WO2021025140A1 - 二重特異性タンパク質 - Google Patents

二重特異性タンパク質 Download PDF

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WO2021025140A1
WO2021025140A1 PCT/JP2020/030304 JP2020030304W WO2021025140A1 WO 2021025140 A1 WO2021025140 A1 WO 2021025140A1 JP 2020030304 W JP2020030304 W JP 2020030304W WO 2021025140 A1 WO2021025140 A1 WO 2021025140A1
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antibody
bispecific
cell
lymphoma
arm
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French (fr)
Japanese (ja)
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史朗 柴山
智也 手塚
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Ono Pharmaceutical Co Ltd
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Ono Pharmaceutical Co Ltd
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Priority to EP20849041.7A priority Critical patent/EP4011918A4/en
Priority to JP2021537397A priority patent/JP7771749B2/ja
Priority to US17/633,092 priority patent/US20220332825A1/en
Publication of WO2021025140A1 publication Critical patent/WO2021025140A1/ja
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Priority to JP2025100692A priority patent/JP2025128349A/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2818—Immunoglobulins [IG], 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 against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2812—Immunoglobulins [IG], 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 against CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure discloses a bispecific protein capable of specifically binding to PD-1 and CD4 (hereinafter, may be abbreviated as "PD-1 / CD4 bispecific protein") and a medicament thereof. Regarding therapeutic applications.
  • PD-1 is an immunosuppressive receptor belonging to the immunoglobulin family, and is a molecule having a function of suppressing the immune activation signal of T cells activated by stimulation from an antigen receptor.
  • PD-1 signals are obtained from autoimmune dilated cardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease, type I diabetes and It is known to play an important role in suppressing autoimmune diseases such as rheumatoid arthritis. Therefore, it has been pointed out that substances that enhance PD-1 signal can be prophylactic or therapeutic agents for autoimmune diseases.
  • T cells express the CD3 antigen on their surface.
  • the expression pattern differs depending on the subtype. For example, in addition to CD3, killer T cells express CD8, helper T cells express CD4, and regulatory T cells express CD4, CD25, FoxP3, CD127, and the like.
  • bispecific antibodies that recognize PD-1 have been known as substances that enhance PD-1 signals (Patent Documents 1 to 3). These bispecific antibodies are genetically engineered links between the antigen recognition site of an antibody that recognizes CD3, which is a member of the T cell receptor complex, and the antigen recognition site of an antibody that recognizes PD-1. It has the effect of enhancing the inhibitory signal of PD-1 to the T cell receptor complex by increasing the frequency with which PD-1 is located near the T cell receptor complex. Furthermore, these patent documents also describe that PD-1 bispecific antibodies can be used for the prevention or treatment of autoimmune diseases.
  • the PD-1 / CD4 bispecific protein of the present invention has not been reported at all so far.
  • An object of the present invention is to provide a new drug for prevention, suppression of symptom progression, suppression of recurrence or treatment of autoimmune diseases, blood cancers and the like.
  • the inventors of the present invention focused on PD-1 / CD4 bispecific protein as a substance capable of solving such a problem, and completed the present invention.
  • a bispecific protein having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4 in the present specification, “specifically to PD-1 and CD4, respectively". It is synonymous with “bispecific protein that can bind”, and this is also abbreviated as “PD-1 / CD4 bispecific protein”).
  • a first arm that specifically binds to PD-1 hereinafter, may be abbreviated as “first arm” and a second arm that specifically binds to CD4 (hereinafter, “second arm”).
  • a bispecific antibody having a bispecific antibody (hereinafter, may be abbreviated as “PD-1 / CD4 bispecific antibody”) or an antibody fragment thereof (hereinafter, these may be abbreviated as “”.
  • PD-1 / CD4 bispecific antibody, etc. may be abbreviated.
  • VH Heavy chain variable region
  • anti-PD-1 antibody light of an antibody
  • VL of anti-PD-1 antibody, VH of anti-PD-1 antibody, VH of anti-CD4 antibody and VL of anti-CD4 antibody were linked in this order via a peptide linker or directly.
  • the PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding item [4] which has a structure.
  • VL of anti-CD4 antibody, VH of anti-PD-1 antibody, VL of anti-PD-1 antibody and VH of anti-CD4 antibody were linked in this order via a peptide linker or directly.
  • the PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding item [4] which has a structure.
  • the peptides are in the order of VL of anti-CD4 antibody, VH of anti-CD4 antibody, IgG constant region (hinge / CH3), VH of anti-PD-1 antibody and VL of anti-PD-1 antibody.
  • the PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding item [4] which has a structure linked via a linker or directly.
  • the peptide linker is Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 1; where "Gly” represents glycine and “Ser” represents serine, hereinafter "(Gly) x 4-Ser”. (Sometimes abbreviated as ".) Or Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2; ) ⁇ 4-Ser) ⁇ 3 ”(However, when multiple peptide linkers are present, they may be the same or different, and at least one of them is the peptide linker.
  • PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the above items [5] to [10].
  • the PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the above items [2] to [4], which is in the form of mAb 2 .
  • the PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding item [15] wherein the binding of the IgG 1 antibody to the Fc receptor is lost or attenuated.
  • the Scaffold molecule is in the form of Adnectin, Affibody®, Anticalin®, Avimer, DARPin, LRRP, Affilin®, Affitin or Fynomer, and the modified peptide is a special cyclic peptide.
  • Addbody® or Mirabody® the bispecific protein according to the preceding paragraph [23].
  • PD-1 / CD4 according to any one of the above items [1] to [24], which transmits a suppressive signal of PD-1 (or is used for transmitting a suppressive signal of PD-1).
  • Bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof is described by the above items [1] to [24.
  • the first arm allows interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both (hereinafter, "PD-1 and PD-L"). (March be abbreviated as "allowing interaction with”), PD-1 / CD4 bispecific protein or PD-1 / CD4 dual according to any one of the preceding paragraphs [1] to [25]. Specific antibody or antibody fragment thereof.
  • PD-1 / CD4 bispecific protein or PD according to any one of the above items [1] to [26], wherein cytokine production or release during administration or within 24 hours after administration is sufficiently reduced.
  • -1 / CD4 bispecific antibody or antibody fragment thereof are examples of the above items [1] to [26]
  • the first arm allowed interaction with PD-1 and PD-L1, and cytokine production or release during or within 24 hours after administration was sufficiently reduced.
  • the PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of [25].
  • PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof [31] Specific binding to each of PD-1 expressed in cancer cells of blood cancer, which is a target cell, and CD4 expressed in lymphocytes (for example, helper T cells or regulatory T cells) which are effector cells.
  • PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [1] to [29].
  • PD-1 / CD4 according to any one of the above items [1] to [29], which specifically binds to PD-1 and CD4 expressed in cancer cells derived from T cells, which are target cells, respectively.
  • Bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof An isolated PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4.
  • the first arm contains the anti-PD-1 antibody VH and VL
  • the second arm contains the anti-CD4 antibody VH and VL.
  • B specifically binds to each of PD-1 and CD4 expressed on lymphocytes (eg, helper T cells or regulatory T cells, etc.), and (c) (i) PD-1 inhibitory signals.
  • a humanized or fully human isolated PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4.
  • the first arm contains the anti-human PD-1 antibody VH and VL
  • the second arm contains the anti-human CD4 antibody VH and VL.
  • (C) (i) transmit a suppressive signal for PD-1, (ii) the first arm allows interaction with PD-1 and PD-L1, and / or (iii) during or during administration. Cytokine production or release within the next 24 hours is sufficiently reduced, and is in the form of (d) (i) bispecific hybrid antibody and / or (ii) IgG 1 or IgG 4 antibody. antibody.
  • An isolated PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4.
  • the first arm contains the anti-PD-1 antibody VH and VL
  • the second arm contains the anti-CD4 antibody VH and VL.
  • B (i) Specific to PD-1 expressed in cancer cells of blood cancer, which is a target cell, and CD4, which is expressed in lymphocytes (for example, helper T cells or regulatory T cells) which are effector cells. Cytokines that bind specifically or (ii) specifically bind to each of PD-1 and CD4 expressed in T cell-derived cancer cells that are target cells, and (c) cytokines during or within 24 hours after administration. The antibody for which production or release is sufficiently reduced.
  • a humanized or fully human isolated PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4.
  • the first arm contains the anti-human PD-1 antibody VH and VL
  • the second arm contains the anti-human CD4 antibody VH and VL.
  • B (i) Specific to PD-1 expressed in cancer cells of blood cancer, which is a target cell, and CD4, which is expressed in lymphocytes (for example, helper T cells or regulatory T cells) which are effector cells. Bind specifically, or (ii) specifically bind to each of PD-1 and CD4 expressed in T cell-derived cancer cells, which are target cells.
  • the active ingredient is PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof selected from any one of the above items [1] to [36].
  • Autoimmune diseases include Bechette's disease, systemic erythematosus, chronic discoid erythematosus, polyarteritis nodosa, systemic sclerosis, progressive systemic sclerosis, chondrosis, polyarteritis nodosa, and skin.
  • polyarteritis nodosa polyarteritis nodosa, microangiitis nodosa
  • aortitis syndrome hyperan arteritis
  • rheumatoid arthritis rheumatoid arthritis
  • juvenile idiopathic arteritis spondyloarthritis
  • mixed connective tissue Diseases Castleman's disease, Schegren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid vasculitis, macroangiitis
  • ANCA-related vasculitis eg, polyarteritis nodosa) (Sickness and polyarteritis nodosa)
  • Cogan syndrome RS3PE syndrome
  • temporal arteritis polyarteritis nodosa
  • fibromyalgia antiphospholipid antibody syndrome
  • autoimmune myocardium Flames IgG
  • PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific selected from any one of the above items [1] to [30], [33] and [34].
  • GVHD transplant-to-host disease
  • Insulin preparations eg, human insulin, insulin glargine, insulin lispro, insulin detemil, insulin aspart, etc.
  • sulfonylureas eg, Glibenclamide, Gliclazide, Glimepiride, etc.
  • haste insulin secretagogues eg, Glypiride
  • An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating type I diabetes which comprises a sex antibody or an antibody fragment thereof as an active ingredient.
  • Steroid drugs eg, cortisone, cortisone acetate, hydrocortisone, sodium hydrocortisone phosphate, sodium hydrocortisone succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, sodium prednisolone succinate, butyl acetate Prednisolone, sodium prednisolone phosphate, halopredon acetate, methylprednisolone, methylprednisolone acetate, sodium methylprednisolone succinate, triamsinolone, triamsinolone acetate, triamsinolone acetonide, dexamethasone, dexamethasone, dexamethasone, dexa
  • Agents Administered with any one or more agents selected from steroids (eg, the steroids described in [2-5] above), immunosuppressants (eg, Cilospolin, Tacrolimus, Fingolimod, etc.) and Belimumab.
  • steroids eg, the steroids described in [2-5] above
  • immunosuppressants eg, Cilospolin, Tacrolimus, Fingolimod, etc.
  • Belimumab PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific selected from any one of the above items [1] to [30], [33] and [34].
  • An agent for preventing, suppressing progression of symptoms, suppressing recurrence and / or treating systemic lupus erythematosus which comprises a sex antibody or an antibody fragment thereof as an active ingredient.
  • Steroids eg, steroids according to [2-5] above
  • anti-rheumatic drugs eg, Methotrexate, Sulfasalazine, Bucillamine, Leflunomide, Mizoribine, Tacrolimus, etc.
  • anti-cytocytosis drugs eg, Infliximab, etc.
  • one or more agents selected from Abatacept which are administered together with any one of the above items [1] to [30], [33] and [34].
  • PD-1 / CD4 bispecific protein or PD selected from any one of the above items [1] to [29], [31], [32], [35] and [36].
  • the blood cancer is one or more cancers selected from multiple myeloma, malignant lymphoma, leukemia, central nervous system primary malignant lymphoma and myeloproliferative syndrome. ..
  • Non-hodgkin lymphoma is B-cell non-hodgkin lymphoma
  • B-cell non-hodgkin lymphoma is precursor B-cell lymphoblastic lymphoma, precursor B-cell acute lymphocytic leukemia, chronic B-lymphocytic leukemia.
  • Pre-B-cell lymphocytic leukemia lymphoplasmacytic lymphoma, nodal marginal B-cell lymphoma, extranodal marginal B-cell lymphoma, primary splenic marginal B-cell lymphoma, hairy cell leukemia , Hairy cell leukemia variant type, follicular lymphoma, pediatric follicular lymphoma, diffuse large B cell lymphoma, diffuse large B cell lymphoma / non-specific type, splenic diffuse red splenic spinal cord small B Cellular lymphoma, lymphoid cell lymphoma, primary mediasular large cell B-cell lymphoma, primary exudative lymphoma, Berkit lymphoma, mantle cell lymphoma, monoclonal B-cell lymphocytosis, splenic B-cell lymphoma / Leukemia / unclassifiable type, monoclonal gamma globulinemia of unknown significance / Ig
  • Non-hodgkin lymphoma is T / NK cell non-hodgkin lymphoma, and T / NK cell non-hodgkin lymphoma is precursor T cell lymphoblastic lymphoma, chronic T lymphocytic leukemia, T cell acute lymphoma.
  • T cell lymphoblastic leukemia T cell type large granule lymphocytic leukemia, large granule NK cell leukemia, rapidly progressive NK cell leukemia, peripheral T cell lymphoma, peripheral T cell lymphoma / unspecified Type, unclassifiable peripheral T-cell lymphoma, vascular immunoblastic T-cell lymphoma, undifferentiated large cell (CD30 positive) lymphoma, vasocentral lymphoma, intestinal T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatospleen Type ⁇ - ⁇ T cell lymphoma, subcutaneous adipose tissueitis-like T cell lymphoma, mycobacterial sarcoma, Cesarie syndrome, Hodgkin-like / Hodgkin-related undifferentiated large cell lymphoma, extranodal NK / T cell lymphoma, adult T cell lymphoma, Pre-T cell lympho
  • Hodgkin lymphoma Malignant lymphoma is Hodgkin lymphoma, and Hodgkin lymphoma is classical Hodgkin lymphoma (eg, nodular sclerosing, mixed cell, lymphocyte-rich and hypocytopenic) or nodular lymphocyte-dominant Hodgkin.
  • the blood cancer is leukemia, and the leukemia is acute myelogenous leukemia, acute premyelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome or chronic myelogenous leukemia.
  • [3-9] The agent according to any one of the preceding items [3-1] to [3-8], wherein the blood cancer is a childhood blood cancer.
  • [3-10] The description in any one of the preceding paragraphs [3-1] to [3-9], wherein the blood cancer is a blood cancer for which the therapeutic effect of another anticancer drug is insufficient or insufficient.
  • Agent. [3-11] The agent according to any one of the preceding items [3-1] to [3-10], wherein the blood cancer is a blood cancer exacerbated after treatment with another anticancer drug.
  • [3-12] The agent according to any one of the above items [3-1] to [3-9], wherein the blood cancer patient has not been treated with another anticancer drug.
  • the other anticancer agents include alkylating agents, platinum preparations, antimetabolites (eg, antimetabolites, pyridine metabolism inhibitors, purine metabolism inhibitors), ribonucleotide reductase inhibitors, nucleotides.
  • One or more drugs selected from analogs, topoisomerase inhibitors, microtube polymerization inhibitors, microtube depolymerization inhibitors, antitumor antibiotics, cytokine preparations, molecular targeted drugs and cancer immunotherapeutic agents [3] -10], [3-11] or [3-14].
  • [3-17] The agent according to the preceding paragraph [3-14] or [3-15], which is administered after administration of any one or more of the other anticancer agents.
  • [3-18] The agent according to the preceding item [3-14] or [3-15], which is administered before administration of any one or more of the other anticancer agents.
  • [3-19] The agent according to any one of the preceding items [3-1] to [3-18], which is administered together with a steroid drug.
  • [3-20] The agent according to any one of the preceding items [3-1] to [3-18], which is administered after administration of a steroid drug.
  • [4-1] PD-1 / CD4 bispecific protein or PD selected from any one of the above items [1] to [29], [31], [32], [35] and [36].
  • a blood cancer cell growth inhibitor containing a 1 / CD4 bispecific antibody or an antibody fragment thereof as an active ingredient.
  • PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific selected from any one of the above items [1] to [30], [33] and [34].
  • GVHD transplant-to-host disease
  • a method for preventing, suppressing symptom progression, suppressing recurrence and / or treating blood cancer which comprises administering an effective amount of 1 / CD4 bispecific antibody or antibody fragment thereof to a patient.
  • GVHD transplant-to-host disease
  • GVHD transplant-to-host disease
  • the PD-1 / CD4 bispecific protein of the present invention can be used for prevention, suppression of symptom progression, suppression of recurrence or treatment of autoimmune diseases and blood cancers.
  • the PD-1 / CD4 bispecific protein of the present invention has reduced induction of cytokine production or release during administration, and thus suppresses the expression of infusion reaction or cytokine release syndrome after administration. Can be expected.
  • PD-1 / CD4 bispecific antibody described in Example 1 (hereinafter, may be abbreviated as “PD-1 / CD4 scDb”) is shown.
  • VH CD4" and VL CD4 represent VH and VL of the anti-CD4 antibody, respectively
  • VH PD-1 and VL PD-1 represent VH and VL of the anti-PD-1 antibody, respectively.
  • L1", “L2” and “L3” represent peptide linkers that link each VH and VL, L1 and L3 are (Gly) ⁇ 4-Ser, and L2 is ((Gly) ⁇ 4-Ser) ⁇ It has a structure represented by 3.
  • PD-1 / CD4 bispecific antibody described in Example 2 (hereinafter, may be abbreviated as “PD-1 / CD4 scDb-Fc”) is shown.
  • IgG 1 (Hinge-CH 3) represents a human IgG 1 constant region having a hinge portion to a CH 3 region.
  • L4 represents a peptide having a structure represented by (Gly) ⁇ 4-Ser or ((Gly) ⁇ 4-Ser) ⁇ 3.
  • Other symbols have the same meanings as those shown in FIG.
  • Flow cytometry showing the specific binding of PD-1 / CD4 scDb to mouse PD-1 is shown.
  • Clone-1 and Clone-A to -H represent PD-1 / CD4 scDb prepared in Example 1, respectively.
  • Flow cytometry showing the specific binding of PD-1 / CD4 scDb-Fc to mouse PD-1 is shown.
  • Clone-2 and Clone-I to -P each represent PD-1 / CD4 scDb-Fc prepared in Example 2.
  • Flow cytometry showing the specific binding of PD-1 / CD4 scDb to mouse CD4 is shown.
  • Flow cytometry showing the specific binding of PD-1 / CD4 scDb-Fc to mouse CD4 is shown.
  • PD-1 (P rogrammed Cell D eath -1) , in one embodiment, a membrane protein having the amino acid sequence shown in GenBank accession number NP_005009.
  • PD-1 is preferably human PD-1.
  • CD4 in one embodiment, has the amino acid sequence indicated by Genbank registration number NP_000607. When referred to herein as "CD4", unless otherwise specified, all isoforms thereof and their modifications of the "second arm specifically binding to CD4" epitope according to the invention are conserved. It may be used as including the body. In the present invention, CD4 is preferably human CD4.
  • isolated is defined as substantially a single pure substance by being identified, separated and / or purified from impurities containing multiple or innumerable components extracted from a host cell. It means that it becomes an ingredient.
  • bispecific protein means a protein having binding specificity for two different antigen molecules or epitopes in one molecule, but any form thereof as long as it has the binding specificity. It may be, for example, a bispecific antibody and an antibody fragment thereof, a Scaffold molecule (Current Opinion in Biotechnology (2006), Vol. 17, No. 6, p.653-658, Current Opinion in Biotechnology (2007)). , Vol. 18, p.1-10, Current Opinion in Structural Biology (1997), Vol. 7, p.463-469 or Protein Science (2006), Vol. 15, p.14-27) and modified types. Peptides (for example, special cyclic peptides (J. Synth. Org.
  • Scaffold molecules include, for example, Adnectin (see WO2001 / 64942), Affibody® (see WO95 / 19374 and WO2000 / 63243), Anticalin® (see WO99 / 16873), Avimer (see Nature Biotechnology (2005), Vol. 23, p. 1556-1561), DARPin (see Nature Biotechnology (2004), Vol. 22, p. 575-582), LRRP (see Nature (2004), Vol. 430) , No.
  • Adnectin see WO2001 / 64942
  • Affibody® see WO95 / 19374 and WO2000 / 63243
  • Anticalin® see WO99 / 16873
  • Avimer see Nature Biotechnology (2005), Vol. 23, p. 1556-1561
  • DARPin see Nature Biotechnology (2004), Vol. 22, p. 575-582
  • LRRP see Nature (2004), Vol. 430
  • the term "monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies that binds to the same specific antigen.
  • bispecific antibody means an antibody having binding specificity to two different antigen molecules or epitopes in one molecule, and the term “bispecific monoclonal antibody” further means substantially. Means a bispecific antibody obtained from an antibody population that is uniformly homogeneous.
  • Bispecific antibody forms include, for example, diabody, bispecific sc (Fv) 2 , bispecific minibody, bispecific F (ab') 2 , bispecific hybrid antibody, Covalently coupled diabody (bispecific DART), bispecificity (FvCys) 2 , bispecificity F (ab'-zipper) 2 , bispecificity (Fv-zipper) 2 , bispecificity There are three-chain antibody and bispecific mAb 2 .
  • a diabody is a dimer of a single-stranded peptide in which VHs and VLs that recognize different antigens are linked with a peptide linker (Proc. Natl. Acad. Sci. USA (1993), Vol. 90, No. .14: See p.6444-6448).
  • Bispecific sc (Fv) 2 is a small molecule modified so that two sets of VH / VL of two antibodies that recognize different antigens are produced in a continuous single-chain form via a peptide linker. It is a chemical antibody (see J. Biological Chemistry (1994), Vol. 269: p. 199-206).
  • Bispecific F (ab') 2 is a low molecular weight antibody in which Fab'fragments of an antibody that recognizes two different antigens are covalently bonded by a disulfide bond or the like.
  • the bispecific minibody is a low molecular weight antibody fragment in which a small molecule antibody fragment modified so that the constant region CH3 domain of an antibody is linked to scFv that recognizes different antigens is covalently bonded by a disulfide bond or the like on the CH3 domain. It is a molecularized antibody (see Biochemistry (1992), Vo.31, No.6, p.1579-1584).
  • a bispecific hybrid antibody is an intact antibody in which a heavy chain / light chain complex of an antibody that recognizes two different antigens is covalently bound by a disulfide bond or the like.
  • the preferred form of the bispecific antibody is preferably a form in which the movable region between the two variable regions is wide, and is, for example, a bispecific hybrid antibody.
  • the bispecific hybrid antibody can be produced, for example, from a hybridoma prepared by the hybrid hybridoma method (see US4474893).
  • the bispecific hybrid antibody can be produced by co-expressing and secreting a total of four types of cDNA encoding the heavy chain and the light chain of an antibody that recognizes different antigens in mammalian cells.
  • the monoclonal antibody used in the present invention is a hybridoma method (for example, Kohler and Milstein et al., Natur (1975), Vol. 256, p.495-97, Hongo et al., Hybridoma (1995), Vol. 14, No. 3 , P.253-260, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press (1988), Vol.
  • an antibody or monoclonal antibody When administered to a human, it can be prepared in the form of a chimeric antibody, a humanized antibody, or a fully human antibody in order to reduce or eliminate its antigenicity.
  • the "chimeric antibody” means an antibody in which the variable region sequence and the constant region sequence are derived from different mammals.
  • the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. ..
  • the chimeric antibody is a gene encoding an antibody variable region isolated by a known method from an antibody-producing hybridoma isolated by the above-mentioned hybridoma method, recombinant DNA method or phage display method, using a known method.
  • Human-derived antibody constant regions can be linked to a gene encoding (see, for example, US4816567 of Cabilly et al.).
  • Humanized antibody means an antibody in which a complementarity determining region (CDR) sequence derived from a germ cell type of another mammal such as a mouse is transplanted onto a human framework sequence.
  • CDR complementarity determining region
  • a gene encoding an antibody CDR region isolated by a known method from an antibody-producing hybridoma isolated by the above method can be obtained from a human-derived antibody framework region using a known method.
  • Can be made by linking to a gene encoding see, eg, US5225539 and US5530101 in Winter, US5585089 and US6180370 in Queen et al.).
  • Human antibody or “fully human antibody” means an antibody derived from a human germ cell type immunoglobulin sequence in both the variable region consisting of the framework region and the CDR region and the constant region.
  • the human antibodies used in the present invention are mice transformed to produce human antibodies, such as Humab mice (eg, US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318 by Lonberg and Kay et al.
  • mice see, eg, WO2002 / 43478 of Ishida et al.
  • Xeno mice see, eg, US5939598, US6075181, US6114598, US6150584 and US6162963
  • Tc mice eg, Tomizuka et al., Proc. Natl. It can be produced by the method using Acad. Sci. USA (2000), p.722-727. It can also be prepared using SCID mice in which human immune cells have been reconstituted (see, eg, US5476996 and US5698767 by Wilson et al.) So that immunization causes a human antibody response.
  • the human antibody used in the present invention can also be produced by the phage display method described above.
  • an "antibody fragment" of a PD-1 / CD4 bispecific antibody is an antibody that is part of a full-length antibody and has an antigen-binding portion to PD-1 and an antigen-binding portion to CD4.
  • the antigen-binding portion means the minimum unit that an antibody can bind to the antigen, and for example, the target antigen can be recognized by the combination of three CDRs each in VH and VL and those CDRs. It consists of a framework area where the CDR is placed.
  • isotype is used to refer to an antibody class (eg, IgM or IgG) encoded by a heavy chain constant region gene.
  • the preferred isotype of the bispecific antibody of the invention is IgG, more preferably IgG 1 or IgG 4 .
  • IgG 1 is one in which the binding to the Fc receptor (specifically, the Fc ⁇ receptor) is abolished or attenuated, or ADCC and / or CDC action is abolished or attenuated.
  • an IgG 1 antibody in which the binding to the Fc receptor is lost or attenuated can be obtained.
  • an antibody lacking the C-terminal amino acid, for example, ricin at position 447 according to the EU numbering system is preferable.
  • the Fc region of the bispecific antibody of the present invention may be substituted with any amino acid in that region so that two different heavy chains can easily associate with each other.
  • leucine at position 351 is replaced with lysine and threonine at position 366 is replaced with lysine by the EU numbering system in the constant region on the heavy chain with the VH of the first arm that specifically binds to PD-1.
  • PD-1 / CD4 double in which leucine at position 351 and leucine at position 368 in the constant region in the heavy chain with VH of the second arm that specifically binds to CD4 was replaced with glutamic acid.
  • Specific antibodies can be mentioned.
  • leucine at position 351 was replaced with aspartic acid and leucine at position 368 was replaced with glutamic acid by the EU numbering system in the constant region in the heavy chain having VH of the first arm that specifically binds to PD-1.
  • PD-1 / CD4 double in which leucine at position 351 and threonine at position 366 in the constant region in the heavy chain with VH of the second arm that specifically binds to CD4 was replaced with lysine.
  • Specific antibodies can also be mentioned.
  • the bispecific antibody of the present invention is IgG 4
  • a variant in which any amino acid in the heavy chain constant region is substituted, deleted or inserted is more preferable so as to suppress swapping in the antibody molecule.
  • an antibody in which serine at position 228 by the EU numbering system is replaced with proline, which is located in the hinge region is preferable.
  • the amino acid position assigned to the CDR of the variable region of an antibody and the framework can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987). ) And (1991)).
  • the amino acids in the constant region are represented according to the EU numbering system according to the amino acid position of Kabat (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).
  • peptide linker refers to two sets of VHs and VLs constituting the bispecific antibody of the present invention or an antibody fragment thereof, and to link them with an IgG 1 constant region (hinge / CH3). It is not particularly limited as long as it does not inhibit the functional expression and formation of a heavy specific antibody or an antibody fragment thereof, but is not limited to, for example, Ser, (Gly) n -Ser, Ser- (Gly) n , ((Gly) 4 -Ser) n , (Ser- (Gly) 4 ) n [n represents an integer from 1 to 6, and other symbols have the same meaning as above.
  • bispecific antibody of the present invention or an antibody fragment thereof has a plurality of peptide linkers, each of them may have the same amino acid sequence or may be different, and at least one of them may have the same amino acid sequence. It may be a peptide linker consisting of an amino acid sequence.
  • first arm specifically bound to PD-1 is referred to as the present invention. It is a protein that specifically binds to PD-1, regardless of whether it is a part of the bispecific protein according to the invention or exists as a single substance, and the bispecific protein is bispecific. In the case of a sex antibody, the first arm is at least PD-1 specific, whether or not it is contained as part of the antibody or antibody fragment, or is not part of it and exists as a single entity.
  • VH and VL of an antibody that binds specifically (hereinafter, may be abbreviated as anti-PD-1 antibody), and means an antibody portion that can specifically bind to PD-1.
  • anti-PD-1 antibody the Fv part of the antibody consisting of VH and VL constituting the antigen binding site of the anti-PD-1 antibody, and the Fab'part and Fab part of the antibody containing the VH and VL are also included in this. included.
  • “specifically binding to PD-1” means at least an affinity higher than 1x10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9 M (dissociation constant (Kd value)).
  • the "antibody” in the "antibody that specifically binds to PD-1" or “anti-PD-1 antibody” is a full-length antibody, that is, from two heavy chains linked by a disulfide bond and two light chains. Means a full-length antibody, preferably a monoclonal antibody thereof.
  • the "first arm that specifically binds to PD-1" in the present invention is, as one embodiment, an interaction with PD-1 and PD-L1, an interaction with PD-1 and PD-L2, or Allow both of these interactions.
  • "allowing interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both of them” means PD-1 / CD4 according to the present invention. Even in the situation where the heavy-specific antibody is present in an excess of 20 times the concentration of soluble PD-L1 or PD-L2, the PD-1 / CD4 bispecific antibody according to the present invention has an interaction between them.
  • the first arm allows these interactions is determined by known methods, such as binding of the soluble PD-1 protein to immobilized PD-L1 protein or PD-L1-expressing cells. For example, via core analysis, ELISA assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer measurement method (FRET), and fluorescence trace measurement technology (FMAT (registered trademark)) are used to measure the effects of It can be judged by doing. Also, the definition of "allowing interactions with PD-1 and PD-L1, interactions with PD-1 and PD-L2, or both of them" does not substantially inhibit those interactions. Sometimes used synonymously with meaning.
  • the "first arm that specifically binds to PD-1" in the present invention interacts with PD-1 and PD-L1, interacts with PD-1 and PD-L2, or. It may include those that inhibit these two interactions.
  • first arm that specifically binds to PD-1 for example, a combination of VH and VL of a known anti-PD-1 antibody can be mentioned, and such a known anti-PD-1 antibody.
  • a known anti-PD-1 antibody examples include Nivolumab, Cemiplimab, Pembrolizumab, Spartanizumab, Tislelizumab, Dostarlimab, Tripalimab, Camrelizumab, Genolimzumab, Sintilimab, Lodapolimab, Retifanlimab, Balstilimab, Serplulimab, Balstilimab, Serplulimab, Balstilimab, Serplulimab, Balstilimab, Serplulimab, ), STI-A1110, ENUM 388D4, ENUM 244C8, GLS010, CS1003, BAT-1306, AK103, BI 754091, LZM009, CMAB819, Sym021, SSI
  • second arm specifically binding to CD4 (hereinafter, may be abbreviated as "second arm”) relates to the present invention.
  • the second arm is at least an antibody that specifically binds to CD4, whether or not it is contained as part of an antibody or antibody fragment, or is not part of it and exists as a single entity.
  • anti-CD4 antibody May be abbreviated as anti-CD4 antibody), which means an antibody moiety capable of specifically binding to CD4, including VH and VL.
  • such a second arm also includes an Fv portion of an antibody consisting of VH and VL that constitutes an antigen-binding site of an anti-CD4 antibody, and a Fab'part and a Fab portion of an antibody containing the VH and VL.
  • “specifically binds to CD4" has at least an affinity (dissociation constant (Kd value)) higher than 1x10 -5 M, preferably 1x10 -7 M, and more preferably 1x10 -9 M. It can bind directly to CD4 with its binding activity and is used as a feature that does not substantially bind to other proteins.
  • the "antibody” in the “antibody that specifically binds to CD4" or "anti-CD4 antibody” is a full-length antibody, that is, a full-length antibody consisting of two heavy chains linked by a disulfide bond and two light chains. It means an antibody, preferably a monoclonal antibody thereof.
  • second arms that specifically bind to CD4 include, for example, a combination of VH and VL of known anti-CD4 antibodies, and such anti-CD4 antibodies include, for example, MTRX. -1011A, TRX-1, Ibalizumab, BT-061, huB-F5, Zanolimumab, 4162W94, Clenoliximab, Keliximab, AD-519, PRO-542, Cedelizumab and IT1208.
  • the preferred isotype of the PD-1 / CD4 bispecific antibody of the present invention is an IgG antibody, and more preferably an IgG 1 or IgG 4 antibody.
  • the antibody when the antibody is an IgG 1 antibody, it may take a form in which a mutation that substantially eliminates the binding to the Fc receptor is introduced, for example. Included are IgG 1 antibodies in which leucine at position 234 was replaced with alanine and / or leucine at position 235 was replaced with alanine by the EU numbering system, respectively, on two heavy chain constant regions or hinge regions. Alternatively, it may be an IgG 1 antibody in which leucine at position 235 is replaced with glycine and / or glycine at position 236 is replaced with arginine according to the EU numbering system on each of the two heavy chain constant regions or hinge regions. Further, antibodies lacking the amino acid at the C-terminal of the heavy chain of these bispecific antibodies, for example, lysine at position 447 according to the EU numbering system, are more preferable.
  • PD-1 / CD4 bispecific antibodies are IgG 1 antibodies
  • a preferred embodiment is the EU in the constant region in a heavy chain with a first arm VH that specifically binds to PD-1.
  • leucine at position 351 is replaced with lysine
  • threonine at position 366 is replaced with lysine
  • Examples thereof include IgG 1 antibody in which aspartic acid is substituted and leucine at position 368 is substituted with glutamic acid.
  • leucine at position 351 was replaced with aspartic acid and leucine at position 368 was replaced with glutamic acid by the EU numbering system in the constant region in the heavy chain having VH of the first arm that specifically binds to PD-1.
  • a IgG 1 antibody in which leucine at position 351 and threonine at position 366 in the constant region in the heavy chain with VH of the second arm that specifically binds to CD4 is replaced with lysine is also preferred. ..
  • the PD-1 / CD4 bispecific antibody is an IgG 4 antibody is located in the hinge region, antibody was substituted with proline 228 serine by the EU numbering system are preferred.
  • the PD-1 / CD4 bispecific protein of the present invention (preferably PD-1 / CD4 bispecific antibody or the like) has a feature of transmitting a PD-1 inhibitory signal in one embodiment.
  • the PD-1 / CD4 bispecific protein of the invention (preferably PD-1 / CD4 bispecific antibody, etc.), in one embodiment, interacts with PD-1 and PD-L1, PD- It has the characteristic of allowing interaction with 1 and PD-L2 or both.
  • the PD-1 / CD4 bispecific protein of the present invention (preferably PD-1 / CD4 bispecific antibody, etc.) had a sufficiently reduced cytokine production or release during or within 24 hours after administration. It has characteristics.
  • the cytokine production or release during or within 24 hours after administration was sufficiently reduced means that, for example, the PD-1 / CD4 bispecific antibody according to the present invention was intravenously administered by infusion or the like.
  • the concentration of cytokines including IL-2, IFN- ⁇ and / or TNF- ⁇ in blood or tissue does not increase, or even if it increases, steroid administration It means that it can be suppressed.
  • the PD-1 / CD4 bispecific proteins of the invention are, in one embodiment, lymphocytes (preferably T cell lymphocytes (eg, helpers). It may specifically bind to PD-1 and CD4 expressed on T cells or regulatory T cells, etc.), and at that time, it may bind to PD-1 and CD4 at the same time or the same lymphocyte. It may bind to PD-1 and CD4 expressed on lymphocytes.
  • lymphocytes preferably T cell lymphocytes (eg, helpers). It may specifically bind to PD-1 and CD4 expressed on T cells or regulatory T cells, etc.), and at that time, it may bind to PD-1 and CD4 at the same time or the same lymphocyte. It may bind to PD-1 and CD4 expressed on lymphocytes.
  • the PD-1 / CD4 bispecific protein of the present invention (preferably PD-1 / CD4 bispecific antibody or the like) is expressed on a cancer cell of a blood cancer which is a target cell in one embodiment.
  • PD-1 and CD4 expressed on lymphocytes that are effector cells may specifically bind to each other. At that time, it may bind to PD-1 and CD4 at the same time.
  • the "target cell” means a cell that undergoes a cytotoxic effect of lymphocytes that are effector cells
  • the "effector cell” means a cell that exerts a cytotoxic effect on the target cell.
  • the PD-1 / CD4 bispecific protein of the present invention expresses PD-1 and T cell-derived cancer cells. It may bind specifically to each of CD4, and at that time, it may bind to PD-1 and CD4 at the same time.
  • the PD-1 / CD4 bispecific protein is preferably a PD-1 / CD4 bispecific antibody and an antibody fragment thereof, and the PD-1 / CD4 bispecific antibody is preferable.
  • PD-1 / CD4 bispecific monoclonal antibody more preferably isolated PD-1 / CD4 bispecific monoclonal antibody, still more preferably isolated human PD-1 / human CD4 bi It is a specific monoclonal antibody.
  • isolated human PD-1 / human CD4 bispecific monoclonal antibody means an isolated bispecific monoclonal antibody against human PD-1 and human CD4.
  • the PD-1 / CD4 bispecific antibody of the present invention and an antibody fragment thereof are described in WO2014 / 051433, WO2013 / 157953 or WO2013 / 157954. But it can be manufactured.
  • a polynucleotide encoding a heavy chain having a VH of an anti-PD-1 antibody (2) a polynucleotide encoding a heavy chain having a VH of an anti-CD4 antibody, and (3) a VL of an anti-PD-1 antibody.
  • An expression vector in which a polynucleotide encoding a light chain having a light chain and (4) a polynucleotide encoding a light chain having a VL of an anti-CD4 antibody was inserted was gene-introduced into mammalian cells and transformed into both. It can be produced by expressing and secreting both heavy and light chains.
  • the host cell expressing the bispecific antibody of the present invention may be any host cell capable of introducing an expression vector into a gene and expressing the introduced expression vector.
  • insect cells such as SF-9 and SF-21 cells, more preferably mouse cells including CHO cells, BHK cells, SP2 / 0 cells and NS-0 myeloma cells, primates such as COS and Vero cells.
  • Examples include mammalian cells such as similar cells, MDCK cells, BRL3A cells, hybridomas, tumor cells, immortalized primary cells, embryonic retinal cells such as W138, HepG2, HeLa, HEK293, HT1080 or PER.C6.
  • an expression vector of mammalian cells and a host may be used so that the antibody is appropriately glycosylated.
  • Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies that match the glycosylation pattern in humans.
  • Protein production in host cells transformed by gene transfer of expression vectors is described, for example, in Current Protocols in Protein Science (1995), Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184. -8, Bendig, 1988 can be referred to, and general guidelines, procedures and practical methods for maximizing host cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach. It can be carried out with reference to (M. Butler, ed., IRL Press, 1991). Expression of antibodies in host cells is described, for example, in published publications such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567 and WO2000 / 63403.
  • the culture conditions of the host cell can be optimized by a known method, and the amount of protein produced can be optimized.
  • the culture is carried out, for example, in a dish, a roller bottle or a reaction tank by batch culture, fed-batch culture, continuous culture, culture with hollow fibers, or the like.
  • Antibodies expressed in host cells and recovered from the cells or cell culture medium by a known method are purified using a known method. Purification methods include immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography and the like. In addition, protein A or protein G affinity chromatography is preferably used (see, eg, US4801687 and US5151504).
  • the PD-1 / CD4 bispecific protein of the present invention is useful for the prevention, suppression of symptom progression and / or treatment of autoimmune diseases, graft-versus-host diseases (GVHD), blood cancers and the like.
  • GVHD graft-versus-host diseases
  • the autoimmune diseases include, for example, Bechet's disease, systemic erythematosus, chronic discoid erythematosus, polyarteritis nodosa, systemic sclerosis, progressive systemic sclerosis, sclerosis, polyarteritis nodosa.
  • Polyarteritis nodosa gestational herpes, linear IgA vesicular dermatosis, acquired epidermal vesicular disease, alopecia alopecia, white spots, white spots vulgaris, Harada disease, autoimmune optic neuropathy, idiopathic aspernia, habitual abortion, Inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease), Celiac disease, atopic dermatitis, optic neuromyelitis, chronic inflammatory demyelinating polyneuritis, polyarteritis nodosa, alveolar proteinosis, autoimmune disease Immune hemorrhagic disease XIII, recurrent polyarteritis nodosa, sarcoidosis, tonic spondylitis, severe asthma, chronic urticaria, transplant immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, food allergy, Examples include systemic obesity cell disease, myotrophic
  • PD-1 / CD4 bispecific proteins used in the treatment of autoimmune diseases are at least partially lymphocytes (preferably T cell lymphocytes). It specifically binds to PD-1 and CD4 expressed on lymphocytes (for example, helper T cells and regulatory T cells), and exerts a therapeutic effect by transmitting a PD-1 inhibitory signal.
  • lymphocytes for example, helper T cells and regulatory T cells
  • the feature that allows interaction with PD-1 and PD-L1 contributes to the prevention, suppression of symptom progression, suppression of recurrence and / or enhancement or persistence of therapeutic effect.
  • the blood cancer includes, for example, multiple myeloma, malignant lymphoma (eg, non-hodgkin lymphoma (eg, B-cell non-hodgkin lymphoma (eg, prodromal B-cell lymphoblastic lymphoma, prodromal B-cell acute)).
  • malignant lymphoma eg, non-hodgkin lymphoma (eg, B-cell non-hodgkin lymphoma (eg, prodromal B-cell lymphoblastic lymphoma, prodromal B-cell acute)).
  • Lymphocytic leukemia chronic B lymphocytic leukemia (small lymphocytic lymphoma or prodromal leukemia), pre-B cell lymphocytic leukemia, lymphocytic lymphoma, nodal marginal B cell lymphoma, extranodal Marginal zone B-cell lymphoma (MALT lymphoma), primary splenic marginal zone B-cell lymphoma, hair cell leukemia, hair cell leukemia variant type, follicular lymphoma, pediatric follicular lymphoma, diffuse large cells Diffuse large B-cell lymphoma, diffuse large B-cell lymphoma / non-specific type, diffuse red splenic spinal cord small B-cell lymphoma, lymphoplasmocyte lymphoma, primary mediasophageal B-cell lymphoma, primary Exudative lymphoma, Berkitt lymphoma, mantle cell lymphoma, monoclonal B-cell lymphocytosis,
  • the PD-1 / CD4 bispecific protein used for blood cancer treatment and the like in the present invention includes at least a part of blood as a target cell. It is specific for PD-1 expressed on cancer cells and CD4 expressed on lymphocytes that are effector cells (preferably T cell lymphocytes (for example, helper T cells or regulatory T cells)). It exerts a therapeutic effect by binding to. In addition, at least a part of them may specifically bind to each of PD-1 and CD4 expressed in T cell-derived cancer cells and exert a therapeutic effect by transmitting a PD-1 inhibitory signal. ..
  • treatment means, for example, curing or ameliorating a certain disease or a symptom thereof
  • prevention means preventing or delaying the onset of a certain disease or a symptom for a certain period of time. That is, “suppression of symptom progression” means suppressing the progression or worsening of symptoms and stopping the progression of the pathological condition. The meaning of “prevention” also includes suppression of recurrence. By “suppressing recurrence” is meant preventing or reducing the likelihood of recurrence of a disease or symptom.
  • the PD-1 / CD4 bispecific protein of the present invention is usually administered systemically or locally in a parenteral form.
  • Specific examples of such an administration method include injection administration, nasal administration, and pulmonary administration.
  • Examples of the injection administration include intravenous injection, intramuscular injection, intraperitoneal injection and the like, and in the case of intravenous injection, administration by infusion is preferable.
  • the dose varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 0.1 ⁇ g / kg to 300 mg / kg per adult, particularly preferably. , 0.1 mg / kg to 10 mg / kg, parenteral administration once to several times daily, or continuous intravenous administration in the range of 30 minutes to 24 hours daily.
  • a dose smaller than the above dose may be sufficient, or administration beyond the range may be necessary.
  • the injection or infusion solution may be an aqueous solution, a suspension or an emulsion. It may be in the form of, or it may be formulated as a solid agent with a pharmaceutically acceptable carrier so that it can be dissolved, suspended or emulsified by adding a solvent at the time of use. Good.
  • Solvents used in infusions for injections or infusions include, for example, distilled water for injection, saline, glucose solutions and isotonic solutions (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, etc. A solution of sodium chloride, propylene glycol, etc.) can be used.
  • examples of the pharmaceutically acceptable carrier include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, preservatives, pH adjusters and antioxidants.
  • Stabilizers include, for example, various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate or Dibutylhydroxytoluene and the like can be used.
  • Dissolution aids include, for example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 20®, polysorbate 80®). ) And HCO-50, etc.) can be used.
  • the suspending agent for example, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • emulsifier for example, gum arabic, sodium alginate, tragant and the like can be used.
  • the soothing agent for example, benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • a buffer for example, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a carbonic acid buffer solution, a citrate buffer solution, a Tris buffer solution, a glutamate buffer solution, an epsilon aminocaproic acid buffer solution, or the like can be used.
  • Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid or borax. Sand or the like can be used.
  • As the preservative for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • As the pH adjuster for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used.
  • antioxidants for example, (1) water-soluble antioxidants such as (1) ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite and sodium bisulfite, (2) ascorbic palmitate, butylated hydroxyanisol, etc.
  • oil-soluble antioxidants such as butylated hydroxytoluene, lecithin, propyl gallate and ⁇ -tocopherol and (3) metal chelating agents such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid and phosphoric acid. Can be done.
  • the infusion solution for injection or infusion can be produced by sterilization in the final step or by aseptic technique, for example, filtering with a filter or the like to sterilize, and then filling in a sterile container.
  • Infusions for injections or infusions are vacuum-dried and lyophilized sterile powders (which may contain pharmaceutically acceptable carrier powders) dissolved in a suitable solvent before use. You can also do it.
  • the PD-1 / CD4 bispecific proteins of the invention are used in the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune disease or graft-versus-host disease (GVHD) or blood cancer. It may be used in combination with a drug.
  • the administration form when used in combination with other drugs may be a combination drug in which both components are mixed in one preparation, or may be administered as separate preparations. There may be.
  • the bispecific antibody of the present invention and another drug are administered separately, they may be co-administered for a certain period of time, and then only the bispecific antibody or only the other drug may be administered.
  • the bispecific antibody of the present invention may be administered first and another drug may be administered after the end of the administration, or the other drug may be administered first and the bispecific of the present invention may be administered after the end of the administration.
  • the sex antibody may be administered later, and each administration method may be the same or different. It can also be provided as a kit of a preparation containing the bispecific antibody of the present invention and a preparation containing another drug.
  • the dose of the other drug can be appropriately selected based on the clinically used dose.
  • other drugs may be administered in combination of any two or more at an appropriate ratio.
  • the other drugs include not only those found so far but also those found in the future.
  • insulin preparations eg, human insulin, insulin glalgin, insulin lispro, insulin detemil, insulin
  • Asparts etc.
  • sulfonylureas eg, Glibenclamide, Gliclazide, Glimepiride, etc.
  • haste insulin secretagogues eg, Nateglinide, etc.
  • biguanide preparations eg, Metformin, etc.
  • insulin resistance improvers eg, Pioglitazone, etc.
  • ⁇ -Glucosidase inhibitors eg, Acarbose and Voglibose
  • diabetic neuropathy treatments eg, Epalrestat, Mexiletine and Imidapril, etc.
  • GLP-1 analogs eg, Liraglutide, Exenatide and Lixisenatide
  • steroid drugs eg, cortisone, cortisone acetate, hydrocortisone, hydrocortisone phosphorus
  • steroid drugs eg, cortisone, cortisone acetate, hydrocortisone, hydrocortisone phosphorus
  • Sodium acid ester sodium hydrocortisone succinate
  • fludrocortisone acetate prednisolone, prednisolone acetate, sodium prednisolone succinate, prednisolone butylacetate, sodium prednisolone phosphate, halopredone acetate, methylprednisolone, methylprednisolone, Methylpredonizolone succinate sodium, triamsinolone, triamsinolone acetate, triamsinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone valerate, dexamethasone, de
  • a steroid drug for example, the steroid drug described above
  • an immunosuppressant for example, Ciclospolin, Tacrolimus and Fingolimod, etc.
  • a steroid drug for example, the steroid drug described above
  • an anti-rheumatic drug for example, Methotrexate
  • Sulfasalazine for example, Bucillamine, Leflunomide, Mizoribine and Tacrolimus, etc.
  • anti-cytocytomous drugs eg, Infliximab, Adalimumab, Tocilizumab, Etanercept, Abatacept, Golimumab, Certolizumab, etc.
  • the bispecific protein of the present invention When applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of other autoimmune diseases, the bispecific protein of the present invention is used in combination with any one or more of the other agents described above. You may.
  • the bispecific protein of the present invention when applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of blood cancer, for example, alkylating agents (for example, dacarbazine, Nimustine, Temozolomide, Fotomustine, Vincristine) , Cyclophosphamide, Ifosfamide, Carmustine, Chlorambucil and Procarbazine, etc.), platinum preparations (eg, Cisplatin, Carboplatin, Nedaplatin and Oxaliplatin, etc.), antimetabolites (eg, folic acid antimetabolites (eg, Pemetrexed, Leucovorin and Methotrexate, etc.), pyridine Antimetabolites (eg TS-1®, 5-fluorouracil, UFT, Carmofur, Doxifluridine, FdUrd, Cytarabine and Capecitabine), purine antimetabolites (eg Fludarabine, Cladribine and
  • examples of the molecular target drug include ALK inhibitors (eg, Crizotinib, Ceritinib, Ensartinib, Alectinib, Lorlatinib, etc.), BCR-ABL inhibitors (eg, Imatinib, Dasatinib, etc.), and Axl inhibitors (eg, ONO).
  • ALK inhibitors eg, Crizotinib, Ceritinib, Ensartinib, Alectinib, Lorlatinib, etc.
  • BCR-ABL inhibitors eg, Imatinib, Dasatinib, etc.
  • Axl inhibitors eg, ONO.
  • CDK inhibitors eg, Dinaciclib, Abemaciclib, Palbociclib and Trilaciclib, etc.
  • Btk inhibitors eg, Ibrutinib and Acalabrutinib, etc.
  • PI3K- ⁇ / ⁇ inhibitors eg, Umbralisib, Parsaclisib and IPI
  • JAK-1 / 2 inhibitors eg Itacitinib and Luxolitinib etc.
  • FAK inhibitors eg Defactinib etc.
  • Syk / FLT3 dual inhibitors eg Mivavotinib etc.
  • ATR inhibitors eg Mivavotinib etc.
  • Ceralasertib etc. Ceralasertib etc.
  • Wee1 kinase inhibitors eg Adavosertib etc.
  • mTOR inhibitors eg Temsirolimus, Everolimus, Vistusert
  • cancer immunotherapeutic agents include anti-PD-1 antibodies (eg, Nivolumab, Cemiplimab, Pembrolizumab, Spartanalizumab, Tislelizumab, Dostarlimab, toripalimab, Camrelizumab, Genolimzumab, Sintilimab, Lodapolimab, Retifanlimab, Balstilimab, Stilimab, Balstilimab, Prolgolimab, Sasanlimab, Cetrelimab, Zimberelimab, Penpulimab, AMP-514, STI-A1110, ENUM 388D4, ENUM 244C8, GLS010, CS1003, BAT-1306, AK103, BI 754091, LZM009, CMAB819, Sym021, SSI-361 , HX008 and CX-188, etc.), anti-PD-L1 antibodies (eg Atezolizumab, Ave,
  • examples of other antibody drugs include anti-IL-1 ⁇ antibody (for example, Canakinumab etc.) and anti-CCR2 antibody (for example, Plozalizumab etc.).
  • Example 1 Preparation of PD-1 / CD4 bispecific protein (PD-1 / CD4 scDb)
  • the PD-1 / CD4 bispecific protein PD-1 / CD4 scDb in this example is an anti-mouse CD4 antibody. It is a single-chain Diabody (hereinafter abbreviated as "scDb") composed of VH and VL of the above and VH and VL of the anti-mouse PD-1 antibody.
  • FIG. 1 shows an outline of the structure.
  • DNA prepared by linking the variable regions of each antibody and the DNA encoding the peptide linker in a predetermined order was inserted into an expression vector, and this was expressed in animal cells (Free Style 293F cells). ..
  • the scDb produced in the culture supernatant was recovered and then purified by Ni-NTA affinity chromatography and size exclusion chromatography.
  • the cDNA encoding each variable region (VH and VL) of the anti-mouse CD4 antibody and the anti-mouse PD-1 antibody constituting scDb of this example were obtained according to a method according to a known gene cloning method. ..
  • J43 was used as an anti-mouse PD-1 antibody
  • YTA3.1.2, YTS191 or GK1.5 was used as an anti-mouse CD4 antibody.
  • Example 2 Preparation of PD-1 ⁇ CD4 bispecific protein (PD-1 / CD4 scDb-Fc)
  • PD-1 / CD4 scDb-Fc in this example is anti-mouse CD4 antibody VH and VL.
  • a polypeptide in which a human IgG 1 constant region (IgG 1 constant region (hinge / CH3)) from the hinge site to the CH3 region is fused to a bispecific scDb composed of VH and VL of a mouse PD-1 antibody.
  • polypeptide A a polypeptide consisting of only an IgG 1 constant region (hinge / CH3)
  • polypeptide B a polypeptide consisting of only an IgG 1 constant region
  • the heterodimer has a mutation that abolishes Fc ⁇ receptor binding.
  • FIG. 2 shows an outline of the structure.
  • the DNA encoding polypeptide A is a DNA encoding each VH and VL of the anti-mouse CD4 antibody and the anti-mouse PD-1 antibody obtained in Example 1 in preparing PD-1 / CD4 scDb. It was prepared by ligating with DNA encoding a peptide linker in a predetermined order.
  • An expression vector into which each DNA sequence encoding polypeptides A and B was inserted was gene-introduced into Free Style 293F cells for expression and produced in the culture supernatant.
  • the culture supernatant was collected and then purified by Protein A affinity chromatography and size exclusion chromatography.
  • Example 3 Evaluation of binding of PD-1 / CD4 bispecific protein Mouse PD-1 / CD4 bispecific protein by flow cytometry using mouse PD-1 expressing CHO-S cells The binding to 1 was evaluated. A 6 ⁇ His tag is added to the C-terminus of PD-1 / CD4 scDb, and Alexa Fluor 488-labeled anti-His tag antibody (MBL, model number D291-A48) is used to detect each recombinant protein bound to the cell. It was used. The results are shown in FIG.
  • PD-1 / CD4 scDb-Fc contains a human IgG 1 constant region, so PE-labeled anti-human IgG-Fc antibody (Thermo Fisher Scientific, model number H10104) was used to detect binding to mouse PD-1. used. The results are shown in FIG.
  • Example 4 In vivo action in an experimental allergic encephalomyelitis mouse model (EAE model) of PD-1 / CD4 bispecific protein
  • EAE model experimental allergic encephalomyelitis mouse model
  • PD-1 / CD4 dual The in vivo action of the specific protein was evaluated.
  • Dead tuberculosis H37Ra (BD Biosciences, model number 231141) and incomplete Freund's adjuvant (BD Biosciences, model number 263910) were mixed to prepare a complete Freund's adjuvant (CFA) containing 4 mg / mL killed tuberculosis H37Ra.
  • CFA complete Freund's adjuvant
  • An emulsion was prepared by mixing 1 mg / mL MOG peptide (ANASPEC, model number AS-60130) and an equal amount of CFA, and used as an inducer for the EAE model. 200 ⁇ L of the inducer was subcutaneously administered to the ridge of the C57BL / 6 mouse, and 200 ⁇ L of 1 ⁇ g / mL pertussis toxin (SIGMA-ALDRICH, model number P7208) was administered to the tail vein on the day and the second day of the immunotreatment. It was administered internally. Next, the C57BL / 6 mice were intraperitoneally administered with Clone-1 or Clone-2 once a day for 5 days from 6 to 10 days after immunization.
  • MOG peptide ANASPEC, model number AS-60130
  • CFA pertussis toxin
  • neurological symptoms were evaluated according to Onuki et al. (Onuki M, et al., Microsc Res Tech 2001; 52: 731-9.). Score the degree of neurological symptoms (normal: score 0; tail relaxation: score 1; partial hindlimb paralysis: score 2; hindlimb paralysis: score 3; forelimb paralysis: 4 and dying or death: score 5), with multiple neurological symptoms If it was observed, a high value was adopted as the neurological symptom score on the evaluation day. Cumulative neurological symptom scores for the observation period from the day of immunization to 30 days after immunization were tabulated. The evaluation result is shown in FIG.
  • Clone-1 and Clone-2 suppressed neurological symptoms in the EAE model.
  • Example 5 Effect of PD-1 / CD4 bispecific protein on blood cytokine concentration in in vivo mice
  • the PD-1 / CD4 bispecific protein of the present invention was administered to mice, and the plasma cytokine concentration was in vivo. The change in was evaluated.
  • C57BL / 6 mice were given 0.5 mg / kg of Clone-1 and 1.0 mg / kg of Clone-2, Clone-J, Clone-L and Clone-O as PD-1 / CD4 bispecific proteins, respectively. It was administered.
  • PD-1 / CD3 bispecific scDb (J43 ⁇ 2C11_scDb) disclosed in Patent Document 1 was intraperitoneally administered at a dose of 0.5 mg / kg.
  • IFN- ⁇ Interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-2 interleukin-2
  • J43 ⁇ 2C11_scDb increased the plasma IFN- ⁇ , TNF- ⁇ , and IL-2 concentrations in mice.
  • none of the PD-1 / CD4 bispecific proteins of the present invention increased plasma IFN- ⁇ , TNF- ⁇ , and IL-2 concentrations.
  • the target molecule was CD4, the induction of cytokine production or release during administration was improved.
  • Example 6 Inhibitory effect of PD-1 / CD4 bispecific protein on B cell lymphoma cell line Using healthy human peripheral blood-derived pan-T cells (STEMCELL, model number ST-70024) (human T cells) , B cell lymphoma The cell growth inhibitory effect on cell lines is evaluated. Human T cells and B cell lymphoma cell line EB-1 or human PD-1 forced expression SU-DHL-4 labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded on a U-bottom 96-well plate. , PD-1 / CD4 bispecific protein is added and co-cultured for 48 hours.
  • DiD-positive viable cells are harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive live cells is counted on a flow cytometer.
  • the decrease rate of DiD-positive viable cells in the PD-1 / CD4 bispecific protein-free sample is calculated as 100%, and the decrease rate of DiD-positive viable cells in the protein-added sample is calculated as the cell growth inhibition rate.
  • Example 7 Cell growth inhibitory effect of PD-1 / CD4 bispecific protein on multiple myeloma cell line Using healthy human peripheral blood-derived pan-T cells (STEMCELL, model number ST-70024) (human T cells) To evaluate the cell growth inhibitory effect on multiple myeloma cell lines. Human T cells and the multiple myeloma cell line RPMI8226 labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded on a U-bottom 96-well plate, and PD-1 / CD4 bispecific recombinant protein was seeded. Is added and co-cultured for 48 hours.
  • Human T cells and the multiple myeloma cell line RPMI8226 labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded on a U-bottom 96-well plate, and PD-1 / CD4 bispecific recombinant
  • the decrease rate of DiD-positive viable cells in the PD-1 / CD4 bispecific protein-free sample is calculated as 100%, and the decrease rate of DiD-positive viable cells in the protein white-added sample is calculated as the cell growth inhibition rate.
  • Example 8 Inhibitory effect of PD-1 / CD4 bispecific protein on adult T-cell lymphoma cell line Pan-T cells derived from healthy human peripheral blood (STEMCELL, model number ST-70024) (human T cells) It will be used to evaluate the inhibitory effect on adult T-cell lymphoma cell lines.
  • Human T cells and adult T-cell lymphoma cell line ILT-Mat labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded on a U-bottom 96-well plate and PD-1 / CD4 bispecific. Recombinant protein is added and co-cultured for 48 hours.
  • DiD-positive viable cells are harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive live cells is counted on a flow cytometer.
  • the decrease rate of DiD-positive viable cells in the PD-1 / CD4 bispecific protein-free sample is calculated as 100%, and the decrease rate of DiD-positive viable cells in the protein-added sample is calculated as the cell growth inhibition rate.
  • Example 9 Inhibitory effect of PD-1 / CD4 bispecific protein on acute T-cell leukemia cell line Pan-T cells derived from healthy human peripheral blood (STEMCELL, model number ST-70024) (human T cells) It will be used to evaluate the cell growth inhibitory effect on acute T-cell leukemia cell lines.
  • Human T cells and human PD-1 forced expression Jurkat (acute T cell leukemia cell line) labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded on a U-bottom 96-well plate, and PD-1 was seeded. Add / CD4 bispecific protein and co-culture for 48 hours.
  • DiD-positive viable cells are harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive live cells is counted on a flow cytometer.
  • the decrease rate of DiD-positive viable cells in the PD-1 / CD4 bispecific protein-free sample is calculated as 100%, and the decrease rate of DiD-positive viable cells in the protein-added sample is calculated as the cell growth inhibition rate.
  • the PD-1 / CD4 bispecific protein of the present invention is useful for prevention, symptom progression suppression, recurrence suppression and / or treatment of autoimmune diseases or graft-versus-host diseases (GVHD) or blood cancers.
  • GVHD graft-versus-host diseases

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See also references of EP4011918A4
TOMIZUKA ET AL., PROC. NATL. ACAD. SCI. USA, 2000, pages 722 - 727
UEHA, SATOSHI: "Combination Cancer Immunotherapy of anti- CD 4 antibody and anti-PD-1/PD-Ll antibody", THE JOURNAL OF THE JAPANESE SOCIETY OF LYMPHORETICULAR TISSUE , vol. 56, 30 November 2015 (2015-11-30), JP, pages 60, XP009526788, ISSN: 1342-9248 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12312405B2 (en) 2020-05-26 2025-05-27 Boehringer Ingelheim International Gmbh Anti-PD-1 antibodies
WO2023143478A1 (en) * 2022-01-27 2023-08-03 Crown Bioscience Inc. Novel anti-cd4 and anti-pd-l1 bispecific antibodies

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EP4011918A1 (en) 2022-06-15
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US20220332825A1 (en) 2022-10-20
EP4011918A4 (en) 2023-08-23
JP2025128349A (ja) 2025-09-02
JPWO2021025140A1 (https=) 2021-02-11

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