Classifications

• • 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
• • 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
  • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• • 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
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • 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
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
  • G01N33/505—Cells of the immune system involving T-cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • 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
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/74—Inducing cell proliferation
• • 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
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4724—Lectins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases.
• the present invention further relates to screening methods of immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases.
• Immune system consists of innate and acquired immunity and defends organisms from invasion of pathogens cooperatively and effectively. It has been clarified that various lectin families are involved in this immune system. Although most lectins, which are responsible for immunity, are membrane-binding proteins, galectin is an exceptional and interesting existence. Galectin is defined as a lectin family having carbohydrate recognition domain (CRD) recognizing ⁇ -galactoside structure. At present, galectin families from galectin-1 to galectin-14 have been identified.
• CCD carbohydrate recognition domain
• galectins having one CRD and two CRDs respectively in their structures are present, and the presence of such three types as prototype (ones having one CRD, galectin-6, -7, and -10; ones having two CRDs which bind as a homodimer, galectin-1, -2, -11, -13, and -14), chimeric type (galectin-3) and tandem-repeat type (galectin-4, -6, -8, -9, -12) is known.
• Galectin-9 which has two CRDs tandem via a bridging peptide in its molecule, belongs to tandem-repeat type galectin.
• Human galectin-9 was isolated from spleen cDNA library using autoantibody from patient with Hodgkin's disease (Sahin U., et al.: Proc. Natl. Acad. Sci. USA 92: 11810-11813, 1995). Independently, mouse and rat genes homologous to human galectin-9 gene were cloned from kidney cDNA library (Wada J. and Kanwar YS., J. Biol. Chem. 272: 6078-6086, 1997).
• galectin does not enter classic secretion pathway like normal secretory proteins, and is secreted from cells actively and passively when immunoreactions should be initiated.
• Galectin secreted extracellularly binds autocrine to secreted cells or paracrine to neighbor cells and induces various immune responses by cross-linking to galectin-binding factor or galectin receptor on immune system cells.
• Soluble galectin can cross-link to galectin-binding factor or galectin receptor on cell surface by at least three modes, namely, cell-cell interaction, agonistic signal transduction and formation of galectin-glycoconjugate lattice.
• galectin Unlike other cytokines, characteristics of galectin are to function as a soluble factor or adhesion molecule for leukocytes and to form galectin-glycoconjugate lattice. In recent years, the immunological importance of this galectin-lattice is of a paid attention.
• leukocytes migrate firstly to inflammatory sites. At the sites, leukocytes phagocytose invaded foreign cells, pathogens and/or dead cells. Further, leukocytes secrete cytotoxic factors, microorganism killing factors and/or cytokines activating immune cascades. On these processes, for individual protection or terminating immune responses, apoptosis is induced in leukocytes and a part of subsets (ThI and Th2) of activated T cells, being damaged. It has been clarified that galectin-9 acts on ThI, a subset of helper T cells, and induces apoptosis.
• leukocytes for example, neutrophils, macrophages and lymphocytes infiltrate from blood into inflammatory sites and infection lesions.
• Galectin-3 is a chemotactic factor for macrophages
• galectin-9 is known to be an eosinophil-specific chemotactic factor.
• Galectin-9 is known to induce apoptosis of thymocytes in mice as its biological activity (Wada J., et al.: J. Clin. Invest. 99: 2452-2461, 1997).
• useful biological activities of galectin-9 the exertion of such various activities as cytotoxic activity against malignant tumor, apoptosis-inducing activity of malignant tumor and apoptosis-inducing activity of activated T cells, especially, apoptosis-inducing activity of
• CD4 positive T cells CD4 positive T cells, immunosuppressive activity, anti-inflammatory action and anti-allergic action is disclosed (JP-2004-244411A1). Further, the patent application of nucleotide molecules encoding galectin-8, galectin-9, galectin-10 and galectin-lOSV proteins, and of the application of those galectins to cancers, autoimmune diseases, inflammatory diseases, asthma, allergic diseases and so on is made (JP-2001-501831T).
• galectin-9 exerts biological activity. Especially, a membrane protein, namely galectin-9-binding molecule (galection-9 receptor) which binds galectin-9 and transduces its signal was not identified.
• galectin-9 receptor The screening and identification methods of galectin-9-binding protein (galectin-9 receptor) include: 1) the method using affinity column immobilized galectin-9 protein; 2) immunoprecipitation method; 3) Western blot technique; 4) cross linking method; 5) Yeast two hybrid system; 6) tandem affinity purification (TAP) method (Puig O., et al.: Methods 24: 218-229, 2001) and surface plasmon resonance method.
• TEP tandem affinity purification
• galectin-9-binding protein (galectin receptor is also included) using galectin-9
• cell extract of MOLT4 a tumor cell line, in which apoptosis is induced by galectin-9, is passed through the column immobilized galectin-9CT (C-terminal region) and proteins recovered from the column are loaded on SDS-PAGE.
• galectin-9-binding molecules have been identified by analyzing internal amino acid sequences of the obtained protein bands on gel fragments (JP-2004-244411A1).
• galectin-9-binding proteins including 4F2 heavy chain antigen (177216), ATPase, Na+/K+ transporting, and alpha 1 polypeptide (21361181) and so forth (JP-2004-24441 IAl).
• 4F2 heavy chain antigen 177216
• ATPase ATPase
• Na+/K+ transporting ATPase
• alpha 1 polypeptide 21361181
• JP-2004-24441 IAl alpha 1 polypeptide
• Tim-3 T-cell immunoglobulin and mucin domain protein; also called Hepatitis A virus cellular receptor 2
• Thl T-helper subtype 1
• a fusion protein (Tim-3-Ig) of Tim-3 and immunoglobulin Fc(Ig) was prepared, and was incubated at 4 0 C by adding the extract of a CD8 + mouse lymphoma cell line, TKl, biotinylated cell surface.
• Tim-3-Ig and a protein(s ) bound specifically to Tim-3 was precipitated with protein G-agarose beads, and after washing the beads extensively, the washed beads were boiled in 1 x SDS-PAGE buffer. The upper layer was collected by centrifugation and when the upper layer was loaded on SDS-PAGE, a specific single protein band was appeared on the gel and was identified as galectin-9 by mass spectrometry analysis.
• galectin-9 transduced its signal via binding to Tim-3 and exerted immunosuppressive activity by inducing apoptosis of ThI cells, demonstrating that galectin-9 is the ligand for its receptor, Tim-3 (Zhu C, et al., Nature Immunology 6: 1245-1252, 2005).
• Tim-3 is the receptor for galectin-9.
• Galectin-9 binds to the receptor on cell surface, Tim-3, in vitro and induces calcium influx into ThI cells, resulting in aggregation and apoptosis. Further, it has been clarified that the in vivo administration of galectin-9 causes selective defects of cells producing interferon ⁇ and suppression of ThI autoimmunity (Zhu C, et al., Nature Immunology 6: 1245-1252, 2005). ThI cells producing interferon- ⁇ as a cytokine and Th2 cells producing IL-4 and IL-5 are present in subsets of CD4-positive helper T cells. ThI cells causes cellular immunity and Th2 cells are involved in immediate immune response by inducing humoral immunity (antibody production). Although steroid hormone is used for treating allergic diseases as an immunosuppressive agent, it has strong adverse effects. Therefore, in its use, consideration against the adverse effects is required. Development of new drugs including galectin-9 with less adverse effects instead of steroid hormone is desired.
• type 1 diabetes which is one of autoimmune diseases, resulting in decrease of insulin secretion by inflammation of Langerhans's islets.
• type 1 diabetes is one of autoimmune diseases, resulting in decrease of insulin secretion by inflammation of Langerhans's islets.
• “Therapeutic vaccine compositions for treating type 1 diabetes” JP-2006-526651T
• Antigens which are targeted by morbific and pathogenic T cells and their use in type 1 diabetes” JP-2006-525813T
• autoimmune diseases in which activated ThI cells are involved; e.g., systemic lupus erythematosus developed by occurrence of systemic autoimmune reactions, as well as those developed upon occurrence of autoimmune phenomena caused at limited specific organs, such as type 1 diabetes, rheumatoid arthritis, Sjogren's syndrome and so on.
• effective prophylactic and therapeutic methods are not still established, and development of effective new drugs including galectin-9 is desired.
• pancreatic Langerhans's islets between non-galectin-9 administration group (PBS-control group) and galectin-9-administration group was compared, distinct expression of insulin in pancreatic islets was observed in galectin-9-administration group, but the marked cell infiltration into pancreatic islets and the marked decrease in insulin secretion were observed in non-administration group, confirming the suppressive effect of galectin-9 on the onset of type 1 diabetes histologically.
• galectin-9 was found to have the tendency inducing apoptosis of mature ThI cells (CD4+Tim-3+) from NOD mouse.
• the present inventors prepared anti-galectin-9 antibody and anti-Tim-3 antibody, which block the signaling pathway of galectin-9 -Tim-3, and investigated the effect of each antibody on the onset of type 1 diabetes in NOD mouse.
• anti-galectin-9 and anti-Tim-3 antibody were found to exert unexpected and strong suppressive effects on the onset of type 1 diabetes in NOD mouse.
• galectin-9 exert actually suppressive effects on the onset of type 1 diabetes in vivo, which is a ThI cell involved-autoimmune disease, confirming that galectin-9 -Tim-3 signaling pathway operates in vivo as well.
• anti-galectin-9 antibody and anti-Tim-3 antibody which block the signaling pathway of galectin-9 -Tim-3, which operates in vivo, had been expected to accelerate the onset of type 1 diabetes in NOD mouse; however, surprisingly enough, each of these antibodies exerted suppressive effects on the onset at almost the same level as galectin-9 and at much higher level than galectin-9, respectively.
• galectin-9 stimulates TNF- ⁇ production by dendritic cells which are antigen presenting cells.
• galectin-9 causes inflammation via TNF- ⁇ production (Anderson AC, et al., Science 318: 1141-1143, 2007). Accordingly, on the process of inflammation, the actions of galectin-9 on different cells exert suppressive effect on activated ThI cells, and stimulatory effect on dendritic cells.
• Tim-3 pathway (s) on dendritic cells is located upstream of inflammation mechanisms, and the blocking of the pathway is important for treatment of autoimmune diseases.
• the substances, which block the signaling pathway of galectin-9 — Tim-3 in the present invention include RNA and DNA aptamers besides antibodies.
• Aptamer is paid attention as a substance which recognizes protein like an antibody and has characteristics that can be synthesized chemically and homogeneously compared to antibody, and can be supplied cheaply and without any antigenicity.
• the active ingredients of immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases in the present invention may be RNA and DNA aptamers with characteristics (1) inhibiting the binding of galectin-9 to Tim-3 by binding to galectin-9, and (2) inhibiting the binding of Tim-3 to galectin-9 by binding to Tim-3.
• RNA and DNA aptamers of (1) described above in the present invention can be obtained by evaluating the effects on the binding of galectin-9 to Tim-3, and by selecting aptamers inhibiting the binding of galectin-9 to Tim-3.
• RNA and DNA aptamers of (2) described above in the present invention can be obtained by evaluating the effects on the binding of Tim3 to galectin-9, and by selecting RNA and DNA aptamers inhibiting the binding of Tim-3 to galectin-9.
• the substances, which block the signaling pathway of galectin-9 — Tim-3 include low molecular substances besides anti-galectin-9 antibody, anti-Tim-3 antibody, and RNA and DNA aptamers in the present invention. Since galectin-9 exerts its biological activity via its receptor, Tim-3, the low molecular substances (1) inhibiting the binding of galectin-9 to Tim-3 by binding galectin-9, (2) having higher binding affinity to Tim-3 than galectin-9 and inhibiting the binding of galectin-9 to Tim-3 competitively, and (3) not only abolishing the binding ability of Tim-3 to galectin-9, but also destructing the receptor function through inducing the change in three dimensional structure of Tim-3 by binding tightly to Tim-3, can be used as low molecular substances blocking the signaling pathway described above. Among them, the substances of (3) are useful as the low molecular substances with high possibility exerting high autoimmune suppressive effects like anti-Tim-3 antibody.
• autoimmune diseases from systemic lupus erythematosus with autoimmune reactions occurring systemically to diseases with autoimmune reactions occurring at restrict sites, for example, type 1 diabetes occurring at pancreatic islets, rheumatoid arthritis occurring at joints, Sjogren syndrome occurring at lacrimal glands and salivary glands, and so forth.
• the prophylactic and therapeutic agents containing active substances in the present invention exert high therapeutic effects on Thl-mediated autoimmune diseases, they are, therefore, useful in prevention and treatment of such Thl-mediated autoimmune diseases besides type 1 diabetes as rheumatoid arthritis, systemic lupus erythematosus , antiphospholipid antibody syndrome, polymyostis, dermatomyositis, systemic sclerosis, Sjogren syndrome, mixed connective tissue disease, adult onset Still's disease, systemic vascultis (aortitis, polyarteritis nodosa, microscopic polyangitis, ANCA-associated vasculitis), Wegner granulomatosis, sarcoidosis, Castleman disease, Behcet's disease, IgA nephropathy, membrane nephropathy, rapidly progressive glomrulonephritis, Basedow-disease (Grave's disease), ulcerative colitis, Crohn's disease, Guillan-Barre syndrome, multiple
• An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases comprising a substance blocking the signaling pathway of galectin-9 — Tim-3 as an active ingredient.
• a method of prophylactic and therapeutic treatment for immunosuppression or autoimmune diseases comprising administering to a subject in need thereof an effective amount of a substance blocking the signaling pathway of galectin-9 — Tim-3.
• 1-3 Use of a substance blocking the signaling pathway of galectin-9 — Tim-3 in the preparation of a medicament used for prophylactic and therapeutic treatment for immuno-suppression or autoimmune diseases.
• (2-2) A method of (1-2) comprising administering to a subject in need thereof an effective amount of anti-Tim-3 antibody.
• RNA and DNA aptamer inhibiting the binding of galectin-9 to Tim-3 through binding specifically to galectin-9 are the active ingredients, respectively.
• autoimmune diseases include helper T cell subtype 1 (ThI) mediated diseases such as type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus , antiphospholipid antibody syndrome, polymyostis, dermatomyositis, systemic sclerosis, Sjogren syndrome, mixed connective tissue disease, adult onset Still's disease, systemic vascultis (aortitis, polyarteritis nodosa, microscopic polyangitis, ANCA-associated vasculitis), Wegner granulomatosis, sarcoidosis, Castleman disease, Behcet's disease, IgA nephropathy, membrane nephropathy, rapidly progressive glomrulonephritis, Basedow-disease (Grave's disease), ulcerative colitis, Crohn's disease and Guillan-Barre syndrome, multiple sclerosis,
• ThI helper T cell subtype 1
• Tim-3 b) The process to detect the binding of a low molecular substance to Tim-3, c) The process to evaluate the inhibition of the binding of Tim-3 to galectin-9 and the abolishment of the reactivity of Tim-3 to anti-Tim-3 antibody by the binding of the low molecular substance to Tim-3.
• Prophylactic and therapeutic agents for a lot of helper T cell subtype 1 (ThI) mediated-autoimmune diseases, including type 1 diabetes can be provided by the substances blocking the signaling pathway of galectin-9 — Tim-3 and suppressing autoimmune.
• anti-Tim-3 antibody is effective for prevention and treatment of autoimmune diseases by suppressing the production of inflammatory cytokines such as TNF- ⁇ through blocking Tim-3 pathway on dendritic cells, and by exerting anti- inflammatory effects through suppressing the downstream ThI reactions.
• anti-Tim-3 antibody enhances the apoptosis- inducing effect of galectin-9 in the presence of ineffective quantities of galectin-9.
• FIG. 3 A figure illustrating the suppressive effect of the administration of galectin-9 on the onset of diabetes in NOD mouse.
• FIG. 5 A figure illustrating the difference in insulin expression in pancreatic islets between galectin-9-administration group and non-administration group of NOD mice (44 week-old).
• FIG. 1 A figure illustrating the cell fractions among CD4+, CD8, CD4+CD25+ and CD4+Tim3+ cells, in which apoptosis is induced by administrating galectin-9 to NOD mouse.
• FIG. 7 A figure illustrating inhibition of the bindings of galectin-9 and BALB type Tim-3, and galectin-9 and B6 type Tim-3 by each anti-galectin-9 antibody (RG9-35) and anti-Tim-3 antibody (RMT3-23).
• FIG- H A figure illustrating inducing action of cultured ThI cell-apoptosis by concomitant addition of a concentration (0.04 ⁇ M; low concentration) of galectin-9, which is ineffective concentration, and anti-Tim-3 antibody (RMT3-23).
• galectin-9 natural type galectin-9 produced by human leukocytes and cell lines, which are well known to have an ability producing galectin-9, can be used.
• recombinant mouse galectin-9 and recombinant human galectin-9 can be produced by cloning the known mouse galectin-9 cDNA (Wada J., and Karwar YS.: J. Biol. Chem. 272:
• galectin-9 s-type human galectin-9 cDNA (Tureci O., et al.: J. Biol. Chem. 272: 6416-6422, 1997) and by gene engineering using mammalian cells or E.coli as host cells.
• the galectin-9 molecules with modifications of deletion, addition and substitution of its partial amino acid sequences, which have the activity, namely the binding ability to Tim-3, can be used.
• human galectin-9 with carbohydrate chains is preferable, but one with a deletion of carbohydrate chains still has almost similar activity.
• Tim-3 Full length Tim-3 with IgV and mucin domains, or soluble Tim-3 (sTim-3) with IgV domain and a deletion of mucin domain can be used as Tim-3.
• sTim-3 cDNA encoding soluble Tim-3 (sTim-3) which has IgV domain and a deletion of mucin domain, can be prepared from the flTim cDNA obtained by cloning DNA encoding full length Tim-3 (Monney L., et al.: Nature 415:536-541, 2002).
• Recombinant Tim-3 flTim-3 and sTim-3 can be produced by expressing these Tim-3 cDNAs using mammalian cells as a host.
• flTim-3 or sTim-3 can be used as flTim-3-Ig or sTim-3 Ig fusion protein, which is prepared by expressing the fusion protein, in which Fc of immunoglobulin (Ig) is linked to fTr ⁇ m-3 or sTim-3.
• the Tim-3 molecules with modifications of deletion, addition and substitution of its partial amino acid sequences, which have receptor activity as Tim-3 such as sTim-3 can be used.
• human Tim-3 (flTim-3 and sTim-3) is preferable.
• Mouse antibody, rat antibody, rabbit antibody, sheep antibody, chimera antibody, humanized antibody, human antibody, and so on can be used appropriately for antibody against galectin-9 (anti-galectin-9 antibody) and antibody against Tim-3 (anti-Timi-3 antibody), being used as prophylactic and therapeutic agents in the present invention.
• Both polyclonal and monoclonal antibody can be used, but monoclonal antibody is preferable in producing stably as a homogenous antibody.
• Polyclonal and monoclonal antibody can be produced by the methods known to ordinary skilled persons in the art.
• Hybridoma producing monoclonal antibody can be obtained by fusing immune cells (for example, spleen cells), which are prepared by the usual immunization method using galectin-9 or Tim-3 (Tim-3-Ig fusion protein can be used) as an antigen, to the known myeloma cell line according to the usual cell fusion method, and by the usual screening method.
• immune cells for example, spleen cells
• Tim-3 Tim-3-Ig fusion protein can be used
• the preparation of hybridoma can be carried out usually according to the known method of Milstein et al. (Kohler G. and Milstein C: Methods Enzymol. 73: 3 - 46, 1981).
• immunization can be performed by forming the conjugates comprising antigen and keyhole limpet hemocyanin (KLH).
• KLH keyhole limpet hemocyanin
• recombinant antibodies which are produced by cloning antibody genes from hybridomas producing the aimed monoclonal antibodies, using the Chinese hamster ovary cells (CHO cells) and NSO cells as hosts, in which their safety has been established, and using genetic engineering, can be used (Carl A., et al.: THERAPEUTIC MONOCLONAL ANTIBODIES, 1990, United Kingdom, published by MACHILLAN PUBLISHERS LTD.).
• the cloning of antibody gene from hybridoma can be carried out by synthesizing cDNA encoding variable region (V region ) of antibody from mRNA obtained from hybridoma using reverse transcriptase.
• DNA encoding the aimed antibody is obtained, this DNA encoding V region is linked to the desired constant region (C region) of antibody.
• C region constant region
• antibody gene is incorporated into expression vector to express under expression regulation region, for example, under regulation of enhancer and promoter.
• Recombinant antibody can be produced by introducing thus prepared expression vector containing antibody gene into an appropriate host, and by culturing the transformed host.
• chimeric or humanized antibody with reduced immunogenicity against human is preferable and human antibody without any antigenicity to human is much more preferable.
• Chimeric antibody is an antibody that variable regions in H chains and L chains of antibody of mammalians except human, for example, mouse, are linked to constant regions of H and L chains of human antibody, respectively.
• DNA encoding variable regions of mouse antibody is linked to DNA encoding constant regions of human antibody.
• Chimeric antibody is obtained by incorporating this chimeric DNA into an appropriate expression vector, introducing this chimeric DNA into a host, and culturing the host. Because this chimeric antibody contains 33% of amino acid sequence residues derived from mouse antibody, when administered to human, antibody against the chimeric antibody may appear.
• Humanized antibody can be prepared by the method grafting complementary determining regions (CDR) of antibody of mammalians except human, for example, mouse, into complementary determining regions of human antibody, namely a general gene manipulation method called CDR grafting.
• CDR complementary determining regions
• the preparation of humanized antibody by CDR grafting can be carried out by synthesizing DNA sequences designed to link CDR of mouse antibody to framework region (FR) of human antibody by PCR method using several oligonucleotides prepared to have overlap regions at terminal sites of the DNA sequences, linking thus obtained DNAs to DNAs encoding constant regions of human antibody, subsequently incorporating the resulted DNAs into an expression vector, and expressing through inducing the expression vector into a host (EP 239400).
• FR regions of human antibody linked via CDR are selected to form good complementary determining regions.
• humanized antibody prepared by CDR grafting still has about 10% of amino acid sequence derived from mouse antibody due to the difference in framework between mouse and human antibody.
• humanized antibody When administered to human, humanized antibody would hardly raise antibody against it, because it has extremely low antigen sites derived from mouse antibody compared to chimeric antibody.
• affinity of humanized antibody against antigen is decreased by CDR grafting compared to that of original mouse antibody, if necessarily, substitution of amino acids in framework regions of variable regions of antibody would be performed so that the complementary determining regions of humanized antibody can form suitable antigen determining sites (Sato, K., et al.: Cancer Res. 53: 851-856, 1993).
• Human antibody can be obtained by the known technology.
• the aimed human antibody can be obtained by immunizing human lymphocytes with an antigen in vitro, fusing the immunized lymphocytes to a human myeloma cell line, for example, U266, and selecting hybridoma to secrete antibody with binding affinity to an antigen (JP-1-059878B1).
• human antibody can be obtained by immunizing transgenic mouse, which possesses all repertoires of human antibody genes, with an antigen (WO92/03918, WO93/12227, WO94/25585, WO96/34096).
• KM mouse producing complete human antibody has been established.
• variable regions of human antibody are expressed by phage display method as a single chain Fv (scFV), and phages binding to antigen can be selected by panning.
• DNA sequence encoding variable regions of human antibody can be determined by analyzing genes of the selected phages. If DNA encoding scFV specific to an antigen is obtained, human antibody can be prepared by incorporating the said DNA sequence into an appropriate expression vector using the known technologies (WO92/01047, WO93/06213, WO95/01438), and by expressing the said gene.
• the cloning method (Evec method) can be used, in which peripheral B lymphocytes derived from normal subjects and patients are infected and immortalized by EB virus, and B lymphocytes producing autoantibody against such proteins as cytokines are cloned by culturing the immortalized B lymphocytes.
• the SICREX (single-cell RT-PCR) method can be also used, in which without culturing peripheral B lymphocytes derived from normal subjects and patients, cDNA is synthesized and amplified from one B lymphocyte, and antibody gene encoding antibody specific to the aimed protein is cloned by using in vitro translation system.
• Autoantibody produced by peripheral B lymphocytes derived from normal subjects and patients has characteristics being not only a complete human antibody, but also an antibody with extremely high affinity to antigen.
• an appropriate combination of host and expression vector can be used.
• animal cells in the case of using eukaryotic cells as a host, animal cells, plant cells and fungal cells can be used.
• host cells (1) mammalian cells, for example, CHO, NSO, COS, BHK, Vero and so on; (2) amphibian cells, for example, xenopus oocyte, or; (3) insect cells, for example, sf9, sf21, Tn5 and so on, are known.
• plant cells cells derived from Nicotiana family, for example, Nicotiana tabacum, are known, and recombinant antibody can be produced by the callus culture, using these cells as a host.
• yeast for example, Saccharomyces cerevisiae in Saccharomyces family
• fungus for example, Aspergillus niger in Aspergillus family
• prokaryotic cells as bacteria, E. coli and Bacillus subtilus are known.
• an expression vector inserted the aimed antibody gene is introduced into E. coli, and subsequently antibody can be prepared from the extract of bacteria obtained by tank culture of the transformed E. coli.
• the substances that block the signaling pathway of galectin-9 — Tim-3 in the present invention include not only anti-galectin-9 and anti-Tim-3 antibody, but also RNA and DNA aptamers.
• Active ingredients of immunosuppressive agents and prophylactic and therapeutic agents for autoimmune may be RNA and DNA aptamers with characteristics (1) binding specifically to galectin-9 and inhibiting the binding of galectin-9 to Tim-3; and (2) binding specifically to Tim-3 and inhibiting the binding of Tim-3 to galectin-9.
• RNA and DNA aptamers in the present invention for example, the SELEX method (WO91/19813, USP5270163, JP27639598, EP0786469B1) using RNA library, the rapid and effective method for obtaining aptamers using microarray developed for analysis of RNA expression (WO2004/087919), and the method for adding DNAs to a target protein immobilized on membrane and recovering DNA binding specifically to the target protein (JP-2005-200823A1) can be used. Furthermore, regarding RNA and DNA aptamers binding specifically to galectin-9, firstly, the SELEX method (WO91/19813, USP5270163, JP27639598, EP0786469B1) using RNA library, the rapid and effective method for obtaining aptamers using microarray developed for analysis of RNA expression (WO2004/087919), and the method for adding DNAs to a target protein immobilized on membrane and recovering DNA binding specifically to the target protein (JP-2005-200823A1) can be used. Furthermore
• Galectin-9 is incubated for certain time in the presence or absence of an aptamer, subsequently,
• reaction mixture is added to the immobilized Tim-3 (flTim-3-Ig or sTim-3 -Ig fusion protein can be used.) and then incubated for certain time.
• immobilized Tim-3 flTim-3-Ig or sTim-3 -Ig fusion protein can be used.
• RNA and DNA aptamer of (1) described above in the present invention can be obtained by selecting an aptamer inhibiting the binding of galectin-9 to Tim-3.
• RNA and DNA aptamers binding specifically to Tim-3 firstly, firstly,
• Tim-3 (flTim-3-Ig or sTim-3-Ig fusion protein can be used.) is incubated for certain time in the presence or absence of an aptamer, subsequently,
• reaction mixture is added to the immobilized galectin-9, and incubated for certain time.
• the substances that block the signaling pathway of galectin-9 — Tim-3 in the present invention include not only anti-galectin-9 antibody, anti-Tim-3 antibody, and RNA and DNA aptamers, but also low molecular substances. Since galectin-9 exerts its biological activity via binding to its receptor, Tim-3, as low molecular substances blocking the signaling pathway described above, (1) a low molecular substance binding to galectin-9 and inhibiting the binding of galectin-9 to Tim-3, (2) a low molecular substance inhibiting the binding of galectin-9 to Tim-3 competitively, and (3) a low molecular substance inhibiting the binding of Tim-3 to galectin-9 and abolishing receptor function by destructing three dimensional structure through binding tightly to Tim-3 are nominated. Among them, a low molecular substance of (3) may be useful for exerting high immunosuppressive activity like anti-Tim-3 antibody.
• a low molecular substance of (1) described above can be screened by (a) bringing each of low molecular substances into contact with galectin-9, (b) detecting the binding ability of a low molecular substance to galectin-9 by determining the decrease in reactivity in a galectin-9 ELISA, and, further, (c) evaluating the binding ability of the low molecular substance bound-galectin-9 to Tim-3 by an ELISA against immobilized Tim-3.
• a low molecular substance of (2) can be screened by (a) bringing galectin-9 into contact with Tim-3 (flTim-3-Ig or sTim-3-Ig fusion protein can be used), (b) bringing each of low molecular substances into contact with the binding complex of galectin-9 and Tim-3, and (c) evaluating the decrease in quantity of galectin-9 bound to Tim-3 by adding a low molecular substance using a galectin-9 ELISA.
• a low molecular substance of (3) can be screened by (a) bringing each of low molecular substances into contact with Tim-3 (flTim-3-Ig or sTim-Ig fusion protein can be used), (b) detecting the binding of a low molecular substance to Tim-3, and subsequently (c) evaluating inhibition of the binding of Tim-3 to galectin-9, and abolishment of the reactivity of Tim-3 to anti-Tim-3 antibody, which is an indicator for detecting destruction of three dimensional structure of Tim-3, by the binding of the low molecular substance to Tim-3, using a Tim-3 ELISA.
• the prophylactic and therapeutic agents which contain the substances blocking the signaling pathway of galectin-9 — Tim-3 as active ingredients in the present invention, are useful in prevention and treatment of autoimmune diseases including type 1 diabetes.
• autoimmune diseases from systemic lupus erythematosus with autoimmune reactions occurring systemically to diseases with autoimmune reactions occurring at restrict sites, for example, type 1 diabetes occurring at pancreatic islets, rheumatoid arthritis occurring at joints, Sjogren syndrome occurring at lacrimal glands and salivary glands and so on.
• anti-galectin-9 antibody and anti-Tim-3 antibody significantly suppress the onset of diabetes in a model mouse (NOD mouse) developing type 1 diabetes spontaneously, which is one of diseases involving the activation of ThI cells.
• anti-Tim-3 antibody exerts the marked autoimmune suppressive effects.
• the prophylactic and therapeutic agents containing the substances of the present invention as an active ingredient exert strong therapeutic effects on Thl-mediated autoimmune diseases, and are useful in prevention and treatment of Thl-mediated autoimmune diseases besides type 1 diabetes such as rheumatoid arthritis, systemic lupus erythematosus , antiphospholipid antibody syndrome, polymyostis, dermatomyositis, systemic sclerosis, Sjogren syndrome, mixed connective tissue disease, adult onset Still's disease, systemic vasculitis (aortitis, polyarteritis nodosa, microscopic polyangitis, NACA associated vasculitis), Wegner granulomatosis, sarcoidosis, Castleman disease, Behcet's disease, IgA nephropathy, membrane nephropathy, rapidly progressive glomrulonephritis, Basedow-disease (Grave's disease), ulcerative colitis, Crohn's disease, Guillan-Barre syndrome, multiple s
• suspending agents can be appropriately added to the prophylactic and therapeutic agents for autoimmune diseases including type 1 diabetes in the present invention (hereafter, described as the formulations in the present invention).
• methylcellulose, polysorbate 80, polysorbate 20, polyoxyethylene sorbitanmonolaurate and so on can be exemplified.
• dextran 40 As a stabilizing agent, dextran 40, methylcellulose, gelatin, human serum albumin and so on can be exemplified.
• an isotonic agent for example, D-mannitol, sorbitol and so on can be shown.
• an absorption-preventing agent human serum albumin, gelatin, low molecular gelatin, polysorbate 80, polysorbate 20, dextran, methylcellulose and so on can be exemplified.
• non-ion surface active agents for example, sorbitan fatty acid esters such as sorbitanmonolaurate, sorbitanmonopalmitate and so on ; glycerin fatty acid esters such as glycerylmonocaprate, glycerylmonomyristate, glycerylmonostearate and so on; polyglyceryl fatty acid esters such as monostearate, decaglycerylmonolinorate and so forth; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylenesorbitanmonostearate, polyoxyethylenesorbitanmonopalmitate and so on, are exemplified.
• sorbitan fatty acid esters such as sorbitanmonolaurate, sorbitanmonopalmitate and so on
• glycerin fatty acid esters such as glycerylmonocaprate, glycerylmonomyristate, glycerylmonostearate and so on
• anionic surface active agent for example, alkyl sulfates such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate and so on; polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylenelauryl sufate and so forth; natural surface active agents, for example, glycerophospholipids such as lecithin and sphingophospholipids such as sphingomyelin and so on, can be shown as typical examples.
• glycerophospholipids such as lecithin and sphingophospholipids such as sphingomyelin and so on
• One kind of these surface active agents, or a combination of more than two kinds of these surface active agents can be added to prepare the formulations in the present invention.
• preferable surface active agents are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60 and 80, and polysorbate 20 and 80 are especially preferable.
• polyoxyethylenepolyoxypropyleneglycol representing polyoxymers is often used.
• N-acetylcysteine N-acetylcysteine, thioctic acid, thiodiglycol, thioglycerol, glutathione and so on can be exemplified.
• antioxidant for example, erythorbic acid, ⁇ -tocopherol, L-ascorbic acid, and their salts, L-ascorbic acid palmitate, L-ascorbic acid stearate and so on can be shown.
• inorganic salts such as sodium phosphate, sodium bicarbonate and so on; organic salts such as sodium citrate, potassium citrate, sodium acetate and so on, can be added as a pH adjuster.
• the formulations containing antibodies of the present invention as active ingredients are usually administered as injection drugs (subcutaneously, intradermally, intramuscularly, intravenously and intraperitoneally).
• the formulations can be also administered as suitable drug forms for percutaneous, transmucosal and nasal administrations.
• the formulations containing low molecular substances in the present invention as active ingredients are administered as injection drugs (subcutaneously, intradermally, intramuscularly and intraperitoneally).
• the formulations can be also administered as drug forms suitable for percutaneous, transmucosal and nasal administrations, preferably, drug forms (tablets, capsules, granules, liquids, suspensions) suitable for oral administration.
• the present invention is not limited by administration routes and drug forms.
• the prophylactic and therapeutic agents containing antibodies in the present invention as active ingredients are suitable for intravenous, subcutaneous and intramuscular injections.
• the dosages in that case, which ordinary skilled persons in the art can appropriately determine in consideration of conditions of patients with autoimmune diseases, are usually selected from ranges of 1 - 1000 mg/body weight/ month.
• the prophylactic and therapeutic agents containing low molecular substances in the present invention as active ingredients are preferably administered intravenously, subcutaneously, or orally and the dosages that ordinary skilled persons in the art can appropriately determine in consideration of the conditions of patients with autoimmune diseases, are usually selected from ranges of 1 - 1000 mg/body weight/day.
• the present invention is explained in detail by following examples, but not limited to these examples.
• Example-1 Expression of galectin-9 in pancreatic islets. 1) Comparison of galectin-9 expression in pancreatic islets between NOD and ICR mouse
• the immunostaining was carried out by the following methods. Paraffin-fixed tissues were deparaffinized by washing with 100% xylene 3 times every 5 min. After that, the tissues were hydrophylized by washing with 100% ethanol three times every 5 min, with 90% ethanol for 5 min, with 80% ethanol for 5 min, with 70% ethanol for 5 min. After washing, endogenous peroxidase in the tissues was blocked with 0.3% hydrogen peroxide and methanol, and subsequently with normal rabbit serum (10%) derived from the secondary antibody. Using AVIDIN-BIOTIN BLOCKING KIT (SP-2001) obtained from Vector Inc. (Burlingame, CA, US), Avidin blocking (15 min) and Biotin blocking (15 min) of the tissues was carried out, respectively.
• the tissues were incubated in 1:50 dilution of goat anti-galectin-9 polyclonal antibody (M-20) (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 0 C. Further, after reacting with 1:50 dilution of biotinylated anti-goat IgG (H+L) antibody (Vector Inc, BA- 7000) as the secondary antibody, the tissues were reacted with the third antibody in ABC Kit from Vector Inc. The tissues were reacted with DAB reagent (Vector Inc.) for one min, subsequently, counter-stained with Mayer's hematoxylin for 5 sec and then colored by immersing in 40 0 C water. Further, the tissues were dehydrated with ethanol and xylene, and subsequently embedded with M X (Matsunami Garasukougyou Company).
• pancreatic islets The results of immunostaining of pancreatic islets are shown in Figure 1. Comparison of immunostaining of pancreatic islets between 7-week old-NOD and -ICR mouse revealed expression of galectin-9 in each mouse as shown in Figure 1. 2) Effects of various cytokines on expression of galectin-9 in MIN6, a pancreatic ⁇ -cell line
• MIN6 pancreatic ⁇ -cell line
• DMEM Dulbecco's modified Eagle's medium
• FBS penicillin, streptomycin, glucose (450 mg/dl)
• glutamine glutamine
• the medium was exchanged with DMEM containing 15% FBS, penicillin, streptomycin, glucose (100 mg/ml) and glutamine, and the cells were stimulated with each of 20 ng/ml of IFN- ⁇ , lng/ml of IL-l ⁇ and 5 ng/ml of TNF- ⁇ , and each of combinations of these cytokines for 24 hr.
• the cells were cultured under the conditions using DMEM (D5796) containing 15% FBS, penicillin, streptomycin, glucose (450 mg/ml) and glutamine.
• the Nylon filter was hybridized with cDNA (1 x 10 6 cpm) at 68 °C for two hours using ExpressHyb Hybridization Solution (Clontech, Palo Alto, Cam US).
• the Nylon filter was washed with 1 x SSC (standard sodium citrate)/0.1% SDS (sodium dodecyl sulfate) four times (24 0 C) and with 0.1 x SSC/0.1% SDS twice (68 0 C), and autoradiography of the filter was carried out (Hyperfilm MP, GE Bioscience). The results are shown in Figure 2
• Example 2 Effect of the administration of galectin-9 on the onset of diabetes in model mouse (NOD mouse) developing type 1 diabetes spontaneously.
• the protocol (1) for administration experiments is shown as follows.
• the onset of diabetes is defined as when blood glucose levels reach more than 250 mg/dl for consecutive 2 weeks by determining the levels once every week.
• mice After developing diabetes (because mice die from ketoacidosis), mice are killed immediately and served for experiments.
• Formalin-fixed tissues were deparaphinized by washing with 100% xylene three times every 5 min. After that, the tissues were hydrophilized by washing with 100% ethanol 3 times every 5 min, with 90% ethanol for 5 min, with 80% ethanol for 5 min, with 70% ethanol for 5 min. After washing, endogenous peroxidase in the tissues was blocked with 0.3% hydrogen peroxide and methanol. The tissues were blocked with normal goat serum (10%).
• AVIDIN-BIOTIN BLOCKING KIT SP-2001 obtained from Vector Inc. (Burlingame, CA, US), Avidin blocking (15 min) and Biotin blocking (15 min) of the tissues was performed.
• the tissues were incubated in 1:500 dilution of Polyclonal Guine Pig Anti-Swine (DAKO Inc.: A 0564) at 4 0 C overnight.
• the tissues were stained with 1:150 dilution of Biotinylated Anti-Guinea Pig IgG
• the tissues were reacted with the third antibody in ABC Kit from Vector Inc.
• the tissues were reacted with DAB reagent (Vector Inc.) for one min, subsequently, counter-stained with Mayer's hematoxylin for 5 sec and then colored by immersing in 40 0 C water. Further, the tissues were dehydrated with ethanol and xylene, and subsequently embedded with M-X (Matsunami Garasukougyou Company, JP).
• pancreatic islets The histological findings of pancreatic islets are shown in Figure 4.
• the comparison revealed the marked cell infiltration into pancreatic islets and the decrease in insulin expression in PBS-administration group. From these findings, the suppressive effect of galectin-9 on the onset of diabetes was confirmed histologically.
• Example 3 Analysis of cells which galectin-9 acts on
• lymphocytes were immediately analyzed by a BD FACSAria Cell Sorter.
• Figure 5 shows a population of CD4+ and CD8+ in NOD and ICR mouse.
• CD4+ cell fractions including CD4+CD25+ and CD4+Tim3+ were investigated.
• the comparison of ICR mouse and NOD mouse revealed a high tendency of CD8 positive cells in NOD mouse.
• significant changes in the population by the administration of galectin-9 were not observed.
• mouse galectin-9 was produced using pTrHis vector (Invitrogen, San Diego, CA, US). Galectin-9 was produced as a fusion protein having c-myc epitope and (His)6 at its C-terminus.
• ToplO bacteria host (Invitrogen Inc.) was transformed with the vector (hereafter, described as "pTrcHis2/G9") containing DNA encoding mouse galectin-9.
• the transformed bacteria colony was cultured in Luria-Bertani's medium and protein synthesis was induced by adding 1 mmol/L of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) to the culture medium.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• Tris-DTT buffer [20 mmol/L Tris (pH 7.4), 5 ml/L ethylenediaminetetraacetic acid (EDTA), 150 mmol/L sodium chloride, 1 mmol/L] containing 1% Triton X-100, 10 mmol/L benzamidine, 10 mmol/L, ⁇ -amino-n-caproic acid and 2 mmol/L phenylmethanesulfonyl fluoride.
• Tris-DTT buffer 20 mmol/L Tris (pH 7.4), 5 ml/L ethylenediaminetetraacetic acid (EDTA), 150 mmol/L sodium chloride, 1 mmol/L] containing 1% Triton X-100, 10 mmol/L benzamidine, 10 mmol/L, ⁇ -amino-n-caproic acid and 2 mmol/L phenylmethanesulfonyl fluoride.
• the cell lysate was centrifuged at 2,000 x g at 4 0 C for 30min.
• the upper layer after centrifugation was added to lactosyl-Sepharose column with a size of 10 ml (Sigma, St. Louis, MO, US).
• fusion protein was eluted from the column with Tris-DTT buffer containing 200 mmol/L lactose.
• the elute fraction was dialyzed against PBS containing 1 mmol/L DTT, and stored at -70 0 C. Analysis by
• mouse Tim-3-Ig fusion proteins (mouse Tim-3-Ig) comprising each of extracellular domains (1-191 amino acid residues) from mouse Tim-3 (BALB-type) (Monney L., et akl.: Nature 415: 536-541, 2002) and mouse Tim-3 (B6 type), a major variant of Tim-3, and Fc region of mouse IgG2a were stably expressed in CHO cells and were prepared by purifying from the culture medium of CHO cells by protein G column chromatography, respectively (Oikawa T., et al.: J. Immunology 177: 4281-4287, 2006).
• Immunization was performed by injecting 100 ⁇ g of recombinant mouse galectin-9 (rmgalectin-9) with complete Freund's adjuvant for the first immunization, and 100 ⁇ g of rmgalectin-9 with incomplete Freund's adjuvant for the second and third immunizations into footpads of SD rats every two weeks for 3 times. 7 days after final immunization, popliteal lymph node cells were fused to P3U1, a myeloma cell line, and antibody activity in culture supernatant of hybridoma was screened by an ELISA against immobilized mouse recombinant galectin-9. By cloning, a hybridoma producing anti-galectin-9 antibody (RG9-35) stably was established.
• hybridomas were prepared similarly after immunization of SD rats as described above.
• Hybridoma producing antibody against each of BALB type Tim-3 and B6 type Tim-3 was screened by FACS using NRK cells expressing full length mouse Tim-3 stably.
• Stable hybridoma producing an antibody (RMT3-23), which recognizes both BALB type Tim-3 and B6 type Tim-3, was established.
• Example 5 Blocking the signaling pathway of galectin-9 - Tim-3 by anti-galectin-9 and anti-Tim-3 antibody.
• anti-galectin-9 antibody (RG9-35) suppressed apoptosis of ThI cells induced by galectin-9, but anti-Tim-3 antibody (RMT3-23) hardly did.
• anti-Tim-3 antibody (RMT3-23) inhibits the binding of Tim-3 to galectin-9, but does not suppress apoptosis of ThI cells induced by galectin-9, suggesting that apoptosis of ThI cells by galectin-9 is not solely mediated by Tim-3.
• Example 6 Effects of anti-galectin-9 and anti-Tim-3 antibodies on the onset of diabetes in NOD mouse. Galectin-9 has suppressive effect on the onset of type 1 diabetes, and its mechanism is considered that apoptosis of ThI cells is mediated by action of galectin-9 on matured ThI cells (CD4+Tim-3+).
• Protocol (2) (a) Anti-galectin-9 antibody or anti-Tim-3 antibody is injected intraperitoneally into NOD mice at a dose of 0.25 mg per mouse twice every week, for 43 weeks from 8- to 51-week old. (b) As in the case of galectin-9-administration experiment as shown in Example 2, the onset of diabetes is defined as when blood glucose levels reach more than 250 mg/dl for consecutive 2 weeks by determining the levels once every week.
• mice After developing diabetes, mice are killed immediately and served for various experiments
• anti-galectin-9 and anti-Tim-3 antibody exerted unexpected effects in vivo experiments. Its mechanism or cause is currently unknown, but is considered as follows.
• the present inventors have confirmed that galctin-9 stimulates TNF- ⁇ production from dendritic cells, being antigen-presenting cells, besides the induction of activated ThI cell-apoptosis (data not shown). In addition, it has been reported that galectin-9 causes inflammation via TNF- ⁇ production from dendritic cells (Anderson AC, et al.: Science 318: 1141-1143, 2007).
• the actions of galectin-9 on different cells exert suppressive effect on activated ThI cells, and have stimulatory effect on dendritic cells. Therefore, it is explained that the suppressive effect on the onset of diabetes by the administration of galectin-9 is almost the same as the therapeutic effect through suppression of inflammatory process via dendritic cells stimulated with galectin-9 by the administration of anti-galectin-9 antibody.
• anti-Tim-3 antibody suggests that (1) a binding site(s) except galectin-9 in the ligand binding sites of Tim-3, namely a signaling pathway besides galectin-9 -Tim-3 is present, and anti-Tim-3 antibody blocks the signaling pathway(s), because anti-Tim-3 antibody (RMT3-23) does not suppress the induction of apoptosis of ThI cells by galectin-9 as shown in Figure 8, and (2) a Tim-3 pathway (s) on dendritic cells locating upstream of inflammation mechanisms is present, and the blocking of the pathway is important for treatment of autoimmune diseases.
• Example 7 Effect of anti-Tim-3 antibody on ThI cell-apoptosis induced by galectin-9.
• anti-Tim-3 antibody (RMT3-23) hardly affected apoptosis of ThI cells cultured in the presence of galectin-9 (0.2 ⁇ M).
• anti-Tim-3 antibody in the presence of effective dose (0.2 ⁇ M) and ineffective dose (0.04 ⁇ M) of galectin-9 for inducing ThI cell-apoptosis was investigated.
• CD4+ T cells were isolated from spleen of female ICR mice, using CD4 microbeads (Miltenyi).
• CD4+ T cells were inoculated into 24-well plates coated with anti-CD3 mAb (2Cl 1, 10 ⁇ g/ml) at a cell density of 1 x 10 6 cells/well, and incubated for 4 days by adding 10 ⁇ g/ml of anti-CD28 mAb (PV-I), 10 ⁇ g/ml of mouse IL-12 and 10 ⁇ g/ml of anti-IL-4 mAb (11B11) into each well.
• the cells recovered from the plates were re-inoculated into 96-well plates at a cell density of 5 x 10 5 cells /well and incubated for 3 days by adding 20 U/ml of human IL-2 into each well.
• FIG. 10 shows effect of anti-Tim-3 antibody (RMT3-23) on apoptosis-inducing action by a high concentration (0.2 ⁇ M) of galectin-9 (rGal-9). ThI cell-apoptosis was induced by adding rGal-9 (0.2 ⁇ M; high concentration).
• Figure 11 shows effect of RMT3-23 on apoptosis-inducing action by a low concentration (0.04 ⁇ M) of galectin-9 (rGal-9). Namely, ThI cell- apoptosis was not induced by the low concentration of rGal-9, but the dose-dependent enhancement of apoptosis was observed by adding anti-Tim-3 antibody.
• anti-Tim-3 antibody was found to enhance the induction of ThI cell-apoptosis in the presence of ineffective concentrations of galectin-9.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Immunology (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Chemical & Material Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Hematology (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Biochemistry (AREA)
• Diabetes (AREA)
• Cell Biology (AREA)
• Rheumatology (AREA)
• Physical Education & Sports Medicine (AREA)
• Analytical Chemistry (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Endocrinology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Neurology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Heart & Thoracic Surgery (AREA)
• Toxicology (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=42356044

Family Applications (1)

Country Status (2)

Cited By (16)

Families Citing this family (1)

Family Cites Families (4)

• 2010
  • 2010-01-26 WO PCT/JP2010/051306 patent/WO2010084999A1/en active Application Filing
  • 2010-01-26 JP JP2011547342A patent/JP5569946B2/ja not_active Expired - Fee Related

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events