST was also digested with EcoRI and NotI and purified. Further, each was treated with LigationHigh (TOYOBO, # LGK-201), and then the same treatment as in Example 2 (after transformation to E. coli) was performed, and the transformed E. coli was applied to a 50 μg ampicillin-containing LB agarose plate. Was plated. PCR was performed from colonies obtained by overnight culture at 37 ° C. so as to contain the insert portion, and the pMX-SST vector containing the desired sequence was confirmed by sequencing. The following oligonucleotides were used as PCR primers for sequencing.

SST3 'side 5'-ggcgcgcagctgtaaacggtag-3' (SEQ ID NO: 29) SST5 'side 5'-cgggggtggaccatcctcta-3' (SEQ ID NO: 30)

Thereafter, Ba / F3 cells containing MANSC1 gene sequences of various chain lengths were prepared in the same manner as in Example 1 (4) (after virus packaging). Furthermore, the reactivity between Ba / F3 cells expressing MANSC1 molecules of various chain lengths and the ACT35-51_1B4A7D antibody was analyzed with a flow cytometer in the same manner as in Example 4 (FIG. 10). As a result, the ACT35-51_1B4A7D antibody does not react with clones expressing MANSC1 molecules up to 1-60, but reacts with clones expressing longer than 1 to 153 region. From these results, it has been clarified that an antibody epitope is contained between positions 61 to 153 from the N-terminus of the MANSC1 molecule.
　（実施例１２）　ACT35-51_1B4A7D抗体のカイネティクス解析
　表面プラズモン共鳴（surface plasmon Resonance、SPR）法によりACT35-51_1B4A7D抗体とMANSC1-Fc融合タンパク質との結合についての解析を行った。 (Example 12) Kinetic analysis of ACT35-51_1B4A7D antibody The binding between ACT35-51_1B4A7D antibody and MANSC1-Fc fusion protein was analyzed by the surface plasmon resonance (SPR) method.
　（１）MANSC1-Fc融合タンパク質の調製
　MANSC1の細胞外ドメインをコードするDNAをPCRにて増幅させた後に、ヒトIgGのFcのヒンジ部分以降が挿入されたカセットベクターであるpACT001　（pcDNA3.1を由来とするベクター、ACTGen社製）に組み込み、MANSC1-Fc融合タンパク質発現ベクターを作製した。なお、MANSC1の細胞外ドメインをコードするDNAは、テンプレートとしてGCIY細胞由来のcDNAを用い、PrimeSTAR
MAX DNA polymerase　 (TaKaRa #R045A)によってPCRを行うことにより増幅した。プライマーは以下のものを利用した。
 
フォワードプライマー：　ccggaattcatccttgacctttgaagacc　（配列番号：３１）
リバースプライマー：　ttttccttttgcggccgccgaagccatttttcaaatggaa　（配列番号：３２）
 
　そして、前記プライマーを用いて増幅した後、制限酵素EcoRI（TaKaRa、#1040A）とNotI(TaKaRa、#1166A)にて増幅断片を処理し、同様の制限酵素処理を行ったpACT001にライゲーション反応により組み込んだ。 (1) Preparation of MANSC1-Fc fusion protein pACT001 (pcDNA3.1) is a cassette vector in which the DNA encoding the extracellular domain of MANSC1 is amplified by PCR and then the Fc hinge part of human IgG is inserted. MANSC1-Fc fusion protein expression vector was prepared by incorporating into the vector derived from ACTGen. The DNA encoding the extracellular domain of MANSC1 uses cDNA derived from GCIY cells as a template, and PrimeSTAR
Amplification was performed by PCR using MAX DNA polymerase (TaKaRa # R045A). The following primers were used.

Forward primer: ccggaattcatccttgacctttgaagacc (SEQ ID NO: 31)
Reverse primer: ttttccttttgcggccgccgaagccatttttcaaatggaa (SEQ ID NO: 32)

After amplification using the above primers, the amplified fragment was treated with restriction enzymes EcoRI (TaKaRa, # 1040A) and NotI (TaKaRa, # 1166A), and incorporated into pACT001 subjected to the same restriction enzyme treatment by ligation reaction. It is.
　得られた発現ベクターをFugene6 Transfection Reagent(Roche、#11 988
387 001)を用いて、能書に従って293T細胞にリポフェクションし、無血清培地（Free-style 293 expression medium）中に一過性にMANSC1-Fc融合タンパク質を産生させた。MANSC1-Fc融合タンパク質を含む培養上清に等量のMAPSII結合バッファー（BIO-RAD）を添加した後、ProteinAセファロース（GEヘルスケア、#17-1279-03）を用いて精製を行った。そして、L-Arginine（PH4.0）により溶出された画分についてPBSにて透析を実施し、得られた精製品をカイネティクス解析に用いた。 The resulting expression vector was transferred to Fugene6 Transfection Reagent (Roche, # 11 988
387 001) was lipofected into 293T cells according to the capacity document, and MANSC1-Fc fusion protein was transiently produced in a serum-free medium (Free-style 293 expression medium). After adding an equal amount of MAPSII binding buffer (BIO-RAD) to the culture supernatant containing the MANSC1-Fc fusion protein, purification was performed using Protein A Sepharose (GE Healthcare, # 17-1279-03). Then, the fraction eluted with L-Arginine (PH4.0) was dialyzed with PBS, and the obtained purified product was used for kinetic analysis.
　（２）ACT35-51_1B4A7D抗体とMANSC1-Fc融合タンパク質との結合の解析
　SPR法によるカイネティクス解析にはProteOn XPR36(BIO-RAD)を用いた。ACT35-51_1B4A7D抗体はアミンカップリング法により20μg/mlの条件でProteOn GLCセンサーチップ (BIO-RAD)に固定化した。MANSC1-Fc融合タンパク質はPBSを用いて10nM、5nM、 2.5nM、 1.25nM、
0.625nMに希釈し、アナライトとした。リガンドであるACT35-51_1B4A7D抗体とアナライトであるMANSC1-Fc融合タンパク質の相互反応をProteOnによりモニターした結果を図１１に示す。また、会合速度定数 (ka)、解離速度定数 (Kd)、解離定数 (KD)はProteOn Manager v.2 (BIO-RAD)を用いて算出した。結果を表１に示す。 (2) Analysis of binding between ACT35-51_1B4A7D antibody and MANSC1-Fc fusion protein ProteOn XPR36 (BIO-RAD) was used for kinetic analysis by the SPR method. The ACT35-51_1B4A7D antibody was immobilized on a ProteOn GLC sensor chip (BIO-RAD) under the condition of 20 μg / ml by the amine coupling method. MANSC1-Fc fusion protein is 10nM, 5nM, 2.5nM, 1.25nM using PBS
Dilute to 0.625 nM to make an analyte. FIG. 11 shows the results of monitoring the interaction between the ligand ACT35-51_1B4A7D antibody and the analyte MANSC1-Fc fusion protein using ProteOn. The association rate constant (ka), dissociation rate constant (Kd), and dissociation constant (KD) were calculated using ProteOn Manager v.2 (BIO-RAD). The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　その結果、ACT35-51_1B4A7D抗体は、MANSC1タンパク質に対して10-10という大変小さい解離定数 (KD)を有していることから、ACT35-51_1B4A7D抗体とMANSC1タンパク質との親和性は高いことが明らかになった。 As a result, the ACT35-51_1B4A7D antibody has a very low dissociation constant (KD) of 10 -10 to the MANSC1 protein, which clearly indicates that the ACT35-51_1B4A7D antibody has a high affinity for the MANSC1 protein. became.
　（実施例１３）　抗MANSC1抗体の解析
　実施例３に記載の方法にて、前述のACT35-51_1A4B7D以外の抗MANSC1抗体を産生するハイブリドーマクローン１４種を得た（表２　参照）。なお、これらハイブリドーマクローンが産生するモノクローナル抗体に関しては、実施例４に記載の方法と同様にして、MANSC1タンパク質を発現するBa/F3細胞株に対する反応性を確認した。得られた結果の一部を図１２～２０に示す。 (Example 13) Analysis of anti-MANSC1 antibody 14 hybridoma clones producing anti-MANSC1 antibodies other than the aforementioned ACT35-51_1A4B7D were obtained by the method described in Example 3 (see Table 2). As for the monoclonal antibodies produced by these hybridoma clones, the reactivity with respect to the Ba / F3 cell line expressing MANSC1 protein was confirmed in the same manner as in the method described in Example 4. Some of the results obtained are shown in FIGS.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　これらのクローンが産生するモノクローナル抗体について、実施例８に記載の方法と同様にして、胃癌細胞株（GCIY)及び乳癌細胞株(ZR75-1)を用いたMTTアッセイを行った。なお、ACT35-51_2C4E8C抗体及びACT35-51_8H12A7H抗体に関しては、胃癌細胞株（GCIY)を用いたMTTアッセイのみを行った。得られた結果を図２１～３４に示す。なお図中、胃癌細胞株（GCIY)においてはWST-1添加3時間後の発色をO.D.値(O.D450nm-O.D.630nm)で示し、乳癌細胞株(ZR75-1)においてはWST-1添加4時間後の発色をO.D.値(O.D450nm-O.D.630nm)で示す。 The monoclonal antibodies produced by these clones were subjected to an MTT assay using a stomach cancer cell line (GCIY) and a breast cancer cell line (ZR75-1) in the same manner as in the method described in Example 8. For the ACT35-51_2C4E8C antibody and the ACT35-51_8H12A7H antibody, only the MTT assay using a gastric cancer cell line (GCIY) was performed. The obtained results are shown in FIGS. In the figure, in the gastric cancer cell line (GCIY), color development 3 hours after addition of WST-1 is shown as an OD value (O.D450nm-OD630nm), and in the breast cancer cell line (ZR75-1), WST-1 addition 4 The color development after time is indicated by OD value (O.D450nm-OD630nm).
　図２１～３４に示した結果から明らかなように、ACT35-51_3C3F3抗体、ACT35-51_5D6C11抗体、ACT35-51_5E2H6抗体、ACT35-51_7D9C8抗体、及びACT35-51_8G11B7においては、乳癌細胞株(ZR75-1)に対する増殖抑制効果が確認された（図２１～２５参照）。また、ACT35-51_2C4E8C抗体、ACT35-51_9G12F12C抗体、ACT35-51_9G12F3E抗体、及びACT35-51_8H12A7H抗体においては、胃癌細胞株（GCIY)に対する増殖抑制効果が確認された（図２６～２９参照）。しかしながら、これら以外のモノクローナル抗体においては、試した２種の細胞株に対する増殖抑制効果が見られなかった（図３０～３４参照）。 As is clear from the results shown in FIGS. 21 to 34, the ACT35-51_3C3F3 antibody, ACT35-51_5D6C11 antibody, ACT35-51_5E2H6 antibody, ACT35-51_7D9C8 antibody, and ACT35-51_8G11B7 against the breast cancer cell line (ZR75-1) A growth inhibitory effect was confirmed (see FIGS. 21 to 25). In addition, the ACT35-51_2C4E8C antibody, ACT35-51_9G12F12C antibody, ACT35-51_9G12F3E antibody, and ACT35-51_8H12A7H antibody were confirmed to have a growth inhibitory effect on the gastric cancer cell line (GCIY) (see FIGS. 26 to 29). However, other monoclonal antibodies did not show growth inhibitory effects on the two cell lines tested (see FIGS. 30 to 34).
　また、胃癌細胞株（GCIY)又は乳癌細胞株(ZR75-1)に対して増殖抑制効果が確認されたモノクローナル抗体に関しては、実施例１０に記載の方法と同様にして、各々の軽鎖及び重鎖の可変領域、CDR領域、シグナルの配列を同定した。得られた結果を図３５～４１に示す。また各々の軽鎖及び重鎖の可変領域、CDR領域、シグナルの配列については表２に記した配列番号にて配列表に示す。 In addition, regarding the monoclonal antibody whose growth inhibitory effect was confirmed against the gastric cancer cell line (GCIY) or the breast cancer cell line (ZR75-1), the light chain and the heavy chain of each were tested in the same manner as in the method described in Example 10. The chain variable region, CDR region, and signal sequence were identified. The obtained results are shown in FIGS. In addition, the variable region, CDR region, and signal sequence of each light and heavy chain are shown in the Sequence Listing with the SEQ ID Nos.
　図４１に示した結果から明らかなように、ACT35-51_9G12F12C抗体、ACT35-51_9G12F3E抗体、及びACT35-51_8H12A7H抗体の重鎖は、塩基配列及びアミノ酸配列が同一のものであった。また、ACT35-51_9G12F12C抗体、ACT35-51_9G12F3E抗体、及びACT35-51_8H12A7H抗体の軽鎖は、アミノ酸配列においては同一のものであったが、塩基配列においては相異していた。 As is clear from the results shown in FIG. 41, the heavy chains of the ACT35-51_9G12F12C antibody, the ACT35-51_9G12F3E antibody, and the ACT35-51_8H12A7H antibody have the same base sequence and amino acid sequence. The light chains of the ACT35-51_9G12F12C antibody, the ACT35-51_9G12F3E antibody, and the ACT35-51_8H12A7H antibody were identical in amino acid sequence, but differed in nucleotide sequence.
　さらに、前述の抗体に関しては、実施例１１に記載の方法と同様にして、各々の抗体のエピトープを同定した。得られた結果を表２に示す。 Furthermore, regarding the above-mentioned antibodies, the epitope of each antibody was identified in the same manner as in the method described in Example 11. The obtained results are shown in Table 2.
　表２に示した結果から明らかなように、ACT35-51_1A4B7D抗体も含め、癌細胞の増殖抑制効果が確認されたモノクローナル抗体のエピトープはMANSC1タンパク質のN末端から61位～153位のアミノ酸配列に含まれることが示された。これに対して、GCIY及びZR75-1に対する増殖抑制効果が確認されなかったモノクローナル抗体に関しては、これらのエピトープはMANSC1タンパク質のN末端から154位～186位、187位～219位、又は220位～250位のアミノ酸配列に含まれることが示された。 As is apparent from the results shown in Table 2, the epitopes of monoclonal antibodies confirmed to have cancer cell growth-inhibiting effects, including the ACT35-51_1A4B7D antibody, are included in the amino acid sequence from positions 61 to 153 from the N-terminus of the MANSC1 protein. It was shown that In contrast, for monoclonal antibodies for which growth inhibitory effects on GCIY and ZR75-1 were not confirmed, these epitopes were located at positions 154 to 186, 187 to 219, or 220 to the N-terminal of the MANSC1 protein. It was shown to be included in the amino acid sequence at position 250.
　これらの結果から、MANSC1タンパク質のN末端から61位～153位に含まれるアミノ酸配列に結合する抗体を、抗癌活性を有する抗体として好適に用いることができることが示唆された。また、MANSC1タンパク質のN末端から33位～117位のアミノ酸配列はMANSCドメインと称され、高度に保存された7つのシステイン配列を含むモチーフ（motif　at　N　terminus　with　seven　cysteines）であるため、このドメインと抗癌活性との関連性も示唆される。 From these results, it was suggested that an antibody that binds to an amino acid sequence contained in positions 61 to 153 from the N-terminus of the MANSC1 protein can be suitably used as an antibody having anticancer activity. In addition, the amino acid sequence from position 33 to position 117 from the N-terminus of the MANSC1 protein is called a MANSC domain, which is a motif containing seven highly conserved cysteine sequences (motif at N terminus with seven cysteines). There is also a suggestion of an association between anticancer activity and anticancer activity.
　本発明のモノクローナル抗体は、優れた抗癌活性を有するため、癌の治療または予防に用いることができる。特に、胃癌やグリオーマや乳癌に対しては、強い細胞増殖抑制効果を示す。本発明のモノクローナル抗体は、非常に悪性度の高く、これまで治療が困難とされてきたスキルス胃癌に対しても優れた効果を有すると考えられることから、医療上極めて有用である。また、本発明のモノクローナル抗体は、癌の診断や癌細胞の検出・選別などへの応用も可能である。 Since the monoclonal antibody of the present invention has excellent anticancer activity, it can be used for treatment or prevention of cancer. In particular, it has a strong cell growth inhibitory effect on gastric cancer, glioma and breast cancer. The monoclonal antibody of the present invention is extremely useful for medical treatment because it is considered to have an excellent effect on Skills gastric cancer, which is very malignant and has been difficult to treat. In addition, the monoclonal antibody of the present invention can be applied to cancer diagnosis and cancer cell detection / selection.
配列番号１３～３２
＜２２３＞　　人工的に合成されたプライマーの配列 SEQ ID NOs: 13-32
<223> Artificially synthesized primer sequences

Claims (25)

  1. 　ヒト由来のMANSC1蛋白質に結合し、かつ、抗癌活性を有する抗体 Antibody that binds to human-derived MANSC1 protein and has anticancer activity
  2. 　ヒト由来のMANSC1蛋白質の細胞外領域に結合する、請求項１に記載の抗体。 The antibody according to claim 1, which binds to an extracellular region of a human-derived MANSC1 protein.
  3. 　癌が胃癌、グリオーマ、または乳癌である、請求項１に記載の抗体。 The antibody according to claim 1, wherein the cancer is gastric cancer, glioma, or breast cancer.
  4. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：３～５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：６～８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：１０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：１２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1, having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NO: 3 to 5 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 6 to 8 or at least one of the amino acid sequences, wherein one or more amino acids are (B) the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence in which the signal sequence has been removed from the amino acid sequence, comprising a heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence; Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or inserted. A light chain variable region comprising an amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence obtained by removing a signal sequence from the amino acid sequence, or at least one of these amino acid sequences, one or more amino acids are It retains the heavy chain variable region containing the amino acid sequence substituted, deleted, added and / or inserted.
  5. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：３３～３５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：３６～３８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：４０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：４２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1, having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence according to SEQ ID NO: 33 to 35 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 36 to 38 or at least one of the amino acid sequences, wherein one or more amino acids are (B) the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence in which the signal sequence has been removed from the amino acid sequence, comprising a heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence; Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 42, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  6. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：４３～４５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：４６～４８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：５０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：５２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1 having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NOs: 43 to 45 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 46-48 or at least one of the amino acid sequences, wherein one or more amino acids are (B) the amino acid sequence set forth in SEQ ID NO: 50 or an amino acid sequence in which the signal sequence has been removed from the amino acid sequence, comprising: a heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence; Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 52, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  7. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：５３～５５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：５６～５８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：６０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：６２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1, having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NOs: 53 to 55 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 56-58 or at least one of the amino acid sequences, wherein one or more amino acids are A heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence (b) the amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence from which the signal sequence has been removed, Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 62, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  8. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：６３～６５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：６６～６８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：７０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：７２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1, having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NOs: 63 to 65 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 66-68 or at least one of the amino acid sequences, wherein one or more amino acids are A heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence (b) the amino acid sequence set forth in SEQ ID NO: 70 or an amino acid sequence from which the signal sequence has been removed, Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 72, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  9. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：７３～７５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：７６～７８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：８０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：８２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1 having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NOs: 73 to 75 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted, and the amino acid sequence set forth in SEQ ID NOs: 76 to 78 or at least one of the amino acid sequences, wherein one or more amino acids are A heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence (b) the amino acid sequence set forth in SEQ ID NO: 80 or an amino acid sequence from which the signal sequence has been removed, Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 82, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  10. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：８３～８５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：８６～８８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：９０に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：９２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1, which has the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NOs: 83 to 85 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NOs: 86 to 88 or at least one of the amino acid sequences, wherein one or more amino acids are A heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence (b) the amino acid sequence set forth in SEQ ID NO: 90 or an amino acid sequence from which the signal sequence has been removed, Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence, the amino acid sequence set forth in SEQ ID NO: 92, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  11. 　下記（ａ）又は（ｂ）に記載の特徴を有する請求項１に記載の抗体
    　（ａ）配列番号：９３～９５に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：９６～９８に記載のアミノ酸配列又は該アミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する
    　（ｂ）配列番号：１０２に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む軽鎖可変領域と、配列番号：１０４に記載のアミノ酸配列若しくは該アミノ酸配列からシグナル配列が除去されたアミノ酸配列、又はこれらのアミノ酸配列の少なくともいずれかにおいて、１若しくは複数のアミノ酸が置換、欠失、付加および／または挿入されたアミノ酸配列を含む重鎖可変領域とを保持する。     The antibody according to claim 1, having the characteristics described in the following (a) or (b): (a) one or more amino acids in the amino acid sequence of SEQ ID NO: 93 to 95 or at least one of the amino acid sequences A light chain variable region comprising an amino acid sequence substituted, deleted, added and / or inserted; and the amino acid sequence set forth in SEQ ID NO: 96-98 or at least one of the amino acid sequences, wherein one or more amino acids are (B) the amino acid sequence set forth in SEQ ID NO: 102 or an amino acid sequence in which the signal sequence has been removed from the amino acid sequence, and a heavy chain variable region comprising a substituted, deleted, added and / or inserted amino acid sequence. Or in at least one of these amino acid sequences, one or more amino acids are substituted, deleted, added and / or One or more of the light chain variable region containing the inserted amino acid sequence and the amino acid sequence set forth in SEQ ID NO: 104, the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or at least one of these amino acid sequences And a heavy chain variable region containing the amino acid sequence in which the amino acids are substituted, deleted, added and / or inserted.
  12. 　ヒト由来のMANSC1蛋白質の６１～１５３位のアミノ酸配列からなるペプチド配列に結合する、請求項１に記載の抗体。 2. The antibody according to claim 1, which binds to a peptide sequence consisting of amino acid sequences at positions 61 to 153 of a human-derived MANSC1 protein.
  13. 　ヒト由来のMANSC1蛋白質における、下記（ａ）～（ｈ）のいずれかに記載の抗体のエピト―プに結合する、請求項１に記載の抗体
    　（ａ）配列番号：１０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：１２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｂ）配列番号：４０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：４２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｃ）配列番号：５０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：５２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｄ）配列番号：６０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：６２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｅ）配列番号：７０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：７２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｆ）配列番号：８０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：８２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｇ）配列番号：９０に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：９２に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体
    　（ｈ）配列番号：１０２に記載のアミノ酸配列を含む軽鎖可変領域と配列番号：１０４に記載のアミノ酸配列を含む重鎖可変領域とを保持する抗体。     The antibody according to claim 1, which binds to the epitope of the antibody according to any one of the following (a) to (h) in a human-derived MANSC1 protein: (a) the amino acid sequence according to SEQ ID NO: 10 An antibody having a light chain variable region comprising the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 (b) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and SEQ ID NO: 42 An antibody having a heavy chain variable region comprising the amino acid sequence described (c) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 52; (D) an antibody having a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 60 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 62 (e) SEQ ID NO: 70 Amino acids described in An antibody having a light chain variable region comprising a sequence and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 72 (f) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 80 and SEQ ID NO: (G) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90 and a heavy chain variable comprising the amino acid sequence set forth in SEQ ID NO: 92 (H) An antibody retaining a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 102 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104.
  14. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：３～５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：１０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：６～８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：１２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or a variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 3 to 5 Peptide (b) A peptide comprising the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or the variable region of the antibody light chain according to claim 1 (c) A peptide comprising the heavy chain of the antibody of claim 1 or a variable region thereof comprising the amino acid sequence of No. 6 to 8 (d) The amino acid sequence of SEQ ID No. 12 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  15. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：３３～３５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：４０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：３６～３８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：４２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or a variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 33 to 35 Peptide (b) The peptide comprising the amino acid sequence of SEQ ID NO: 40 or the light chain of the antibody or the variable region thereof according to claim 1, comprising the amino acid sequence obtained by removing the signal sequence from the amino acid sequence (c) The peptide comprising the heavy chain of the antibody according to claim 1 or the variable region thereof, comprising the amino acid sequence of No. 36 to 38 (d) The amino acid sequence of SEQ ID No. 42 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  16. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：４３～４５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：５０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：４６～４８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：５２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or the variable region thereof, comprising the amino acid sequence according to SEQ ID NOs: 43 to 45 Peptide (b) Peptide consisting of the amino acid sequence of SEQ ID NO: 50 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or the variable region thereof (c) A peptide comprising the heavy chain of the antibody according to claim 1 or a variable region thereof comprising the amino acid sequence of No. 46 to 48 (d) The amino acid sequence of SEQ ID No: 52 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  17. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：５３～５５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：６０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：５６～５８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：６２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or the variable region thereof, comprising the amino acid sequence according to SEQ ID NOs: 53 to 55 Peptide (b) The peptide comprising the amino acid sequence of SEQ ID NO: 60 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or the variable region of the antibody light chain according to claim 1 (c) The peptide consisting of the heavy chain of the antibody according to claim 1 or the variable region thereof comprising the amino acid sequence of No .: 56 to 58 (d) The amino acid sequence of SEQ ID No: 62 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  18. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：６３～６５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：７０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：６６～６８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：７２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or the variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 63-65 Peptide (b) The peptide consisting of the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or the peptide comprising the light chain of the antibody or a variable region thereof (c) A peptide comprising the heavy chain of the antibody according to claim 1 or a variable region thereof comprising the amino acid sequence of Nos. 66 to 68 (d) The amino acid sequence of SEQ ID No: 72 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  19. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：７３～７５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：８０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：７６～７８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：８２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or the variable region thereof, comprising the amino acid sequence according to SEQ ID NOs: 73 to 75 Peptide (b) The peptide comprising the amino acid sequence of SEQ ID NO: 80 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence or the variable region of the antibody light chain according to claim 1 (c) A peptide comprising the heavy chain of an antibody according to claim 1, or a variable region thereof, comprising the amino acid sequence according to SEQ ID NO: 76 to 78 (d) the amino acid sequence according to SEQ ID NO: 82 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  20. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：８３～８５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：９０に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：８６～８８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：９２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1 or the variable region thereof, comprising the amino acid sequence according to SEQ ID NOs: 83 to 85 Peptide (b) The peptide consisting of the amino acid sequence of SEQ ID NO: 90 or the amino acid sequence of which the signal sequence has been removed from the amino acid sequence, comprising the light chain of an antibody or a variable region thereof (c) A peptide comprising the heavy chain of the antibody according to claim 1 or a variable region thereof, comprising the amino acid sequence of No. 86 to 88 (d) The amino acid sequence of SEQ ID No. 92 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  21. 　下記（ａ）～（ｄ）のうちのいずれかに記載のペプチド
    　（ａ）配列番号：９３～９５に記載のアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｂ）配列番号：１０２に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の軽鎖またはその可変領域からなるペプチド
    　（ｃ）配列番号：９６～９８に記載のアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド
    　（ｄ）配列番号：１０４に記載のアミノ酸配列または該アミノ酸配列からシグナル配列が除去されたアミノ酸配列を含む、請求項１に記載の抗体の重鎖またはその可変領域からなるペプチド。     The peptide according to any one of the following (a) to (d): (a) comprising the light chain of the antibody according to claim 1, or the variable region thereof, comprising the amino acid sequence according to SEQ ID NOs: 93 to 95 The peptide (b) comprising the amino acid sequence of SEQ ID NO: 102 or the amino acid sequence obtained by removing the signal sequence from the amino acid sequence, or the peptide comprising the light chain of the antibody or a variable region thereof (c) A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof comprising the amino acid sequence of No. 96 to 98 (d) The amino acid sequence of SEQ ID No. 104 or a signal sequence derived from the amino acid sequence A peptide comprising the heavy chain of an antibody according to claim 1 or a variable region thereof, comprising the removed amino acid sequence.
  22. 　請求項１～１３のいずれかに記載の抗体または請求項１４～２１のいずれかに記載のペプチドをコードするDNA。 A DNA encoding the antibody according to any one of claims 1 to 13 or the peptide according to any one of claims 14 to 21.
  23. 　請求項１～１３のいずれかに記載の抗体を産生する、または、請求項２２に記載のDNAを含む、ハイブリドーマ。 A hybridoma that produces the antibody according to any one of claims 1 to 13 or contains the DNA according to claim 22.
  24. 　請求項１～１３のいずれかに記載の抗体を有効成分とする、抗癌剤。 An anticancer agent comprising the antibody according to any one of claims 1 to 13 as an active ingredient.
  25. 　癌が胃癌、グリオーマ、または乳癌である、請求項２４に記載の抗癌剤。 The anticancer agent according to claim 24, wherein the cancer is gastric cancer, glioma, or breast cancer.
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