WO2010084999A1 - Immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases - Google Patents

Immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases Download PDF

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WO2010084999A1
WO2010084999A1 PCT/JP2010/051306 JP2010051306W WO2010084999A1 WO 2010084999 A1 WO2010084999 A1 WO 2010084999A1 JP 2010051306 W JP2010051306 W JP 2010051306W WO 2010084999 A1 WO2010084999 A1 WO 2010084999A1
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galectin
tim
binding
antibody
low molecular
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PCT/JP2010/051306
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French (fr)
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Jun Wada
Motoko Kanzaki
Hideo Yagita
Takeo Tanai
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Protegene, Inc.
National University Corporation Okayama University
Juntendo Educational Foundation
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Priority to JP2011547342A priority Critical patent/JP5569946B2/en
Publication of WO2010084999A1 publication Critical patent/WO2010084999A1/en

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    • 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
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    • G01N33/5047—Cells of the immune system
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    • 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
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • 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
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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.

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Abstract

Immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases, which contain the substances (for example, anti-galectin-9 antibody, anti-Tim-3 antibody, RNA and DNA aptamers and low molecular substances) blocking the signaling pathway (galectin-9 -Tim-3 signaling pathway) consisting of galectin-9 as a ligand and Tim-3 as a receptor, as active ingredients, and the screening methods of low molecular substances, being active ingredients of immunosuppressive agents and prophylactic and therapeutic agents for autoimmune diseases.

Description

[DESCRIPTION]
[Title of Invention]
IMMUNOSUPPRESSIVE AGENTS AND PROPHYLACTIC AND THERAPEUTIC
AGENTS FOR AUTOIMMUNE DISEASES [Technical Field]
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. [Background Art]
[Citation List]
[Patent Literature]
[PTL l] JP-2004-244411A1
[PTL 2] JP-2001-501831T [PTL 3] JP-2006-526651T
[PTL 4] JP-2006-525813T
[Non Patent Literature]
[NPL 1] Sahin U., et al: Proc. Natl. Acad. Sci. USA 92: 11810-11813, 1995.
[NPL 2] Wada J. and Kanwar YS. : J. Biol. Chem. 272: 6078-6086, 1997. [NPL 3] Wada J., et al.: J. Clin. Invest. 99: 2452-2461, 1997.
[NPL 4] Puig O., et al.: Methods 24: 218-229, 2001.
[NPL 5] Zhu C, et al.: Nature Immunology 6: 1245-1252, 2005.
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 β-galactoside structure. At present, galectin families from galectin-1 to galectin-14 have been identified. Among galectin families, 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).
It is known that after biosynthesis, 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. 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.
On the process of immunoreactions and inflammatory reactions, 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. In addition, leukocytes, for example, neutrophils, macrophages and lymphocytes infiltrate from blood into inflammatory sites and infection lesions. Galectin-3 is a chemotactic factor for macrophages, and 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). In addition, as 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, 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).
The mechanism by which galectin-9 exerts biological activity was not well known. Especially, a membrane protein, namely galectin-9-binding molecule (galection-9 receptor) which binds galectin-9 and transduces its signal was not identified.
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.
To screen and identify 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). According to this document, a dozen kinds of galectin-9-binding proteins have been identified, including 4F2 heavy chain antigen (177216), ATPase, Na+/K+ transporting, and alpha 1 polypeptide (21361181) and so forth (JP-2004-24441 IAl). The existence of such various kinds of galectin-9-binding proteins suggests that the binding specificity of galectin-9 is extremely low.
On the other hand, Tim-3 (T-cell immunoglobulin and mucin domain protein; also called Hepatitis A virus cellular receptor 2), which is T-helper subtype 1 (Thl)-specific membrane surface protein, is a protein regulating ThI response, and its ligand was unknown. In 2005, galectin-9 was identified as a ligand for Tim-3 (Zhu C, et al., Nature Immunology 6: 1245-1252, 2005). According to the literature, a fusion protein (Tim-3-Ig) of Tim-3 and immunoglobulin Fc(Ig) was prepared, and was incubated at 4 0C by adding the extract of a CD8+ mouse lymphoma cell line, TKl, biotinylated cell surface. Subsequently, the complex of 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. Further, 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).
Since a dozen kinds of galectin-9-binding proteins identified in the prior art described above (JP-2004-24441 IAl) do not include Tim-3, this literature reported in 2005 firstly clarified that 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 γ and suppression of ThI autoimmunity (Zhu C, et al., Nature Immunology 6: 1245-1252, 2005). ThI cells producing interferon-γ 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.
Further, the activation of dendritic cells and ThI cells locating downstream of dendritic cells and the production of ThI cytokines is important for the onset of type 1 diabetes, which is one of autoimmune diseases, resulting in decrease of insulin secretion by inflammation of Langerhans's islets. In prior arts targeting treatment of abnormal immune of type 1 diabetes, "Therapeutic vaccine compositions for treating type 1 diabetes" (JP-2006-526651T) and "Antigens which are targeted by morbific and pathogenic T cells and their use in type 1 diabetes" (JP-2006-525813T) are disclosed. These prior arts do not identify a concrete target of abnormal immune in type 1 diabetes and are less effective, thereby, they do not reach practical use.
There are many 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. However, effective prophylactic and therapeutic methods are not still established, and development of effective new drugs including galectin-9 is desired. [Summary of Invention] [Technical Problem] The inventors of the present invention have found out that substances blocking the signal transduction via binding between galectin-9 as a ligand and its receptor, Tim-3, ("galectin-9 -Tim-3 signaling pathway", or "signaling pathway of galectin-9 -Tim-3") exert unexpected and strong autoimmune suppressive activity in in vivo experiments, and intend to provide drugs and methods for prevention and treatment of many autoimmune diseases including type 1 diabetes, in which helper T cells subtype 1 (ThI) is involved, by suppressing autoimmune by the said substances.
[Solution to Problem] Because it was clarified that Tim-3 is the receptor for galectin-9, the present inventors investigated whether galectin-9 suppresses Thl-mediated-autoimmune in vivo, as shown in Examples described later. The suppressive effect of galectin-9 on the onset of diabetes in mouse model (NOD mice) developing spontaneously type 1 diabetes, one of Thl-mediated autoimmune diseases, was investigated. Consequently, as expected, comparing to PBS-administration group (control), galectin-9 -administration group exhibited significant (p = 0.048) suppressive effect on the onset, namely autoimmune suppressive effect. In addition, when the expression of insulin in 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.
Moreover, when target cells of galectin-9 were investigated by using Flow Cytometry, galectin-9 was found to have the tendency inducing apoptosis of mature ThI cells (CD4+Tim-3+) from NOD mouse.
As shown in Examples described later, to confirm that substances blocking the signaling pathway of galectin-9 -Tim-3 accelerate the onset of type 1 diabetes in 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. As a result, surprisingly, 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. Moreover, comparing to the suppressive effect on the onset of type 1 diabetes in galectin-9-administration group, anti-galectin-9 antibody-administration group performed at the same time exerted almost the same effects, but anti-Tim-3 antibody-administration group was found to exert much higher efficacy, leading to accomplishment of the present invention. It has been clarified that the signal transduction of galectin-9 -Tim-3 induces apoptosis of ThI cells, and suppresses autoimmunity (Zhu C, et al., Nature Immunology 6: 1245-1252, 2005). Based on this fact, it is suggested that galectin-9 suppresses autoimmune by inducing apoptosis of ThI cells. As shown in Examples described later, 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.
Accordingly, 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.
Although the mechanisms by which these unexpected effects were exerted in vivo, or the reasons are not well known, the present inventors consider as follows.
Besides induction of apoptosis of activated ThI cells by galectin-9, the present inventors confirmed that galectin-9 stimulates TNF-α production by dendritic cells which are antigen presenting cells. In addition, it has been reported that galectin-9 causes inflammation via TNF-α 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. 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 by suppression of inflammatory process via galectin-9-stimulated dendritic cells by the administration of anti-galectin-9 antibody. On the other hand, the exertion of much stronger therapeutic effects by anti-Tim-3 antibody suggest 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); and
(2) a 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.
Moreover, 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.
Concerning RNA and DNA aptamers binding specifically to galectin-9, 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. In addition, concerning RNA and DNA aptamers binding specifically 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.
Moreover, 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.
There are many 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.
Because 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 sclerosis, aplastic anemia, rejection after transplantation and graft versus host disease (GVHD) and so on. Namely, the present invention provides the followings:
(1) 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. (1-2) 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.
(1-4) A substance blocking the signaling pathway of galectin-9 — Tim-3 for use in prophylactic and therapeutic treatment for immuno-suppression or autoimmune diseases.
(2) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), comprising anti-Tim-3 antibody as an active ingredient.
(2-2) A method of (1-2) comprising administering to a subject in need thereof an effective amount of anti-Tim-3 antibody.
(2-3) Use of anti-Tim-3 antibody in the preparation of a medicament used for prophylactic and therapeutic treatment for immuno-suppression or autoimmune diseases. (2-4) An anti-Tim-3 antibody for use in prophylactic and therapeutic treatment for immuno-suppression or autoimmune diseases.
(3) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), wherein anti-galectin-9 antibody is the active ingredient.
(4) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (2), wherein said anti-Tim-3 antibody has a characteristic to enhance helper T cell subtype 1 (ThI cell) -apoptosis induced by galectin-9 in the presence of ineffective concentrations of galectin-9.
(5) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), wherein RNA and DNA aptamer inhibiting the binding of galectin-9 to Tim-3 through binding specifically to galectin-9 are the active ingredients, respectively.
(6) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), wherein RNA and DNA aptamer inhibiting the binding of Tim-3 to galectin-9 through binding specifically to Tim-3 are the active ingredients, respectively. (7) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), wherein a low molecular substance inhibiting the binding of galectin-9 to Tim-3 through binding specifically to galectin-9 is the active ingredient. (8) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), wherein a low molecular substance inhibiting the binding of galectin-9 to Tim-3 competitively through binding specifically to Tim-3 is the active ingredient.
(9) An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases described in (1), wherein a low molecular substance abolishing the binding ability of Tim-3 to galectin-9 by destructing three dimensional structure of Tim-3 through binding tightly to Tim-3 is the active ingredient.
(10) An immunosuppressive agent and prophylactic agent and therapeutic agent for autoimmune diseases described in (1), wherein the 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, aplastic anemia, rejection after transplantation and graft versus host disease (GVHD) and so on.
(11) The screening method of a low molecular substance inhibiting the binding of galectin-9 to Tim-3 by binding to galectin-9 as described in (7), which comprises the following processes:
(a) The process to bring each of low molecular substance samples into contact with galectin-9, (b) The process to detect the binding of a low molecular substance to galectin-9,
(c) The process to evaluate the inhibition of the binding of galectin-9 to Tim-3 through the binding of the low molecular substance to galectin-9.
(12) The screening method of a low molecular substance inhibiting the binding of galectin-9 to Tim-3 competitively through binding to Tim-3 as described in (8), which comprises the following processes: a) The process to bring galectin-9 into contact with Tim-3, b) The process to bring each of low molecular substance samples into contact with the galectin-9 bound-Tim-3, c) The process to evaluate the decrease in quantity of galectin-9 bound to Tim-3 by adding a low molecular substance.
(13) The screening method of a low molecular substance abolishing the binding ability of Tim-3 to galectin-9 by destructing three dimensional structure through binding tightly to Tim-3 as described in (9), which comprises the following processes: a) The process to bring each of low molecular substance samples into contact with
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.
[Advantageous Effects of Invention]
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. For example, anti-Tim-3 antibody is effective for prevention and treatment of autoimmune diseases by suppressing the production of inflammatory cytokines such as TNF-α through blocking Tim-3 pathway on dendritic cells, and by exerting anti- inflammatory effects through suppressing the downstream ThI reactions. Furthermore, in induction of ThI cell-apoptosis by galectin-9, anti-Tim-3 antibody enhances the apoptosis- inducing effect of galectin-9 in the presence of ineffective quantities of galectin-9.
Besides type 1 diabetes, the application of prophylactic and therapeutic agents to 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, 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 sclerosis, aplastic anemia, rejection after transplantation and graft versus host disease (GVHD) is assumed. [Brief Description of Drawings]
[Fig- 1]
A figure illustrating the expression of galectin-9 in respective pancreatic islets of NOD mouse, being a spontaneous mouse model of type 1 diabetes, and ICR mouse.
[Fig. 2]
A figure illustrating the effects of various kinds of cytokines on expression of galectin-9 of
MIN6, an established pancreatic β cell line.
[Fig. 3] A figure illustrating the suppressive effect of the administration of galectin-9 on the onset of diabetes in NOD mouse.
[Fig. 4]
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. 5]
A figure illustrating a population of CD4+cells CD8+cells, CD4+CD25+cells and
CD4+Tim3+ cells in NOD mouse and ICR mouse, and the effects of galectin-9 on the population. [Fig. 6]
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. 8]
A figure illustrating the suppression of galectin-9-induced ThI cell death by anti-galectin-9 antibody (RG9-35), but not by anti-Tim-3 antibody (RMT3-23).
[Fig. 9]
A figure illustrating the suppressive effect of each administration of anti-galectin-9 antibody
(RG9-35) and anti-Tim-3 antibody (RMT3-23) on the onset of diabetes in NOD mouse, especially the marked suppressive effect of the administration of anti-Tim-3 antibody (RMT3-23) on the onset of type 1 diabetes.
[Fig. 10]
A figure illustrating no suppressive effect of anti-Tim-3 antibody (RMT3-23) on inducing action of cultured ThI cell-apoptosis by galectin-9 (0.2 μM; high concentration) .
[Fig- H] A figure illustrating inducing action of cultured ThI cell-apoptosis by concomitant addition of a concentration (0.04 μM; low concentration) of galectin-9, which is ineffective concentration, and anti-Tim-3 antibody (RMT3-23).
[Description of Embodiments]
As 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. In addition, 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:
6078-6086, 1997) and 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. Among them, human galectin-9 with carbohydrate chains is preferable, but one with a deletion of carbohydrate chains still has almost similar activity.
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. In addition, 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 fTrϊm-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. Among them, 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. 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). In the case of antigens with low immunogenicity like peptides, immunization can be performed by forming the conjugates comprising antigen and keyhole limpet hemocyanin (KLH). Furthermore, as antibody drugs, 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.). Usually, 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.
If DNA encoding the aimed antibody is obtained, this DNA encoding V region is linked to the desired constant region (C region) of antibody. Thus obtained 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.
In the case of using antibody as a therapeutic agent for human, chimeric or humanized antibody with reduced immunogenicity against human is preferable and human antibody without any antigenicity to human is much more preferable.
The modified antibodies such as chimeric and humanized antibodies can be prepared by using the known method. 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. As a concrete preparation method, 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. 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. However, 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. 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. Generally, since 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. For Example, 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). In addition, 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). For example, KM mouse producing complete human antibody has been established. Moreover, there is a phage display method that makes it possible to obtain human antibody by panning using phage library of human antibody. For example, 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. Furthermore, 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. In contrast to this, 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.
In the case of producing recombinant antibody, an appropriate combination of host and expression vector can be used. In the case of using eukaryotic cells as a host, animal cells, plant cells and fungal cells can be used. As 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. As 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.
As fungal cells, yeast, for example, Saccharomyces cerevisiae in Saccharomyces family and fungus, for example, Aspergillus niger in Aspergillus family, are known. In the case of using prokaryotic cells, as bacteria, E. coli and Bacillus subtilus are known. For example, 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.
As the concrete methods for obtaining 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,
(a) Galectin-9 is incubated for certain time in the presence or absence of an aptamer, subsequently,
(b) The 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.
(c) After that, the quantities of galectin-9 bound to Tim-3 are determined by galectin-9 ELISA, and 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. In addition, regarding RNA and DNA aptamers binding specifically to Tim-3, firstly,
(a) 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,
(b) The reaction mixture is added to the immobilized galectin-9, and incubated for certain time.
(c) After that, the quantities of Tim-3 bound to galectin-9 is determined by Tim-3 ELISA, and RNA and DNA aptamers of (2) described above in the present invention can be obtained by selecting aptamers inhibiting the binding of Tim-3 to galectin-9.
Furthermore, 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.
As a concrete screening method, using low molecular substance libraries, 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. Further, 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. In addition, using low molecular substance libraries, 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. There are many 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.
In the present invention, it has been demonstrated that 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. Especially, anti-Tim-3 antibody exerts the marked autoimmune suppressive effects.
Thus, 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 sclerosis, aplastic anemia, rejection after transplantation and graft versus host disease (GVHD) and so on.
Depending on administration methods and drug types, suspending agents, solubilizing agents, stabilizing agents, isotonicity agents, adsorption-preventing agents, surface active agents, sulfur-containing reducing agents, antioxidants, diluents, excipients, pH adjusters, buffers, and so on 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).
As a suspending agent, methylcellulose, polysorbate 80, polysorbate 20, polyoxyethylene sorbitanmonolaurate and so on can be exemplified.
As a stabilizing agent, dextran 40, methylcellulose, gelatin, human serum albumin and so on can be exemplified.
As an isotonic agent, for example, D-mannitol, sorbitol and so on can be shown. As an absorption-preventing agent, human serum albumin, gelatin, low molecular gelatin, polysorbate 80, polysorbate 20, dextran, methylcellulose and so on can be exemplified.
As a surface active agent, 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. Moreover, as an 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. 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.
Among these many surface active agents, 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. In addition, polyoxyethylenepolyoxypropyleneglycol representing polyoxymers is often used.
As a sulfur-containing reducing agent, N-acetylcysteine, thioctic acid, thiodiglycol, thioglycerol, glutathione and so on can be exemplified.
As an antioxidant, for example, erythorbic acid, α-tocopherol, L-ascorbic acid, and their salts, L-ascorbic acid palmitate, L-ascorbic acid stearate and so on can be shown. Moreover, as a component of buffers, 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). However, the formulations can be also administered as suitable drug forms for percutaneous, transmucosal and nasal administrations. In addition, the formulations containing low molecular substances in the present invention as active ingredients are administered as injection drugs (subcutaneously, intradermally, intramuscularly and intraperitoneally). Further, 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. In addition, 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. Various changes and modifications in the present invention are possible and these modifications are also included in the range of the present invention. [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 0C. 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 0C water. Further, the tissues were dehydrated with ethanol and xylene, and subsequently embedded with M X (Matsunami Garasukougyou Company).
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 β-cell line
Subsequently, MIN6, a pancreatic β-cell line, was stimulated with various cytokines, and effects of these cytokines on expression of galectin-9 were analyzed by northern blot. The concrete experimental method is as follows.
For culturing MIN6 cells, DMEM (Dulbecco's modified Eagle's medium) (D5795) (sigma, St. Louis, MO, US) containing 15% FBS, penicillin, streptomycin, glucose (450 mg/dl), and glutamine was used. MIN6 cells were inoculated into each well in 16- well cell culture cluster (Costar, Corning, NY, US) at a cell density of 1 x 106 cells/ml, and incubated for 3 days. Furthermore, 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-γ, lng/ml of IL-lβand 5 ng/ml of TNF-α, and each of combinations of these cytokines for 24 hr. To check the effect of high glucose concentration, the cells were cultured under the conditions using DMEM (D5796) containing 15% FBS, penicillin, streptomycin, glucose (450 mg/ml) and glutamine. Total RNA was extracted from the cultured cells by using RNAeasy Mini (Qiagen, Hilden, Germany), loaded on electrophoresis using 1% agarose gel containing 2.2 mol/L formaldehyde, and subsequently transferred to Hybond N+Nylon (GE Bioscience, Buckinghamshire, UK). Mouse galectin-9 cDNA was labeled with [alpha-32P]dCTP using Rediprime II DNA Labelling System (GE Bioscience). The Nylon filter was hybridized with cDNA (1 x 106 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 0C) and with 0.1 x SSC/0.1% SDS twice (68 0C), and autoradiography of the filter was carried out (Hyperfilm MP, GE Bioscience). The results are shown in Figure 2
When MIN6 cells were stimulated with the combination of interferon-γ (IFN-γ) and interleukin-lβ(IL-lβ) or TNF-α, or the combination of TNF-α and IL-I, the expression of galectin-9 was observed and the inducing effects on the expression were found to be synergistic. From these results, it was found that galectin-9 is present in pancreatic islets, and its expression is induced by IFN-γ, which is a cytokine produced by ThI cells.
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.
Protocol (1)
(a) Recombinant galectin-9 (1 mg/kg body weight) and PBS-DTT as a control group are administered to NOD mice (n = 20, female) intraperitoneally once every week from 7 to 42 weeks, respectively. (The production and preparation of recombinant mouse galectin-9 was carried out according to Example 4-1 described below.)
(b) 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.
(c) After developing diabetes (because mice die from ketoacidosis), mice are killed immediately and served for experiments.
1) Effect of the administration of galectin-9 on the onset of diabetes
Effect of the administration of recombinant galectin-9 on the onset of type 1 diabetes in NOD mouse is shown in Figure 3, using Kaplan-Meyer's survival curves. As shown in Figure 3, the galectin-9-administration group exhibited significantly (p = 0.048) high suppressive effect on the onset of diabetes compared to the control group at 40-week-old.
2) Effect of the administration of galectin-9 on insulin expression in pancreatic islets Staining of insulin in pancreatic islets was carried out as follows.
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%). 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 performed. The tissues were incubated in 1:500 dilution of Polyclonal Guine Pig Anti-Swine (DAKO Inc.: A 0564) at 4 0C overnight. The tissues were stained with 1:150 dilution of Biotinylated Anti-Guinea Pig IgG
(H+L) (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 0C water. Further, the tissues were dehydrated with ethanol and xylene, and subsequently embedded with M-X (Matsunami Garasukougyou Company, JP).
The histological findings of pancreatic islets are shown in Figure 4. The comparison of insulin expression in pancreatic islets between galectin-9-administration group and PBS-administration group (control group), which did not develop diabetes until 44-week old, revealed the distinct insulin expression in galectin-9-administration group. However, 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
Subsequently, investigation was carried out by using flow cytometry to determine on which cells galectin-9 act. The protocol is shown as follows.
The analyzing protocol for flow cytometry.
(a) 24hr before analyzing, recombinant mouse galectin-9 (1 mg/kg/body weight) and PBS as a control group were administered to 7-week-old NOD mice and ICR mice.
(The production and preparation of recombinant mouse galectin-9 was carried out according to Example 4-1) described below.)
(b) After killing the mice, spleen cells were isolated, and lymphocytes were isolated by using Lymphocyte-M.
(c) After staining, lymphocytes were immediately analyzed by a BD FACSAria Cell Sorter.
1) A population of CD4+ and CD8+ cells in NOD and ICR mouse, and effect of galectin-9 -administration on the population
The analyzing results by FACS are shown in Figure 5.
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. On the other hand, in both ICR and NOD mouse, significant changes in the population by the administration of galectin-9 were not observed.
2) Apoptosis-induced cells by the administration of galectin-9
Apoptosis of each cell fraction of CD4+, CD8+, CD4+CD25 and CD4+Tim3+ was analyzed using Annexin V. The results are shown in Figure 6. In each cell fraction (n=5), the tendency of increased apoptosis of CD4+Tim3+ cells, namely matured ThI (CD4+Tim3+) cells, by the administration of galectin-9 was observed in NOD mouse. However, such tendency was not observed in ICR mouse. Example 4: Preparation of antibody Anti-galectin-9 and anti-Tim-3 antibody, which block the signaling pathway of galectin-9 - Tim-3, were prepared, respectively.
1) Preparation of recombinant mouse galectin-9
(a) Based on the known methods (Wada J., et al.: J. Biol. Chem. 272: 6078-6086, 1997; Wada J., et al.: J. Clin. Invest. 99: 2452-2461, 1997), 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.
(b) 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-β-D-thiogalactopyranoside (IPTG) to the culture medium. Bacteria was lysed with 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, ε-amino-n-caproic acid and 2 mmol/L phenylmethanesulfonyl fluoride.
The cell lysate was centrifuged at 2,000 x g at 4 0C for 30min. The upper layer after centrifugation was added to lactosyl-Sepharose column with a size of 10 ml (Sigma, St. Louis, MO, US). After washing proteins unabsorbed to the column, 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 0C. Analysis by
SDS-PAGE using 12.5% gel revealed that the sample (the elute) showed a single band and had a molecular weight of 39 kDa, being consistent with molecular weight which is calculated from constitutive amino acids of the fusion protein.
2) Preparation of recombinant mouse Tim-3-Ig fusion protein As mouse Tim-3, 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).
1) Preparation of anti-galectin-9 antibody
Immunization was performed by injecting 100 μg of recombinant mouse galectin-9 (rmgalectin-9) with complete Freund's adjuvant for the first immunization, and 100 μg 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.
2) Preparation of anti-Tim-3 antibody
Using each of mouse Tim-3-Ig fusion proteins (BALB type and B6 type) as an antigen, 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.
3) Production and purification of antibody Antibodies, RG9-35 (rat IgG2a, K) and RMT3-23 (rat IgG2a, K), were respectively produced by inoculating hybridomas producing each antibody into peritoneum of nude mice, and purified from the ascites by caprylic acid/ ammonium sulfate-precipitation method.
Example 5: Blocking the signaling pathway of galectin-9 - Tim-3 by anti-galectin-9 and anti-Tim-3 antibody.
1) Inhibitory effect of anti-galectin-9 and anti-Tim-3 antibodies on the binding of galectin-9 and Tim-3
The inhibitory effects of anti-galectin-9 antibody (RG9-35) and anti-Tim-3 antibody (RMT3-23) on the binding of Tim-3-Ig to immobilized galectin-9 is shown in Figure 7. Both RG9-35 and RMT3-23 were found to inhibit the bindings of BALB-type Tim-3-Ig and B6-type Tim-3-Ig to galectin-9.
2) Inhibitory effects of anti-galectin-9 and anti-Tim-3 antibodies on galectin-9-induced ThI cell-apoptosis
The results investigating the effects of antibodies on apoptosis of ThI cells stimulated with galectin-9 are shown in Figure 8.
As shown in Figure 8, anti-galectin-9 antibody (RG9-35) suppressed apoptosis of ThI cells induced by galectin-9, but anti-Tim-3 antibody (RMT3-23) hardly did. As shown in Figure 7, 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+).
Subsequently, using anti-galectin-9 antibody (RG9-35) and anti-Tim-3 antibody (RMT3-23), whether these antibodies exert reversely stimulatory effect on the onset of diabetes was investigated.
As shown in Example 5-1), these antibodies have been shown to inhibit the binding of galectin-9 and Tim-3.
A protocol for administration experiment is shown as follows. 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.
(c) After developing diabetes, mice are killed immediately and served for various experiments
The results of administration experiments are shown in Figure 9. Unexpectedly, anti-Tim-3 antibody-administration group had extremely high suppressive effect on the onset of diabetes, compared to PBS-administration group as a control, galectin-9-administration and anti-galectin-9 antibody-administration group, anti-Tim-3 antibody-administration group was found to exert significant suppressive effect ( PBS v.s. Tim-3 Ab: p < 0.0001; Gal-9 v.s. Tim-3 Ab: p = 0.0025; Gal Ab v.s. Tim-3 Ab: p = 0.0067) on the onset of diabetes, respectively.
Compared to PBS-administration group as a control, galectin-9-adminisration group exerted significant suppressive effect (PBS v.s, Gal-9: p = 0.0248) on the onset of diabetes, and the reproducibility of the results in Example 2 was confirmed. In addition, significant difference was not observed in suppressive effect on the onset of diabetes between galectin-9-administration group and anti-galectin-9 antibody-administration group (p =0.634).
Thus, 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-α 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-α production from dendritic cells (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 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.
On the other hand, the exertion of much stronger therapeutic effects by 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.
As is clear from the results of Example 5-2 (Figure 8), anti-Tim-3 antibody (RMT3-23) hardly affected apoptosis of ThI cells cultured in the presence of galectin-9 (0.2 μM). In the present example, the effect of anti-Tim-3 antibody in the presence of effective dose (0.2 μM) and ineffective dose (0.04 μM) of galectin-9 for inducing ThI cell-apoptosis was investigated.
The concrete experimental procedures are as follows.
1. CD4+ T cells were isolated from spleen of female ICR mice, using CD4 microbeads (Miltenyi).
2. The CD4+ T cells were inoculated into 24-well plates coated with anti-CD3 mAb (2Cl 1, 10 μg/ml) at a cell density of 1 x 106 cells/well, and incubated for 4 days by adding 10 μg/ml of anti-CD28 mAb (PV-I), 10 μg/ml of mouse IL-12 and 10 μg/ml of anti-IL-4 mAb (11B11) into each well.
3. The cells recovered from the plates were re-inoculated into 96-well plates at a cell density of 5 x 105 cells /well and incubated for 3 days by adding 20 U/ml of human IL-2 into each well.
4. 24 hours before analysis, recombinant mouse galectin-9 (rGal-9) (0.2 μM or 0.04 μM) and anti-Tim-3 antibody (RMT3-23) (10 μg/ml or 100 μg/ml) were added to each well, and apoptosis of the cells was analyzed using a FACS Aria. Figure 10 shows effect of anti-Tim-3 antibody (RMT3-23) on apoptosis-inducing action by a high concentration (0.2 μM) of galectin-9 (rGal-9). ThI cell-apoptosis was induced by adding rGal-9 (0.2 μM; high concentration). Addition of anti-Tim-3 antibody alone did not induce the apoptosis at any concentrations tested. In the case of concomitant addition of a high concentration of rGal-9 and anti-Tim-3 antibody, neither suppression nor stimulation of apoptosis was found. Furthermore, no suppression of apoptosis was found even in the case of addition of anti-galectin-9 antibody to a high concentration of rGal-9.
Figure 11 shows effect of RMT3-23 on apoptosis-inducing action by a low concentration (0.04 μM) 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.
From this result, in induction of ThI cell-apoptosis by galectin-9, anti-Tim-3 antibody was found to enhance the induction of ThI cell-apoptosis in the presence of ineffective concentrations of galectin-9.

Claims

[CLAIMS]
[Claims 1] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases, comprising a substance blocking the signaling pathway of
Tim-3-galectin-9 as an active ingredient, wherein galectin-9 is a ligand and Tim-3 is a receptor.
[Claims 2] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, comprising anti-Tim-3 antibody as an active ingredient.
[Claims 3] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, comprising anti-galectin-9 antibody as an active ingredient.
[Claims 4] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 2, wherein said anti-Tim-3 antibody has a characteristic to enhance helper T cell subtype 1 (ThI cell) -apoptosis induced by galectin-9 in the presence of ineffective concentrations of galectin-9.
[Claims 5] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, wherein RNA and DNA aptamer inhibiting the binding of galectin-9 to Tim-3 through binding specifically to galectin-9 are the active ingredients, respectively.
[Claims 6] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of in claim 1, wherein RNA and DNA aptamer inhibiting the binding of
Tim-3 to galectin-9 through binding specifically to Tim-3 are the active ingredients, respectively.
[Claims 7] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, wherein a low molecular substance inhibiting the binding of galectin-9 to Tim-3 through binding specifically to galectin-9 is the active ingredient.
[Claims 8] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, wherein a low molecular substance inhibiting the binding of galectin-9 to Tim-3 competitively through binding specifically to Tim-3 is the active ingredient.
[Claims 9] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, wherein a low molecular substance abolishing the binding ability of Tim-3 to galectin-9 by destructing three dimensional structure of Tim-3 through binding tightly to Tim-3 is the active ingredient.
[Claims 10] An immunosuppressive agent and prophylactic and therapeutic agent for autoimmune diseases of claim 1, wherein autoimmune diseases include 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 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 and Guillan-Barre syndrome, multiple sclerosis, aplastic anemia, rejection after transplantation and graft versus host disease (GVHD) and so on.
[Claims 11] The screening method of a low molecular substance inhibiting the binding of galectin-9 to Tim-3 by binding to galectin-9 as recited in claim 7, which comprises the following processes:
1) The process to bring each of low molecular substance samples into contact with galectin-9,
2) The process to detect the binding of a low molecular substance to galectin-9,
3) The process to evaluate the inhibition of the binding of galectin-9 to Tim-3 through the binding of the low molecular substance to galectin-9.
[Claims 12] The screening method of a low molecular substance inhibiting the binding of galectin-9 to Tim-3 competitively through binding to Tim-3 as recited in claim 8, which comprises the following processes:
1) The process to bring galectin-9 into contact with Tim-3,
2) The process to bring each of low molecular substance samples into contact with the Tim-3 bound galectin-9, 3) The process to evaluate the decrease in quantity of galectin-9 bound to Tim-3 by adding a low molecular substance.
[Claims 13] The screening method of a low molecular substance abolishing the binding ability of Tim-3 to galectin-9 by destructing three dimensional structure through binding tightly to Tim-3 as recited in claim 9, which comprises the following processes:
1) The process to bring each of low molecular substance samples into contact with Tim-3,
2) The process to detect the binding of a low molecular substance to Tim-3,
3) 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.
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