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WO2011052753A1 - Anticorps de liaison à une protéine mansc1, présentant une activité anticancéreuse - Google Patents

Anticorps de liaison à une protéine mansc1, présentant une activité anticancéreuse Download PDF

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Publication number
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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Japanese (ja)
Inventor
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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Actgen
ACTGEN Inc
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Actgen
ACTGEN Inc
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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.

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Abstract

La présente invention porte sur un ADN complémentaire (ADNc) qui provient d'une bibliothèque d'ADNc dérivée d'une lignée cellulaire cancéreuse et code pour une protéine exprimée sur une surface cellulaire ou secrétée par une cellule que l'on crible au moyen du procédé SST-REX. Un anticorps monoclonal contre ladite protéine codée par l'ADNc ainsi criblée est construit et son activité anticancéreuse est examinée in vitro et in vivo. Il en résulte la découverte d'un anticorps monoclonal apte à se lier à une protéine MANSC1, qui présente une excellente activité anticancéreuse. En outre, une région qui contient un épitope de cet anticorps dans la protéine MANSC1 est identifiée et les structures des régions variables à chaîne légère et à chaîne lourde de cet anticorps peuvent être déterminées avec succès.
PCT/JP2010/069377 2009-10-29 2010-10-29 Anticorps de liaison à une protéine mansc1, présentant une activité anticancéreuse Ceased WO2011052753A1 (fr)

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US9309305B2 (en) 2011-06-10 2016-04-12 National Research Council Of Canada Anti-ricin antibodies and uses thereof
CN107530426A (zh) * 2015-04-30 2018-01-02 东丽株式会社 癌的治疗和/或预防用药物组合物

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