Bispecific proteins

Abstract

The present invention addresses the problem of providing a new drug that, with respect to autoimmune diseases or blood cancer, is for preventing, suppressing the advance of symptoms, suppressing recurrence, or treating such diseases.　The inventors of the present invention conducted extensive studies, and came to focus on a PD-1 / CD4 dual-specific protein as a substance that can solve the foregoing problem, leading to completion of the present invention. The PD-1 / CD4 dual-specific protein according to the present invention is configured to reduce the induction of generation or release of cytokine that occurs when the protein is administered. As a result, it can be expected that infusion reaction or cytokine release syndrome that could occur upon administration of the protein is suppressed.

Description

[Technical Field]

[0001] The present disclosure relates to a bispecific protein capable of specifically binding to PD-1 and CD4, respectively (hereinafter sometimes abbreviated as "PD-1 / CD4 bispecific protein") and its pharmaceutical therapeutic applications. [Background technology]

[0002] PD-1 is an immunosuppressive receptor belonging to the immunoglobulin family that suppresses immune activation signals of T cells activated by antigen receptor stimulation. Analysis of PD-1 knockout mice and other studies has shown that PD-1 signaling plays an important role in suppressing autoimmune diseases such as autoimmune dilated cardiomyopathy, lupus-like syndrome, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease, type 1 diabetes, and rheumatoid arthritis. Therefore, it has been suggested that substances that enhance PD-1 signaling could be used as preventive or therapeutic agents for autoimmune diseases.

[0003] All T cell subtypes express the CD3 antigen on their surface. However, the expression patterns of other antigens vary 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, and CD127.

[0004] Bispecific antibodies that recognize PD-1 are known to date as substances that enhance PD-1 signaling (Patent Documents 1 to 3). These bispecific antibodies are formed by genetically linking the antigen recognition site of an antibody that recognizes CD3, a member of the T cell receptor complex, with the antigen recognition site of an antibody that recognizes PD-1. These bispecific antibodies have the effect of enhancing the inhibitory signal of PD-1 on the T cell receptor complex by increasing the frequency at 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 to prevent or treat autoimmune diseases.

[0005] On the other hand, the PD-1 / CD4 bispecific protein of the present invention has never been reported to date. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2003 / 011911 Pamphlet [Patent Document 2] International Publication No. 2004 / 072286 Pamphlet [Patent Document 3] International Publication No. 2013 / 022091 Brochure Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a new drug for preventing, inhibiting the progression of symptoms, inhibiting recurrence or treating autoimmune diseases, blood cancers and the like. [Means for solving the problem]

[0008] As a result of extensive research, the inventors of the present invention focused on PD-1 / CD4 bispecific proteins as substances that can solve these problems, and completed the present invention.

[0009] That is, the present invention is as follows. [1] A bispecific protein having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4 (herein, this is synonymous with a "bispecific protein capable of specifically binding to PD-1 and CD4, respectively," and may also be abbreviated as a "PD-1 / CD4 bispecific protein"). [2] A bispecific antibody having a first arm (hereinafter sometimes abbreviated as "first arm") that specifically binds to PD-1 and a second arm (hereinafter sometimes abbreviated as "second arm") that specifically binds to CD4 (hereinafter sometimes abbreviated as "PD-1 / CD4 bispecific antibody") or an antibody fragment thereof (hereinafter sometimes abbreviated as "PD-1 / CD4 bispecific antibody, etc."). [3] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph [2], wherein the first arm comprises a heavy chain variable region (hereinafter abbreviated as "VH") and a light chain variable region (hereinafter abbreviated as "VL") of an antibody that specifically binds to PD-1 (hereinafter referred to as "anti-PD-1 antibody"). [4] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph [2] or [3], wherein the second arm comprises the VH and VL of an antibody that specifically binds to CD4 (hereinafter referred to as the "anti-CD4 antibody"). [5] A PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph [4], which has a structure in which, from the N-terminus, the VL of an anti-PD-1 antibody, the VH of an anti-CD4 antibody, the VL of an anti-CD4 antibody, and the VH of an anti-PD-1 antibody are linked, either directly or via a peptide linker. [6] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph [4], which has a structure in which the VL of an anti-PD-1 antibody, the VH of an anti-CD4 antibody, the VH of an anti-CD4 antibody, and the VL of an anti-CD4 antibody are linked, in this order from the N-terminus, via a peptide linker or directly. [7] The PD-1 / CD4 bispecific antibody or antibody fragment thereof described in the preceding paragraph [4], which has a structure in which, from the N-terminus, the VL of an anti-CD4 antibody, the VH of an anti-PD-1 antibody, the VL of an anti-PD-1 antibody, and the VH of an anti-CD4 antibody are linked, either directly or via a peptide linker. [8] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [5] to [7], wherein the VH of the anti-PD-1 antibody according to the preceding paragraph [5], the VL of the anti-CD4 antibody according to the preceding paragraph [6], or the VH of the anti-CD4 antibody according to the preceding paragraph [7] is further linked, via a peptide linker or directly, to the N-terminus of a polypeptide comprising a human IgG constant region from the hinge region to the CH3 region (hereinafter sometimes abbreviated as "IgG constant region (hinge / CH3)"). [9] The PD-1 / CD4 bispecific antibody or antibody fragment thereof described in the preceding paragraph [4], which has a structure in which, from the N-terminus, the VL of an anti-CD4 antibody, the VH of an anti-CD4 antibody, the IgG constant region (hinge / CH3), the VH of an anti-PD-1 antibody, and the VL of an anti-PD-1 antibody are linked via a peptide linker or directly.

[10] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph [8] or [9], further comprising a polypeptide having an IgG constant region (hinge / CH3).

[11] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [5] to

[10] , wherein the peptide linker is Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 1; where "Gly" represents glycine and "Ser" represents serine; hereinafter, this may be abbreviated as "(Gly) × 4-Ser") or Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2; hereinafter, this may be abbreviated as "((Gly) × 4-Ser) × 3") (however, when multiple peptide linkers are present, they may be the same or different, and at least one of them may be the peptide linker).

[12] Diabody, bispecific sc(Fv)2, bispecific minibody, bispecific F(ab′)2, bispecific hybrid antibody, covalent diabody (bispecific DART), bispecific (FvCys)2, bispecific F(ab′-zipper)2, bispecific (Fv-zipper)2, bispecific three-chain antibody, or bispecific mAb 2The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the above [2] to [4], wherein the PD-1 / CD4 bispecific antibody or antibody fragment thereof is in the form of:

[13] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [2] to [4], which is in the form of a bispecific hybrid antibody.

[14] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [2] to [4] and

[13] , which is an IgG antibody.

[15] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[14] , wherein the IgG antibody is an IgG1 antibody or an IgG4 antibody.

[16] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[15] , in which the binding of the IgG1 antibody to an Fc receptor is eliminated or weakened.

[17] A PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[15] or

[16] , in which the ADCC and / or CDC activity of the IgG1 antibody is lost or attenuated.

[18] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [8] to

[10] , wherein the isotype of the IgG constant region (hinge / CH3) is IgG1.

[19] The PD-1 / CD4 bispecific protein, PD-1 / CD4 bispecific antibody, or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[18] , wherein PD-1 and CD4 are human PD-1 and human CD4, respectively.

[20] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [2] to

[19] , which is a monoclonal antibody.

[21] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [2] to

[20] , which is a humanized or fully human antibody.

[22] The PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding items [2] to

[21] , which is an isolated antibody.

[23] The bispecific protein according to the preceding paragraph [1], wherein the bispecific protein is a scaffold molecule or a modified peptide.

[24] The bispecific protein according to the preceding paragraph

[23] , wherein the scaffold molecule is in the form of an Adnectin, Affibody (registered trademark), Anticalin (registered trademark), Avimer, DARPin, LRRP, Affilin (registered trademark), Affitin or Fynomer, and the modified peptide is in the form of a special cyclic peptide, Addbody (registered trademark) or Mirabody (registered trademark).

[25] A PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[24] , which transmits an inhibitory signal of PD-1 (or is used to transmit an inhibitory signal of PD-1).

[26] The PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[25] , wherein the first arm permits interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both (hereinafter sometimes abbreviated as "permits interaction with PD-1 and PD-L").

[27] The PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[26] , which has sufficiently reduced cytokine production or release during administration or within 24 hours after administration.

[28] The PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[25] , wherein the first arm allows interaction with PD-1 and PD-L1, and cytokine production or release during administration or within 24 hours after administration is sufficiently reduced.

[29] The PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to the preceding paragraph

[27] or

[28] , wherein the cytokine is at least IL-2, IFN-γ, or TNF-α.

[30] A PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[29] , which specifically binds to PD-1 and CD4 expressed on lymphocytes (e.g., helper T cells or regulatory T cells).

[31] The PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[29] , which specifically binds to PD-1 expressed on cancer cells of blood cancer, which are target cells, and to CD4 expressed on lymphocytes, which are effector cells (e.g., helper T cells or regulatory T cells).

[32] The PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof according to any one of the preceding paragraphs [1] to

[29] , which specifically binds to PD-1 and CD4, respectively, expressed on cancer cells derived from T cells, which are target cells.

[33] 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, (a) the first arm comprises a VH and VL of an anti-PD-1 antibody, and the second arm comprises a VH and VL of an anti-CD4 antibody; (b) specifically binds to PD-1 and CD4, respectively, expressed on lymphocytes (e.g., helper T cells or regulatory T cells, etc.); and (c) The antibody (i) transmits an inhibitory signal of PD-1, (ii) the first arm allows interaction with PD-1 and PD-L1, interaction with PD-1 and PD-L2, or both, and / or (iii) sufficiently reduces cytokine production or release during or within 24 hours after administration.

[34] An isolated humanized or fully human PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4, (a) the first arm comprises a VH and VL of an anti-human PD-1 antibody, and the second arm comprises a VH and VL of an anti-human CD4 antibody; (b) specifically binds to PD-1 and CD4 expressed on lymphocytes (e.g., helper T cells or regulatory T cells, etc.), (c) (i) transmits an inhibitory signal of PD-1, (ii) the first arm allows interaction with PD-1 and PD-L1, and / or (iii) cytokine production or release is sufficiently reduced during or within 24 hours after administration; and (d) The antibody (i) is in the form of a bispecific hybrid antibody, and / or (ii) is an IgG1 antibody or an IgG4 antibody.

[35] 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, (a) the first arm comprises a VH and VL of an anti-PD-1 antibody, and the second arm comprises a VH and VL of an anti-CD4 antibody; (b) (i) specifically binds to PD-1 expressed on cancer cells of blood cancer, which are target cells, and CD4 expressed on lymphocytes, which are effector cells (e.g., helper T cells or regulatory T cells), respectively, or (ii) specifically binds to PD-1 and CD4 expressed on cancer cells derived from T cells, which are target cells, respectively; and (c) The antibody, which sufficiently reduces cytokine production or release during administration or within 24 hours after administration.

[36] An isolated humanized or fully human PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4, (a) the first arm comprises a VH and VL of an anti-human PD-1 antibody, and the second arm comprises a VH and VL of an anti-human CD4 antibody; (b) (i) specifically binds to PD-1 expressed on cancer cells of blood cancer, which are target cells, and CD4 expressed on lymphocytes, which are effector cells (e.g., helper T cells or regulatory T cells), respectively, or (ii) specifically binds to PD-1 and CD4 expressed on cancer cells derived from T cells, which are target cells, respectively; (c) cytokine production or release is sufficiently reduced during or within 24 hours after administration; and (d) The antibody (i) is in the form of a bispecific hybrid antibody, and / or (ii) is an IgG1 antibody or an IgG4 antibody. [1-1] A pharmaceutical composition comprising, as an active ingredient, a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[36] . [1-2] The pharmaceutical composition according to [1-1] above, further comprising a pharmaceutically acceptable carrier. [2-1] An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease, comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[30] ,

[33] , and

[34] . [2-2] Autoimmune diseases include Behçet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjögren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, and rheumatoid arthritis. vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE syndrome, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related disorders (e.g., primary sclerosing cholangitis and autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, Goodpasture's syndrome, rapidly progressive glomerulonephritis Adrenal nephritis, lupus nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (Graves' disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic hypoadrenocorticism), idiopathic Addison's disease, type 1 diabetes, slowly progressive type 1 diabetes (adult latent autoimmune diabetes), localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis , epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, sarcoidosis, giant cell arteritis, amyotrophic lateral sclerosis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, habitual abortion, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria, transplant immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis. [2-3] An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease or graft-versus-host disease (GVHD), comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[30] ,

[33] , and

[34] . [2-4] Insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylureas (e.g., glibenclamide, gliclazide, and glimepiride, etc.), rapid-acting insulin secretagogues (e.g., nateglinide, etc.), biguanides (e.g., metformin, etc.), insulin sensitizers (e.g., pioglitazone, etc.), α-glucosidase inhibitors (e.g., acarbose and voglibose, etc.), diabetic neuropathy medications (e.g., epalrestat, mexiletin, etc.), an agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating type 1 diabetes, characterized in that it is administered in combination with one or more drugs selected from the group consisting of PD-1 / CD4 bispecific proteins or PD-1 / CD4 bispecific antibodies or antibody fragments thereof selected from any one of the preceding items [1] to

[30] ,

[33] , and

[34] . [2-5] Steroid drugs (e.g., cortisone, cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone Rednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone valerate, dexamethasone cipecilate, dexamethasone palmitate, dexamethasone propionate, dexamethasone sodium phosphate, dexamethasone metasulfobenzoate sodium, paramethasone, paramethasone acetate, betamethasone, beta an agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating multiple sclerosis, the agent comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[30] ,

[33] , and

[34] , which is administered in combination with one or more drugs selected from the group consisting of methasone dipropionate, betamethasone valerate, betamethasone acetate, betamethasone butyrate propionate, and betamethasone sodium phosphate), interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, ciclospolin, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, and alemtuzumab; [2-6] An agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating systemic lupus erythematosus, characterized by being administered in combination with one or more drugs selected from a steroid drug (e.g., a steroid drug described in the preceding paragraph [2-5]), an immunosuppressant (e.g., ciclospolin, tacrolimus, fingolimod, etc.), and belimumab, the agent comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[30] ,

[33] , and

[34] . [2-7] An agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating rheumatoid arthritis, characterized by being administered in combination with one or more drugs selected from a steroid drug (e.g., a steroid drug described in the preceding paragraph [2-5]), an antirheumatic drug (e.g., methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), an anticytokine drug (e.g., infliximab, adalimumab, tocilizumab, etanercept, golimumab, certolizumab, etc.), and abatacept, the agent comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[30] ,

[33] , and

[34] . [2-8] An agent for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease, characterized by being administered in combination with one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7], and comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody, etc., selected from any one of the preceding paragraphs [1] to

[30] ,

[33] , and

[34] . [2-9] A preventive, symptom progression suppressive, recurrence suppressive and / or therapeutic agent for each of the diseases described in the preceding paragraphs [2-4] to [2-8], characterized by being administered to a patient receiving any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7]. [2-10] A preventive, symptom progression suppressive, recurrence suppressive and / or therapeutic agent for each of the diseases described in the preceding paragraphs [2-4] to [2-8], characterized in that it is administered after the administration of any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7]. [2-11] A preventive, symptom progression suppressive, recurrence suppressive and / or therapeutic agent for each of the diseases described in the preceding paragraphs [2-4] to [2-8], characterized in that it is administered before the administration of any one or more of the drugs listed in the preceding paragraphs [2-4] to [2-7]. [3-1] An agent for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating blood cancer, comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[29] ,

[31] ,

[32] ,

[35] , and

[36] . [3-2] The agent described in the preceding paragraph [3-1], wherein the blood cancer is one or more cancers selected from multiple myeloma, malignant lymphoma, leukemia, primary central nervous system malignant lymphoma, and myeloproliferative syndrome. [3-3] The agent according to the preceding item [3-2], wherein the blood cancer is malignant lymphoma, and the malignant lymphoma is non-Hodgkin's lymphoma or Hodgkin's lymphoma. [3-4] The agent according to the preceding item [3-3], wherein the malignant lymphoma is non-Hodgkin's lymphoma, and the non-Hodgkin's lymphoma is B-cell non-Hodgkin's lymphoma or T / NK-cell non-Hodgkin's lymphoma. [3-5] Non-Hodgkin's lymphoma is B-cell non-Hodgkin's lymphoma, and B-cell non-Hodgkin's lymphoma includes precursor B-cell lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, chronic B-lymphocytic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, nodal marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, primary splenic marginal zone B-cell lymphoma, hairy cell leukemia, hairy cell leukemia-variant, follicular lymphoma, pediatric follicular lymphoma, and diffuse large B-cell lymphoma. Lymphoma, diffuse large B-cell lymphoma, unspecified type, splenic diffuse red pulp small B-cell lymphoma, lymphoplasmacytic lymphoma, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, mantle cell lymphoma, monoclonal B-cell lymphocytosis, splenic B-cell lymphoma / leukemia, unclassified type, monoclonal gammopathy of undetermined significance, IgM type, μ heavy chain disease, λ heavy chain disease, α heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraskeletal plasma Cell tumor, monoclonal immunoglobulin deposition disease, large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center lymphoma, T-cell / histiocytocyte-rich large B-cell lymphoma, primary central nervous system diffuse large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma - leg type, EBV-positive diffuse large B-cell lymphoma - unspecified type, EBV-positive mucocutaneous ulcer, chronic inflammation-associated diffuse large B-cell lymphoma, lymphomatoid granulomatosis, intravascular large B-cell lymphoma the agent described in the preceding item [3-4], which is selected from the group consisting of diffuse large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, primary body cavity effusion lymphoma, HHV8-positive diffuse large B-cell lymphoma, unspecified type, Burkitt-like lymphoma with 11q aberration, high-grade B-cell lymphoma with MYC and / or BCL2 and / or BCL6 rearrangements, and high-grade B-cell lymphoma, unspecified type or B-cell lymphoma, unclassifiable type with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma. [3-6] Non-Hodgkin's lymphoma is T / NK cell non-Hodgkin's lymphoma, and T / NK cell non-Hodgkin's lymphoma is precursor T-cell lymphoblastic lymphoma, chronic T-lymphocytic leukemia, T-cell acute lymphoblastic leukemia (T-cell lymphoblastic leukemia), T-cell large granular lymphocytic leukemia, large granular NK cell leukemia, aggressive NK cell leukemia, peripheral T-cell lymphoma, peripheral T-cell lymphoma, unspecified, peripheral T-cell lymphoma, unspecified. tumor, angioimmunoblastic T-cell lymphoma, anaplastic large cell (CD30 positive) lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, enteropathic T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sézary syndrome, Hodgkin-like / Hodgkin-related anaplastic large cell lymphoma, extranodal NK / T-cell lymphoma, adult T-cell lymphoma, T-cell prolymphocytic leukemia, chronic NK-cell lymphoma hyperplasia reproductive dysplasia, pediatric systemic EBV-positive T-cell lymphoma, variola bullosa-like lymphoproliferative dysplasia, extranodal NK / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma, gastrointestinal indolent T-cell lymphoproliferative dysplasia, hepatosplenic T-cell lymphoma, primary cutaneous CD30-positive T-cell lymphoproliferative dysplasia, lymphomatoid papulosis, primary cutaneous anaplastic large cell lymphoma, primary cutaneous gamma delta T-cell lymphoma, primary cutaneous The agent described in the preceding item [3-4], which is CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous acral CD8-positive T-cell lymphoma, primary cutaneous CD4-positive small / medium-sized T-cell lymphoproliferative disorder, follicular T-cell lymphoma, nodal peripheral T-cell lymphoma with follicular helper T-cell phenotype, anaplastic large cell lymphoma ALK-positive type, anaplastic large cell lymphoma ALK-negative type, or breast implant-associated anaplastic large cell lymphoma. [3-7] The agent according to the preceding item [3-3], wherein the malignant lymphoma is Hodgkin's lymphoma, and the Hodgkin's lymphoma is classical Hodgkin's lymphoma (e.g., nodular sclerosing, mixed cellularity, lymphocyte-rich, and lymphocytopenic) or nodular lymphocyte-predominant Hodgkin's lymphoma. [3-8] The agent according to the preceding item [3-2], wherein the blood cancer is leukemia, and the leukemia is acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, or chronic myeloid leukemia. [3-9] The agent according to any one of the preceding items [3-1] to [3-8], wherein the blood cancer is pediatric blood cancer. [3-10] The agent according to any one of the preceding items [3-1] to [3-9], wherein the blood cancer is a blood cancer for which the therapeutic effect of other anticancer drugs is insufficient or not sufficient. [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 that has worsened after treatment with another anticancer drug. [3-12] The agent according to any one of the preceding items [3-1] to [3-9], wherein the patient with blood cancer has no history of treatment with other anticancer drugs. [3-13] The agent according to any one of the preceding items [3-1] to [3-12], wherein the blood cancer is recurrent and / or refractory. [3-14] The agent according to any one of the above items [3-1] to [3-13], which is used in combination with another anticancer drug. [3-15] The agent according to the preceding paragraph [3-10], [3-11] or [3-14], wherein the other anticancer drug is one or more drugs selected from alkylating agents, platinum preparations, antimetabolites (e.g., folate antagonists, pyridine metabolism inhibitors, purine metabolism inhibitors), ribonucleotide reductase inhibitors, nucleotide analogs, topoisomerase inhibitors, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, antitumor antibiotics, cytokine preparations, molecular targeted drugs and cancer immunotherapy drugs. [3-16] The agent according to the preceding item [3-14] or [3-15], which is administered to a patient receiving one or more of the other anticancer drugs. [3-17] The agent according to the preceding item [3-14] or [3-15], which is administered after administration of one or more of the other anticancer drugs. [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 drugs. [3-19] The agent according to any one of the above items [3-1] to [3-18], which is administered together with a steroid drug. [3-20] The agent according to any one of the above items [3-1] to [3-18], which is administered after administration of a steroid drug. [4-1] An inhibitor of blood cancer cell proliferation, comprising as an active ingredient a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[29] ,

[31] ,

[32] ,

[35] and

[36] . [5-1] An intravenous formulation comprising a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding items [1] to

[36] and a pharmaceutically acceptable carrier. [5-2] An intravenous injection preparation according to the preceding paragraph [5-1] for the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of autoimmune disease, graft-versus-host disease (GVHD) or blood cancer. [5-3] The intravenous injection preparation according to the preceding paragraph [5-1] or [5-2], which is for infusion. [6-1] A method for preventing, suppressing the progression of symptoms, suppressing recurrence, and / or treating an autoimmune disease or graft-versus-host disease (GVHD), comprising administering to a patient an effective amount of a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[30] ,

[33] , and

[34] . [6-2] A method for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating blood cancer, comprising administering to a patient an effective amount of a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody or an antibody fragment thereof selected from any one of the preceding paragraphs [1] to

[29] ,

[31] ,

[32] ,

[35] , and

[36] . [6-3] A PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof selected from any one of the preceding items [1] to

[30] ,

[33] , and

[34] , for use in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of autoimmune disease or graft-versus-host disease (GVHD). [6-4] A PD-1 / CD4 bispecific protein or PD-1 / CD4 bispecific antibody or antibody fragment thereof selected from any one of the preceding items [1] to

[29] ,

[31] ,

[32] ,

[35] and

[36] , for use in the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of blood cancer. [6-5] Use of a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody, or an antibody fragment thereof, selected from any one of the preceding items [1] to

[30] ,

[33] , and

[34] in the manufacture of a pharmaceutical for the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of an autoimmune disease or graft-versus-host disease (GVHD). [6-6] Use of a PD-1 / CD4 bispecific protein or a PD-1 / CD4 bispecific antibody, or an antibody fragment thereof, selected from any one of the preceding paragraphs [1] to

[29] ,

[31] ,

[32] ,

[35] , and

[36] in the manufacture of a medicament for the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of blood cancer. [Effects of the Invention]

[0010] The PD-1 / CD4 bispecific protein of the present invention can be used for the prevention, suppression of symptom progression, suppression of recurrence, or treatment of autoimmune diseases, blood cancers, and the like.

[0011] The PD-1 / CD4 bispecific protein of the present invention has reduced induction of cytokine production or release upon administration, and is therefore expected to suppress the onset of infusion reactions or cytokine release syndrome after administration. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 shows the structure of the PD-1 / CD4 bispecific antibody (hereinafter sometimes abbreviated as "PD-1 / CD4 scDb") described in Example 1. In the figure, "VH CD4" and "VL CD4" represent the VH and VL of the anti-CD4 antibody, respectively, and "VH PD-1" and "VL PD-1" represent the VH and VL of the anti-PD-1 antibody, respectively. "L1," "L2," and "L3" represent peptide linkers connecting each VH and VL; L1 and L3 have a structure represented by (Gly)x4-Ser, and L2 has a structure represented by ((Gly)x4-Ser)x3. [Figure 2] Figure 1 shows the structure of the PD-1 / CD4 bispecific antibody (hereinafter sometimes abbreviated as "PD-1 / CD4 scDb-Fc") described in Example 2. In the figure, "IgG1 (Hinge-CH3)" represents the human IgG1 constant region spanning from the hinge to the CH3 region. "L4" represents a peptide having a structure represented by (Gly)x4-Ser or ((Gly)x4-Ser)x3. Other symbols have the same meanings as those shown in Figure 1. [Figure 3] 1 shows flow cytometry results demonstrating the specific binding of PD-1 / CD4 scDb to mouse PD-1. In the figure, Clone-1 and Clone-A to -H represent the PD-1 / CD4 scDb prepared in Example 1, respectively. [Figure 4] Figure 1 shows flow cytometry analysis demonstrating the specific binding of PD-1 / CD4 scDb-Fc to mouse PD-1. In the figure, Clone-2 and Clone-I to -P represent the PD-1 / CD4 scDb-Fc clones prepared in Example 2, respectively. [Figure 5] 1 shows flow cytometry demonstrating the specific binding of PD-1 / CD4 scDb to mouse CD4. [Figure 6] 1 shows flow cytometry demonstrating the specific binding of PD-1 / CD4 scDb-Fc to mouse CD4. [Figure 7] 1 shows the effects of both PD-1 / CD4 bispecific proteins (Clone-1 and Clone-2) in an experimental allergic encephalomyelitis mouse model (EAE model). [Figure 8]Figure 1 shows plasma cytokine concentrations in mice administered with PD-1 / CD4 bispecific proteins. In the figure, "*" and "**" indicate significance at p<0.05 and p<0.01, respectively, in a t-test against vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0013] PD-1( P Programmed Cell D eath -1 ) is, in one embodiment, a membrane-bound protein consisting of the amino acid sequence shown by GenBank accession number NP_005009. Unless otherwise specified, the term "PD-1" used herein may also include all of its isoforms and variants thereof that conserve the epitope of the "first arm that specifically binds to PD-1" according to the present invention. In the present invention, human PD-1 is preferred as PD-1.

[0014] In one embodiment, CD4 has the amino acid sequence shown by Genbank accession number NP_000607. Unless otherwise specified, the term "CD4" used herein may encompass all isoforms thereof and variants thereof that conserve the epitope of the "second arm that specifically binds to CD4" according to the present invention. In the present invention, human CD4 is preferred as CD4.

[0015] As used herein, "isolated" means extracted from a host cell and identified, separated, and / or purified from contaminants, including multiple or numerous components, to provide a substantially single, pure component.

[0016] As used herein, the term "bispecific protein" refers to a protein that has binding specificities for two different antigen molecules or epitopes in a single molecule. However, the bispecific protein may take any form as long as it has the binding specificity. Examples of such bispecific proteins include bispecific antibodies and antibody fragments thereof, scaffold molecules (see Current Opinion in Biotechnology (2006), Vol. 17, No. 6, pp. 653-658, Current Opinion in Biotechnology (2007), Vol. 18, pp. 1-10, Current Opinion in Structural Biology (1997), Vol. 7, pp. 463-469, or Protein Science (2006), Vol. 15, pp. 14-27), and modified peptides (e.g., special cyclic peptides (J. Synth. Org. Chem., Jpn. (2017), Vol. 75, No. 11, pp. 1171-1178), Addbody (registered trademark), Mirabody (registered trademark), etc., but are not limited to these. Further, examples of such scaffold molecules include Adnectin (see WO2001 / 64942), Affibody (registered trademark) (see WO95 / 19374 and WO2000 / 63243), Anticalin (registered trademark) (see WO99 / 16873), Avimer (see Nature Biotechnology (2005), Vol. 23, pp. 1556-1561), DARPin (see Nature Biotechnology (2004), Vol. 22, pp. 575-582), LRRP (see Nature (2004), Vol. 430, No. 6996, pp. 174-180), Affilin (registered trademark) (see WO2001 / 04144 and WO2004 / 106368), Affitin (Journal of molecular biology (2008), Vol. 383, No. 5, pp. 1058-1068) and Fynomer (WO2011 / 023685), but are not limited thereto.

[0017] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies that binds to the same specific antigen.

[0018] As used herein, the term "bispecific antibody" refers to an antibody having binding specificities for two different antigen molecules or epitopes in a single molecule, and the term "bispecific monoclonal antibody" further refers to a bispecific antibody obtained from a substantially homogeneous antibody population.

[0019] Forms of bispecific antibodies include, for example, diabodies, bispecific sc(Fv)2, bispecific minibodies, bispecific F(ab')2, bispecific hybrid antibodies, covalent diabodies (bispecific DART), bispecific (FvCys)2, bispecific F(ab'-zipper)2, bispecific (Fv-zipper)2, bispecific three-chain antibodies, and bispecific mAbs. 2 etc.

[0020] A diabody is a dimer of single-chain peptides in which VH and VL domains that recognize different antigens are linked by a peptide linker (see Proc. Natl. Acad. Sci. USA (1993), Vol. 90, No. 14: pp. 6444-6448).

[0021] Bispecific sc(Fv)2 is a minibody engineered to produce two pairs of VH / VL antibodies that recognize different antigens in the form of a single chain connected via a peptide linker (see J. Biological Chemistry (1994), Vol. 269: pp. 199-206).

[0022] Bispecific F(ab')2 is a minibody in which Fab' fragments of antibodies that recognize two different antigens are covalently linked via disulfide bonds or the like.

[0023] A bispecific minibody is a minibody in which low-molecular-weight antibody fragments, each of which has been modified so that the antibody constant region CH3 domain is linked to scFvs that recognize different antigens, are covalently linked via disulfide bonds or the like in the CH3 domain (see Biochemistry (1992), Vol. 31, No. 6, pp. 1579-1584).

[0024] A bispecific hybrid antibody is an intact antibody in which heavy chain / light chain complexes of antibodies that recognize two different antigens are covalently bound by disulfide bonds or the like.

[0025] In the present invention, a preferred form of bispecific antibody is one in which the flexible region between the two variable regions is large, such as a bispecific hybrid antibody.

[0026] Bispecific hybrid antibodies can be produced, for example, from hybridomas prepared by the hybrid hybridoma method (see U.S. Pat. No. 4,474,893). Alternatively, bispecific hybrid antibodies can be produced by co-expressing and secreting in mammalian cells four types of cDNAs encoding heavy and light chains of antibodies that recognize different antigens.

[0027] The monoclonal antibodies used in the present invention can be produced by hybridoma methods (e.g., Kohler and Milstein et al., Nature (1975), Vol. 256, pp. 495-97; Hongo et al., Hybridoma (1995), Vol. 14, No. 3, pp. 253-260; Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press (1988), Vol. 2) and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, NY, 1981), recombinant DNA methods (see, e.g., US4,816,567), phage display methods (see, e.g., US5,223,409, US5,403,484 and US5,571,698 to Ladner et al., US5,427,908 and US5,580,717 to Dower et al., US5,969,108 and US6,172,197 to McCafferty et al., and US5,885,793, US6,521,404, US6,544,731, US6,555,313, US6,582,915 and US6,593,081 to Griffiths et al.).

[0028] When administered to humans, antibodies or monoclonal antibodies can be produced in the form of chimeric antibodies, humanized antibodies, or fully human antibodies in order to reduce or eliminate their antigenicity.

[0029] A "chimeric antibody" refers to an antibody whose variable region sequences and constant region sequences are derived from different mammals, such as an antibody whose variable region sequences are derived from a mouse antibody and whose constant region sequences are derived from a human antibody. Chimeric antibodies can be produced by linking genes encoding antibody variable regions isolated by known techniques from antibody-producing hybridomas isolated by the above-mentioned hybridoma method, recombinant DNA method, or phage display method to genes encoding antibody constant regions derived from humans using known methods (see, for example, US Pat. No. 4,816,567 by Cabilly et al.).

[0030] The term "humanized antibody" refers to an antibody in which complementarity-determining region (CDR) sequences derived from the germline of another mammal, such as a mouse, have been grafted onto human framework sequences. Humanized antibodies can also be produced by ligating genes encoding antibody CDR regions isolated by known techniques from antibody-producing hybridomas isolated by the above-described method to genes encoding antibody framework regions of human origin, using known methods (see, for example, US Pat. Nos. 5,225,539 and 5,530,101 to Winter, and US Pat. Nos. 5,585,089 and 6,180,370 to Queen et al.).

[0031] By "human antibody" or "fully human antibody" is meant an antibody in which both the variable and constant regions, consisting of the framework and CDR regions, are derived from human germline immunoglobulin sequences. The human antibody used in the present invention can be produced by a mouse transformed to produce a human antibody, such as a Humab mouse (see, e.g., US5545806, US5569825, US5625126, US5633425, US5789650, US5877397, US5661016, US5814318, US5874299, and US5770429 by Lonberg and Kay et al.), a KM mouse (see, e.g., WO2002 / 43478 by Ishida et al.), a Xeno mouse (see, e.g., US5939598, US6075181, US6114598, US6150584, and US6162963), or a Tc mouse (see, e.g., Tomizuka et al., Proc. Natl. Acad. Sci. USA (2000), Human antibodies can be produced by methods using SCID mice (see, for example, US Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.) in which human immune cells have been reconstituted so that a human antibody response occurs upon immunization. Furthermore, human antibodies used in the present invention can also be produced by the phage display method described above.

[0032] As used herein, the "antibody fragment" of a PD-1 / CD4 bispecific antibody refers to a portion of a full-length antibody that has an antigen-binding portion for PD-1 and an antigen-binding portion for CD4, such as F(ab')2. Here, the antigen-binding portion refers to the smallest unit of an antibody capable of binding to its antigen, and is composed, for example, of three CDRs each in VH and VL, and framework regions that position the CDRs so that the combination of these CDRs recognizes the antigen of interest.

[0033] As used herein, the term "isotype" refers to the antibody class (e.g., IgM or IgG) encoded by the heavy chain constant region genes. The preferred isotype for the bispecific antibodies of the present invention is IgG, more preferably IgG1 or IgG4. In one embodiment, the IgG1 antibody has eliminated or attenuated Fc receptor (specifically, Fcγ receptor) binding, or eliminated or attenuated ADCC and / or CDC activity. Specifically, an IgG1 antibody with eliminated or attenuated Fc receptor binding can be obtained by substituting, deleting, or inserting any amino acid in its heavy chain constant region. For example, an IgG1 antibody in which leucine at position 234 (EU numbering system) is substituted with alanine and / or leucine at position 235 is substituted with alanine in each of the two heavy chain constant regions or hinge regions can be used. Alternatively, the bispecific antibody may be an antibody in which leucine at position 235 according to the EU numbering system is substituted with glycine and / or glycine at position 236 is substituted with arginine in each of the two heavy chain constant regions or hinge regions of the bispecific antibody. To reduce antibody heterogeneity, an antibody lacking the C-terminal amino acid, for example, lysine at position 447 according to the EU numbering system, is preferred.

[0034] The Fc region of a bispecific antibody of the present invention may have any amino acid substitution in that region to facilitate association of the two different heavy chains. Examples include a PD-1 / CD4 bispecific antibody in which the leucine at position 351 (EU numbering system) is substituted with lysine and the threonine at position 366 is substituted with lysine in the constant region of the heavy chain having a VH of the first arm that specifically binds to PD-1, and in which the leucine at position 351 is substituted with aspartic acid and the leucine at position 368 is substituted with glutamic acid in the constant region of the heavy chain having a VH of the second arm that specifically binds to CD4. Other examples include PD-1 / CD4 bispecific antibodies in which the leucine at position 351, according to the EU numbering system, is substituted with aspartic acid and the leucine at position 368 is substituted with glutamic acid in the constant region of a heavy chain having a VH in a first arm that specifically binds to PD-1, and the leucine at position 351 is substituted with lysine and the threonine at position 366 is substituted with lysine in the constant region of a heavy chain having a VH in a second arm that specifically binds to CD4.

[0035] Furthermore, when the bispecific antibody of the present invention is IgG4, variants in which any amino acid in the heavy chain constant region has been substituted, deleted, or inserted to suppress swapping within the antibody molecule are more preferred. For example, antibodies in which serine at position 228 according to the EU numbering system, located in the hinge region, has been substituted with proline are preferred. Herein, the amino acid positions assigned to the CDRs and frameworks of antibody variable regions can be defined according to Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., (1987) and (1991)) and the amino acids in the constant region are represented according to the EU numbering system based on Kabat amino acid positions (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242).

[0036] As used herein, the term "peptide linker" is not particularly limited as long as it links the two pairs of VH and VL constituting the bispecific antibody or antibody fragment of the present invention together, and also links them to the IgG1 constant region (hinge / CH3), and inhibits the functional expression and formation of the bispecific antibody or antibody fragment thereof. Examples of the peptide linker include, but are not limited to, Ser, (Gly), n -Ser, Ser-(Gly) n , ((Gly)4-Ser) n , (Ser-(Gly)4) n [n represents an integer of 1 to 6, and the other symbols have the same meanings as defined above.], and more preferably (Gly)x4-Ser and ((Gly)x4-Ser)x3. When a bispecific antibody or antibody fragment thereof of the present invention has multiple peptide linkers, each may be composed of the same or different amino acid sequence as described above, and at least one of them may be a peptide linker composed of the amino acid sequence described above.

[0037] First arm specifically binds to PD-1 As used herein, a "first arm that specifically binds to PD-1" (hereinafter sometimes abbreviated as "first arm") refers to a protein that specifically binds to PD-1, regardless of whether it is a part of a bispecific protein of the present invention or exists alone. When the bispecific protein is a bispecific antibody, the first arm refers to an antibody portion that comprises at least the VH and VL of an antibody that specifically binds to PD-1 (hereinafter sometimes abbreviated as anti-PD-1 antibody), and is capable of specifically binding to PD-1, regardless of whether it is contained in a part of an antibody or antibody fragment, or whether it exists alone as a part of an antibody or antibody fragment. For example, such a first arm also includes the Fv portion of an antibody consisting of the VH and VL that constitute the antigen-binding site of an anti-PD-1 antibody, and the Fab' portion and Fab portion of an antibody comprising said VH and VL. Here, "specifically binds to PD-1" refers to an antibody portion that specifically binds to PD-1 at a concentration of at least 1 x 10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9This term is used to describe the ability to directly bind to PD-1 with a binding activity having a higher affinity (dissociation constant (Kd value)) than M, and to not substantially bind to at least other receptor members belonging to the so-called CD28 family of receptors, such as CD28, CTLA-4, and ICOS. Furthermore, the "antibody" in "antibody that specifically binds to PD-1" or "anti-PD-1 antibody" refers to a full-length antibody, i.e., a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, preferably a monoclonal antibody.

[0038] In one embodiment, the "first arm that specifically binds to PD-1" of the present invention allows interaction between PD-1 and PD-L1, interaction between PD-1 and PD-L2, or both of these interactions. Here, "allowing interaction between PD-1 and PD-L1, interaction between PD-1 and PD-L2, or both of these interactions" means that, even in the presence of a PD-1 / CD4 bispecific antibody of the present invention at a concentration 20-fold excess over soluble PD-L1 or PD-L2, the interaction is maintained at 50% or more, preferably 70% or more, and more preferably 80% or more of the level of the interaction in the absence of the PD-1 / CD4 bispecific antibody of the present invention. Whether the first arm permits these interactions can be determined by known methods, such as measuring the effect of the first arm on the binding of soluble PD-1 protein to immobilized PD-L1 protein or PD-L1-expressing cells using, for example, Biacore analysis, ELISA assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer assay (FRET), or fluorescence micrometric analysis technology (FMAT®). The term "permits the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both" is sometimes used synonymously with the meaning of not substantially inhibiting these interactions.

[0039] In another embodiment, the "first arm that specifically binds to PD-1" of the present invention may include one that inhibits the interaction between PD-1 and PD-L1, the interaction between PD-1 and PD-L2, or both of these interactions.

[0040] Furthermore, other examples of the "first arm that specifically binds to PD-1" include combinations of VH and VL of known anti-PD-1 antibodies. Examples of such known anti-PD-1 antibodies include Nivolumab, Cemiplimab, Pembrolizumab, Spartalizumab, Tislelizumab, Dostarlimab, Toripalimab, Camrelizumab, Genolimzumab, Sintilimab, Lodapolimab, Retifanlimab, Balstilimab, Serplulimab, Budigalimab, Prolgolimab, Sasanlimab, Cetrelimab, Zimberelimab, Penpulimab, AMP-514 (MEDI0680), STI-A1110, ENUM 388D4, ENUM 244C8, GLS010, CS1003, BAT-1306, AK103, BI 754091, LZM009, CMAB819, Sym021, SSI-361, JY034, HX008, ISU106, and CX-188, and further, the following published patent applications: US20180222982, US20180312564, WO2004 / 056875, WO2006 / 121168, WO2008 / 156712, WO2010 / 029434, WO2010 / 029435, WO2010 / 036959, WO2011 / 110604, WO2011 / 11 0621, WO2014 / 179664, WO2014 / 194302, WO2014 / 206107, WO2015 / 035606, WO2015 / 036394 , WO2015 / 085847, WO2015 / 112800, WO2016 / 014688, WO2016 / 015685, WO2016 / 068801, WO 2016 / 077397, WO2016 / 092419, WO2016 / 106159, WO2016 / 127179, WO2016 / 197497, WO2016 / 210129, WO2017 / 011580, WO2017 / 016497, WO2017 / 019846, WO2017 / 024465, WO2017 / 024 515, WO2017 / 025016, WO2017 / 025051, WO2017 / 040790, WO2017 / 055443, WO2017 / 055547,WO2017 / 058115, WO2017 / 058859, WO2017 / 079112, WO2017 / 079116, WO2017 / 087599, WO 2017 / 096026, WO2017 / 106656, WO2017 / 107885, WO2017 / 124050, WO2017 / 125815, WO20 17 / 132827, WO2017 / 133540, WO2017 / 166804, WO2017 / 198741, WO2017 / 201766, WO2017 / 214182, WO2018 / 020476, WO2018 / 026248, WO2018 / 034226, WO2018 / 036472, WO2018 / 05 2818, WO2018 / 053106, WO2018 / 068336, WO2018 / 085358, WO2018 / 085468, WO2018 / 0871 43, WO2018 / 091661, WO2018 / 113258, WO2018 / 119474, WO2018 / 162944, WO2018 / 192089 Examples of first arms that specifically bind to PD-1 include combinations of VH and VL of anti-PD-1 antibodies described in the patent publications identified by WO2018 / 210230, WO2018 / 217227, WO2018 / 226580, WO2019 / 005635, and WO2019 / 051164. Examples of first arms that specifically bind to PD-1 also include combinations of VH of the above anti-PD-1 antibodies and a common light chain.

[0041] The second arm specifically binds to CD4 As used herein, a "second arm that specifically binds to CD4" (hereinafter sometimes abbreviated as "second arm") refers to a protein that specifically binds to CD4, regardless of whether it is a part of the bispecific protein of the present invention or exists as a free-standing protein. When the bispecific protein is a bispecific antibody, the second arm refers to an antibody portion that comprises at least the VH and VL of an antibody that specifically binds to CD4 (hereinafter sometimes abbreviated as anti-CD4 antibody), and is capable of specifically binding to CD4, regardless of whether it is contained in a part of an antibody or antibody fragment, or whether it exists as a free-standing protein rather than as a part of the antibody or antibody fragment. For example, such a second arm also includes the Fv portion of an antibody consisting of the VH and VL that constitute the antigen-binding site of an anti-CD4 antibody, and the Fab' portion and Fab portion of an antibody comprising said VH and VL. Here, "specifically binds to CD4" refers to an antibody portion that comprises at least 1 x 10 -5 M, preferably 1x10 -7 M, more preferably 1x10 -9 It is used to describe the characteristic of being able to directly bind to CD4 with a binding activity having a higher affinity (dissociation constant (Kd value)) than M, and not substantially binding to other proteins. Furthermore, the "antibody" in "antibody that specifically binds to CD4" or "anti-CD4 antibody" refers to a full-length antibody, i.e., a full-length antibody consisting of two heavy chains and two light chains linked by disulfide bonds, preferably a monoclonal antibody.

[0042] Other examples of the "second arm that specifically binds to CD4" include combinations of VH and VL of known anti-CD4 antibodies, such as MTRX-1011A, TRX-1, ibalizumab, BT-061, huB-F5, zanolimumab, 4162W94, clenoliximab, keliximab, AD-519, PRO-542, cedelizumab, and IT1208.

[0043] A preferred isotype for the PD-1 / CD4 bispecific antibody of the present invention is an IgG antibody, and more preferably an IgG1 or IgG4 antibody.

[0044] When the bispecific antibodies of the present invention are IgG1 antibodies, they may be in a form into which a mutation that substantially eliminates Fc receptor binding has been introduced, such as an IgG1 antibody in which leucine at position 234 (EU numbering system) has been substituted with alanine and / or leucine at position 235 (EU numbering system) has been substituted with alanine in each of the two heavy chain constant regions or hinge regions. Alternatively, the bispecific antibodies may be IgG1 antibodies in which leucine at position 235 (EU numbering system) has been substituted with glycine and / or glycine at position 236 (EU numbering system) has been substituted with arginine in each of the two heavy chain constant regions or hinge regions. Furthermore, antibodies in which the C-terminal amino acid of the heavy chain of these bispecific antibodies, for example, lysine at position 447 (EU numbering system) has been deleted are more preferred.

[0045] Furthermore, when these PD-1 / CD4 bispecific antibodies are IgG1 antibodies, preferred embodiments include IgG1 antibodies in which the leucine at position 351 (EU numbering system) is substituted with lysine and the threonine at position 366 (EU numbering system) in the constant region of a heavy chain having a VH in a first arm that specifically binds to PD-1 is substituted with lysine, and the leucine at position 351 (EU numbering system) is substituted with aspartic acid and the leucine at position 368 (EU numbering system) is substituted with glutamic acid in the constant region of a heavy chain having a VH in a first arm that specifically binds to CD4 is substituted with aspartic acid and the leucine at position 368 (EU numbering system) is substituted with glutamic acid. Similarly preferred are IgG1 antibodies in which the leucine at position 351 (EU numbering system) is substituted with aspartic acid and the leucine at position 368 (EU numbering system) in the constant region of a heavy chain having a VH in a first arm that specifically binds to PD-1 is substituted with lysine, and the leucine at position 351 (EU numbering system) is substituted with lysine and the threonine at position 366 (EU numbering system) in the constant region of a heavy chain having a VH in a second arm that specifically binds to CD4 is substituted with lysine.

[0046] On the other hand, when the PD-1 / CD4 bispecific antibody is an IgG4 antibody, an antibody in which serine at position 228 according to the EU numbering system, located in the hinge region, is substituted with proline is preferred.

[0047] In one embodiment, the PD-1 / CD4 bispecific protein of the present invention (preferably, the PD-1 / CD4 bispecific antibody, etc.) is characterized by transmitting an inhibitory signal of PD-1.

[0048] In one embodiment, the PD-1 / CD4 bispecific proteins of the invention (preferably, PD-1 / CD4 bispecific antibodies, etc.) have characteristics that allow for interaction between PD-1 and PD-L1, interaction between PD-1 and PD-L2, or both.

[0049] The PD-1 / CD4 bispecific proteins of the present invention (preferably, PD-1 / CD4 bispecific antibodies, etc.) are characterized by sufficiently reduced cytokine production or release during administration or within 24 hours after administration. Here, "sufficiently reduced cytokine production or release during administration or within 24 hours after administration" means, for example, that the concentrations of cytokines, including IL-2, IFN-γ, and / or TNF-α, in the blood or tissues do not increase during or within 24 hours after intravenous administration of the PD-1 / CD4 bispecific antibodies of the present invention by infusion or the like, or that, if they increase, they are at a level that can be suppressed by steroid administration.

[0050] In one embodiment, the PD-1 / CD4 bispecific proteins of the present invention (preferably, PD-1 / CD4 bispecific antibodies, etc.) may specifically bind to PD-1 and CD4 expressed on lymphocytes (preferably, T-cell lymphocytes (e.g., helper T cells or regulatory T cells, etc.)), respectively, and may simultaneously bind to PD-1 and CD4, or may bind to PD-1 and CD4 expressed on the same lymphocyte.

[0051] In one embodiment, the PD-1 / CD4 bispecific protein of the present invention (preferably, a PD-1 / CD4 bispecific antibody, etc.) may specifically bind to PD-1 expressed on target cells, i.e., cancer cells of blood cancer, and to CD4 expressed on effector cells, i.e., lymphocytes (preferably, T-cell lymphocytes (e.g., helper T cells or regulatory T cells, etc.)), and may simultaneously bind to PD-1 and CD4. Here, "target cells" refers to cells that are subject to the cytotoxic effect of effector lymphocytes, and "effector cells" refers to cells that exert cytotoxic effects on target cells.

[0052] In one embodiment, the PD-1 / CD4 bispecific protein of the present invention (preferably, the PD-1 / CD4 bispecific antibody, etc.) may specifically bind to each of PD-1 and CD4 expressed on T cell-derived cancer cells, and may simultaneously bind to PD-1 and CD4.

[0053] In the present invention, the PD-1 / CD4 bispecific protein is preferably a PD-1 / CD4 bispecific antibody or an antibody fragment thereof, and the PD-1 / CD4 bispecific antibody is preferably a PD-1 / CD4 bispecific monoclonal antibody, more preferably an isolated PD-1 / CD4 bispecific monoclonal antibody, and even more preferably an isolated human PD-1 / human CD4 bispecific monoclonal antibody. Here, "isolated human PD-1 / human CD4 bispecific monoclonal antibody" refers to an isolated bispecific monoclonal antibody against human PD-1 and human CD4.

[0054] Methods for producing and purifying PD-1 / CD4 bispecific antibodies The PD-1 / CD4 bispecific antibodies and antibody fragments thereof of the present invention can also be produced by the methods disclosed in WO2014 / 051433, WO2013 / 157953, or WO2013 / 157954.

[0055] For example, expression vectors containing (1) a polynucleotide encoding a heavy chain having the VH of an anti-PD-1 antibody, (2) a polynucleotide encoding a heavy chain having the VH of an anti-CD4 antibody, (3) a polynucleotide encoding a light chain having the VL of an anti-PD-1 antibody, and (4) a polynucleotide encoding a light chain having the VL of an anti-CD4 antibody can be inserted into mammalian cells to transform them, and both the heavy and light chains can be expressed and secreted to produce the antibodies.

[0056] Here, host cells expressing the bispecific antibodies of the present invention may be any host cells capable of transfecting an expression vector and expressing the introduced expression vector. Preferred examples include insect cells such as SF-9 and SF-21 cells, and more preferred are mouse cells including CHO cells, BHK cells, SP2 / 0 cells, and NS-0 myeloma cells, primate cells such as COS and Vero cells, MDCK cells, BRL 3A cells, hybridomas, tumor cells, immortalized primary cells, and mammalian cells such as W138, HepG2, HeLa, HEK293, HT1080, or embryonic retinal cells such as PER.C6. In selecting an expression system, mammalian cell expression vectors and hosts may be used to ensure appropriate glycosylation of the antibody. Human cell lines, preferably PER.C6, are advantageously used to obtain antibodies with glycosylation patterns consistent with those in humans.

[0057] Protein production in host cells transformed by gene introduction of an expression vector can be carried out with reference to, for example, Current Protocols in Protein Science (1995), Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT, ISBN 0-471-11184-8, Bendig, 1988. Furthermore, general guidelines, procedures, and practical methods for maximizing the productivity of host cell culture can be carried out with reference to Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press, 1991). Expression of antibodies in host cells is described in publications such as EP0120694, EP0314161, EP0481790, EP0523949, US4816567, and WO2000 / 63403.

[0058] Here, the culture conditions of the host cells can be optimized by known methods to optimize the protein production. Culture can be performed, for example, in dishes, roller bottles, or reactors, by batch culture, fed-batch culture, continuous culture, hollow fiber culture, or the like. For large-scale (continuous) production of recombinant proteins by cell culture, it is preferable to grow cells in suspension. It is also preferable to culture the cells in the absence of animal- or human-derived serum or serum components derived from animals or humans.

[0059] Antibodies expressed in host cells and recovered from the cells or cell culture medium by known methods are purified using known methods, including immunoprecipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction chromatography, etc. Protein A or Protein G affinity chromatography is also suitable (see, e.g., US4801687 and US5151504).

[0060] [Medicinal uses] The PD-1 / CD4 bispecific proteins of the present invention are useful for preventing, inhibiting the progression of symptoms of, and / or treating autoimmune diseases, graft-versus-host disease (GVHD), blood cancers, and the like.

[0061] In the present invention, examples of autoimmune diseases include Behcet's disease, systemic lupus erythematosus, chronic discoid lupus erythematosus, multiple sclerosis, systemic sclerosis, progressive systemic sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Castleman's disease, Sjogren's syndrome, adult Still's disease, vasculitis, allergic granulomatous vasculitis, hypersensitivity vasculitis, rheumatoid arthritis, and rheumatoid arthritis. Id vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis), Cogan's syndrome, RS3PE syndrome, temporal arteritis, giant cell arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related disorders (e.g., primary sclerosing cholangitis and autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, nonalcoholic steatohepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpasture's syndrome, and rapidly progressive autoimmune glomerulonephritis, lupus nephritis, anti-glomerular basement membrane nephritis, megaloblastic anemia, autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, hyperthyroidism (Graves' disease (Graves' disease)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease, idiopathic Addison's disease (chronic adrenal insufficiency), type 1 diabetes, slowly progressive type 1 diabetes, localized scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, Examples of such diseases include vitiligo, vitiligo vulgaris, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent abortion, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), celiac disease, atopic dermatitis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, pulmonary alveolar proteinosis, autoimmune hemorrhagic disease XIII, relapsing polychondritis, sarcoidosis, ankylosing spondylitis, severe asthma, chronic urticaria, transplantation immunity, familial Mediterranean fever, eosinophilic sinusitis, dilated cardiomyopathy, food allergy, systemic mastocytosis, amyotrophic lateral sclerosis, and inclusion body myositis.

[0062] PD-1 / CD4 bispecific proteins (preferably PD-1 / CD4 bispecific antibodies, etc.) used in the treatment of autoimmune diseases exert their therapeutic effects by specifically binding, at least in part, to PD-1 and CD4 expressed on lymphocytes (preferably T lymphocytes (e.g., helper T cells and regulatory T cells)), respectively, and transducing PD-1 inhibitory signals. Furthermore, the ability to interact with PD-1 and PD-L1 is expected to contribute to the prevention of autoimmune diseases, the suppression of symptom progression, the suppression of recurrence, and / or the enhancement or prolongation of therapeutic effects.

[0063] In the present invention, examples of blood cancers include multiple myeloma, malignant lymphomas (e.g., non-Hodgkin's lymphomas (e.g., B-cell non-Hodgkin's lymphomas (e.g., precursor B-cell lymphoblastic lymphoma, precursor B-cell acute lymphoblastic leukemia, chronic B-lymphocytic leukemia (small lymphocytic lymphoma or precursor cell leukemia)), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, nodal marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma (MALT lymphoma), primary splenic marginal zone B-cell lymphoma, hairy cell leukemia, hairy cell leukemia variant, follicular lymphoma, lymphoma, childhood follicular lymphoma, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma, unspecified type, splenic diffuse red pulp small B-cell lymphoma, lymphoplasmacytic lymphoma, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, mantle cell lymphoma, monoclonal B-cell lymphocytosis, splenic B-cell lymphoma / leukemia unclassifiable, monoclonal gammopathy of undetermined significance, IgM type, μ heavy chain disease, λ heavy chain disease, α heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraskeletal plasmacytoma, monoclonal immunoglobulin B-cell deposition disease, large B-cell lymphoma with IRF4 rearrangement, primary cutaneous follicle center lymphoma, T-cell / histiocytocyte-rich large B-cell lymphoma, primary central nervous system diffuse large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma - leg type, EBV-positive diffuse large B-cell lymphoma - unspecified type, EBV-positive mucocutaneous ulcer, chronic inflammation-associated diffuse large B-cell lymphoma, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, primary body cavity effusion lymphoma, HHV8-positive diffuse large B-cell lymphoma B-cell lymphoma, unspecified type, Burkitt-like lymphoma with 11q aberration, high-grade B-cell lymphoma with MYC and / or BCL2 and / or BCL6 rearrangements, high-grade B-cell lymphoma, unspecified type, and B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, unclassified type), T / NK-cell non-Hodgkin lymphoma (e.g., precursor T-cell lymphoblastic lymphoma, chronic T-lymphocytic leukemia, T-cell acute lymphoblastic leukemia (T-cell lymphoblastic leukemia), T-cell large granular lymphocytic leukemia, large granular NK-cell leukemia,Rapidly progressive NK cell leukemia, peripheral T cell lymphoma, peripheral T cell lymphoma / unspecified type, unclassifiable peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, undifferentiated large cell (CD30 positive) lymphoma, angiocentric lymphoma, Intestinal T-cell lymphoma, enteropathic T-cell lymphoma, hepatosplenic γ-δ T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sézary syndrome, Hodgkin-like / Hodgkin-associated anaplastic large cell lymphoma, extranodal NK / T-cell lymphoma tumor, adult T-cell lymphoma, T-cell prolymphocytic leukemia, chronic NK-cell lymphoproliferative dysplasia, pediatric systemic EBV-positive T-cell lymphoma, variola bullosa-like lymphoproliferative dysplasia, extranodal NK / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma, gastrointestinal indolent T-cell lymphoproliferative dysplasia, hepatosplenic T-cell lymphoma, primary cutaneous CD30-positive T-cell lymphoproliferative dysplasia, lymphomatoid papulosis, primary cutaneous anaplastic large cell lymphoma, primary Primary cutaneous gamma delta T-cell lymphoma, primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous acral CD8-positive T-cell lymphoma, primary cutaneous CD4-positive small / medium-sized T-cell lymphoproliferative disorder, follicular T-cell lymphoma, nodal peripheral T-cell lymphoma with follicular helper T-cell phenotype, anaplastic large cell lymphoma ALK-positive, anaplastic large cell lymphoma ALK-negative, and breast implant-associated anaplastic large cell lymphoma, and Hodgkin's lymphoma (e.g., classical Hodgkin's lymphoma (e.g., nodular sclerosing, mixed cellularity, lymphocyte-rich, and lymphocytopenic) or nodular lymphocyte-predominant Hodgkin's lymphoma)), leukemia (e.g., acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia (lymphoblastic lymphoma), chronic lymphocytic leukemia (small lymphocytic lymphoma), myelodysplastic syndrome, and chronic myeloid leukemia), primary central nervous system malignant lymphoma, and myeloproliferative syndrome.

[0064] The PD-1 / CD4 bispecific protein (preferably a PD-1 / CD4 bispecific antibody or the like) used in the present invention for the treatment of blood cancers or the like exerts its therapeutic effect by specifically binding, at least in part, to PD-1 expressed on target blood cancer cells and to CD4 expressed on effector lymphocytes (preferably T-cell lymphocytes (e.g., helper T cells or regulatory T cells)). Furthermore, at least in part may exert its therapeutic effect by specifically binding to PD-1 and CD4 expressed on T-cell-derived cancer cells and transmitting PD-1 inhibitory signals.

[0065] In the present invention, "treatment" means, for example, curing or ameliorating a certain disease or its symptoms, "prevention" means preventing the onset of a certain disease or symptom or delaying it for a certain period of time, and "suppression of symptom progression" means suppressing the progression or worsening of symptoms to stop the progression of the pathology. Note that "prevention" also includes suppression of recurrence. "Suppression of recurrence" means preventing the recurrence of a certain disease or symptom or reducing the possibility of recurrence.

[0066] The PD-1 / CD4 bispecific proteins of the present invention are typically administered systemically or locally parenterally. Specific examples of such administration methods include injection, nasal administration, and pulmonary administration. Injection includes, for example, intravenous, intramuscular, and intraperitoneal injection. Intravenous administration is preferably by infusion. The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but typically ranges from 0.1 μg / kg to 300 mg / kg per dose per adult, and more preferably ranges from 0.1 mg / kg to 10 mg / kg. The dosage is administered parenterally once or several times daily, or by continuous intravenous administration for 30 minutes to 24 hours per day. As mentioned above, the dosage varies depending on various conditions. Therefore, a smaller dosage than the above may be sufficient, or a larger dosage may be required.

[0067] [formulation] When the PD-1 / CD4 bispecific proteins of the present invention are formulated and used as injections or infusions for intravenous drip infusion, the injections or infusions may be in the form of an aqueous solution, suspension, or emulsion, or may be formulated as a solid together with a pharmaceutically acceptable carrier so that they can be dissolved, suspended, or emulsified by adding a solvent when used. Solvents that can be used for injections or infusions for intravenous drip infusion include, for example, distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.).

[0068] Examples of pharmaceutically acceptable carriers include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, and antioxidants. Examples of stabilizers that can be used include 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, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol and polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 20 (registered trademark), Polysorbate 80 (registered trademark), and HCO-50). Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of emulsifiers that can be used include gum arabic, sodium alginate, and tragacanth. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, and epsilon aminocaproic acid buffer. Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Examples of antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.Examples of antioxidants that can be used include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid.

[0069] Injections or infusion solutions for intravenous drip can be produced by sterilizing in the final step or by an aseptic procedure, for example, by sterilizing by filtration with a filter or the like, and then filling into a sterile container. Alternatively, injections or infusion solutions for intravenous drip can be prepared by vacuum-drying or freeze-drying to obtain sterile powders (which may contain a powder of a pharmaceutically acceptable carrier), which can be dissolved in an appropriate solvent just before use.

[0070] [Combined or combined drugs] The PD-1 / CD4 bispecific proteins of the present invention may be used in combination with other drugs used for the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of autoimmune diseases, graft-versus-host disease (GVHD), or hematological cancers. When used in combination with other drugs (combined use), the administration form of the bispecific antibodies of the present invention may be a combination drug containing both components in a single formulation, or may be administered as separate formulations. This combination can complement the preventive, suppressive, suppressive, and / or therapeutic effects of the other drug, or maintain or reduce the dosage or frequency of administration. When administering the bispecific antibodies of the present invention and the other drug separately, they may be co-administered for a certain period of time, and then the bispecific antibody or the other drug may be administered alone. Alternatively, the bispecific antibodies of the present invention may be administered first, followed by administration of the other drug after the completion of that administration, or the other drug may be administered first, followed by administration of the bispecific antibodies of the present invention after the completion of that administration. The administration methods for each may be the same or different. A formulation containing the bispecific antibodies of the present invention and a formulation containing the other drug may also be provided as a kit. The dosage of the other drug can be appropriately selected based on the clinically used dose. Furthermore, any two or more of the other drugs may be administered in combination at an appropriate ratio. Furthermore, the other drugs include not only those discovered so far but also those that will be discovered in the future.

[0071] For example, when the bispecific proteins of the present invention are applied to the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of type 1 diabetes, they may be used in combination with insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylureas (e.g., glibenclamide, gliclazide, glimepiride, etc.), rapid-acting insulin secretagogues (e.g., nateglinide, etc.), biguanides (e.g., metformin, etc.), insulin sensitizers (e.g., pioglitazone, etc.), α-glucosidase inhibitors (e.g., acarbose, voglibose, etc.), drugs for treating diabetic neuropathy (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analogues (e.g., liraglutide, exenatide, lixisenatide, etc.), DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, etc.), and the like.

[0072] Furthermore, for example, when the bispecific proteins of the present invention are applied to the prevention, inhibition of symptom progression, inhibition of recurrence, and / or treatment of multiple sclerosis, steroid drugs (e.g., cortisone, cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone, triamcinolone acetate, triamcinolone acetonide, dexamethasone, dexamethasone acetate, dexamethasone valerate, dexamethasone cipesilate, dexamethasone palmitate, betamethasone dipropionate, betamethasone valerate, betamethasone acetate, betamethasone butyrate propionate, and betamethasone sodium phosphate), interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone, azathioprine, cyclophosphamide, ciclospolin, methotrexate, cladribine, adrenocorticotropic hormone (ACTH), corticotropin, mizoribine, tacrolimus, fingolimod, alemtuzumab, and the like.

[0073] Furthermore, for example, when the bispecific proteins of the present invention are applied to the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of systemic lupus erythematosus, they may be used in combination with a steroid drug (e.g., the steroid drugs described above) or an immunosuppressant (e.g., ciclospolin, tacrolimus, fingolimod, etc.).

[0074] For example, when the bispecific proteins of the present invention are applied to the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of rheumatoid arthritis, they may be used in combination with steroid drugs (e.g., the steroid drugs described above), antirheumatic drugs (e.g., methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), or anticytokine drugs (e.g., infliximab, adalimumab, tocilizumab, etanercept, abatacept, golimumab, certolizumab, etc.).

[0075] When applied to the prevention, suppression of symptom progression, suppression of recurrence and / or treatment of other autoimmune diseases, the bispecific proteins of the invention may be used in combination with any one or more of the other agents described above.

[0076] On the other hand, when the bispecific proteins of the present invention are applied to the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of blood cancer, various therapeutic agents such as alkylating agents (e.g., Dacarbazine, Nimustine, Temozolomide, Fotemustine, Bendamustine, Cyclophosphamide, Ifosfamide, Carmustine, Chlorambucil, and Procarbazine), platinum compounds (e.g., Cisplatin, Carboplatin, Nedaplatin, and Oxaliplatin), antimetabolites (e.g., antifolates (e.g., Pemetrexed, Leucovorin, and Methotrexate)), pyridine metabolism inhibitors (e.g., TS-1 (registered trademark), 5-fluorouracil, and the like), and the like are useful in the prevention, suppression of symptom progression, suppression of recurrence, and / or treatment of blood cancer. l, UFT, Carmofur, Doxifluridine, FdUrd, Cytarabine, and Capecitabine, etc.), purine metabolism inhibitors (e.g., Fludarabine, Cladribine, and Nelarabine, etc.), ribonucleotide reductase inhibitors, nucleotide analogs (e.g., Gemcitabine, etc.), topoisomerase inhibitors (e.g., Irinotecan, Nogitecan, and Etoposide, etc.), microtubule polymerization inhibitors (e.g., Vinblastine, Vincristine, Vindesine, Vinorelbine, and Eribulin, etc.), microtubule depolymerization inhibitors (e.g., Docetaxel and Paclitaxel, etc.), antitumor antibiotics (e.g., Bleomycin, Mitomycin, C, Doxorubicin, Daunorubicin, Idarubicin, Etoposide, Mitoxantrone, Vinblastine, Vincristine, Peplomycin, Amrubicin, Aclarubicin, Epirubicin, etc.), cytokine preparations (e.g., IFN-α2a, IFN-α2b, PEG-IFN-α2b, natural IFN-β, interleukin-2, etc.), molecular targeted drugs, cancer immunotherapy drugs, other antibody drugs, etc.

[0077] Examples of molecular targeted drugs include ALK inhibitors (e.g., Crizotinib, Ceritinib, Ensartinib, Alectinib, and Lorlatinib), BCR-ABL inhibitors (e.g., Imatinib and Dasatinib), Axl inhibitors (e.g., ONO-7475 and BGB324), CDK inhibitors (e.g., Dinaciclib, Abemaciclib, Palbociclib, and Trilaciclib), Btk inhibitors (e.g., Ibrutinib and Acalabrutinib), PI3K-δ / γ inhibitors (e.g., Umbralisib, Parsaclisib, and IPI-549), JAK-1 / 2 inhibitors (e.g., Itacitinib and Ruxolitinib), FAK inhibitors (e.g., Defactinib), and Syk / FLT3 inhibitors. Dual inhibitors (e.g., Mivavotinib, etc.), ATR inhibitors (e.g., Ceralasertib, etc.), Wee1 kinase inhibitors (e.g., Adavosertib, etc.), mTOR inhibitors (e.g., Temsirolimus, Everolimus, Vistusertib, and Irinotecan, etc.), HDAC inhibitors (e.g., Vorinostat, Romidepsin, Entinostat, Chidamide, Mocetinostat, Citarinostat, Panobinostat, and Valproate, etc.), EZH2 inhibitors (e.g., Tazemetostat, etc.), STAT3 inhibitors (e.g., Napabucasin, etc.), DNMT inhibitors (e.g., Azacitidine, etc.), BCL-2 inhibitors (e.g., Navitoclax, Venetoclax, etc.), SMO inhibitors (e.g., Vismodegib, etc.), anti-CD20 antibodies (e.g., Rituximab, Blontuvetmab, Epitumomab, Ibritumomab, etc.), tiuxetan, Ocaratuzumab, Ocrelizumab, Technetium ( 99mTc) nofetumomab merpentan, tositumomab, veltuzumab, ofatumumab, ublituximab, obinutuzumab and nofetumomab, etc.), anti-CD30 antibodies (e.g., brentuximab vedotin and iratumumab, etc.), anti-CD38 antibodies (e.g., daratumumab, isatuximab, mezagitamab, AT13 / 5 and MOR202, etc.), anti-DR5 antibodies (e.g., DS-8273a, etc.), anti-TNFRSF10B antibodies (e.g., benufutamab, conatumumab, drozitumab, lexatumumab, tigatuzumab, eftozanermin, alfa and DS-8273a, etc.), anti-CD40 antibodies (e.g., bleselumab, dacetuzumab, iscalimab, lucatumumab, mitazalimab, ravagalimab, selicrelumab, teneliximab, ABBV-428 and APX005M, etc.), anti-CD70 antibodies (e.g., cusatuzumab, vorsetuzumab, vorsetuzumab mafodotin and ARGX-110, etc.), anti-TNFRSF10A antibodies (e.g., mapatumumab, etc.), anti-CD79b antibodies (e.g., iladatuzumab, iladatuzumab vedotin and polatuzumab vedotin, etc.), TNFSF11 antibodies (e.g., denosumab, etc.), anti-NCAM1 antibodies (e.g., lorvotuzumab mertansine, etc.), anti-CD37 antibodies (e.g., lilotomab, lutetium ( 177lu) lilotomab satetraxetan, Naratuximab, Naratuximab and anti-CD200 antibodies (e.g., Samalizumab, etc.), anti-CD30-CD16A bispecific antibodies (e.g., AFM13, etc.), anti-IL3RA-CD3 bispecific antibodies (e.g., Flotetuzumab and Vibecotamab, etc.), anti-GPRC5D-CD3 bispecific antibodies (e.g., Talquetamab), anti-CD3-CD19 bispecific antibodies (e.g., Duvortuxizumab and Blinatumomab, etc.), anti-TNFRSF17-CD3 bispecific antibodies (e.g., Teclistamab), anti-CLEC12A-CD3 bispecific antibodies (e.g., Tepoditamab), and anti-CD20-CD3 bispecific antibodies (e.g., Plamotamab, Odronextamab, Mosunetuzumab, Glofitamab, Epcoritamab, and REGN1979, etc.).

[0078] Furthermore, examples of cancer immunotherapeutic drugs include anti-PD-1 antibodies (e.g., Nivolumab, Cemiplimab, Pembrolizumab, Spartalizumab, Tislelizumab, Dostarlimab, Toripalimab, Camrelizumab, Genolimzumab, Sintilimab, Lodapolimab, Retifanlimab, Balstilimab, Serplulimab, Budigalimab, 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, JY034, ISU106, HX008, and CX-188, etc.), anti-PD-L1 antibodies (e.g., Atezolizumab, Avelumab, Durvalumab, Manelimab, Pacmilimab, Envafolimab, Cosibelimab, BMS-9 36559, STI-1010, STI-1011, STI-1014, SHR-1316, CS1001, MSB2311, BGB-A333, KL-A167, AK106, AK104, ZKAB001, FAZ053, CBT-502, and JS003, etc.), PD-1 antagonists (e.g., AUNP-12, BMS-M1 to BMS-M10, BMS-1 , BMS-2, BMS-3, BMS-8, BMS-37, BMS-200, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, Incyte-1 to Incyte-6 compounds, CAMC-1 to CAMC-4 compounds, RG_1 and DPPA-1, etc.), PD-L1 / VISTA antagonists (e.g., CA-170, etc.), PD-L1 / TIM3 antagonists (e.g., CA-327, etc.), anti-PD-L2 antibodies, PD-L1 fusion proteins, PD-L2 fusion proteins (e.g., AMP-224, etc.), anti-CTLA-4 antibodies (e.g., Ipilimumab, Zalifrelimab, Nurulimab, and Tremelimumab, etc.), anti-LAG-3 antibodies (e.g., Relatlimab,Ieramilimab, Fianlimab, Encelimab, and Mavezelimab, etc.), anti-TIM3 antibodies (e.g., MBG453 and Cobolimab, etc.), anti-KIR antibodies (e.g., Lirilumab, IPH2101, LY3321367, and MK-4280, etc.), anti-BTLA antibodies, anti-TIGIT antibodies (e.g., Tiragolumab, Etigilimab, Vibostolimab, and BMS-986207, etc.), anti-VISTA antibodies (e.g., Onvatilimab, etc.), anti-CD137 antibodies (e.g., Urelumab and Utomilumab, etc.), anti-CSF-1R antibodies, CS F-1R inhibitors (e.g., Cabiralizumab, Emactuzumab, LY3022855, Axatilimab, MCS-110, IMC-CS4, AMG820, Pexidartinib, BLZ945, and ARRY-382, etc.), anti-OX40 antibodies (e.g., MEDI6469, Ivuxolimab, MEDI0562, MEDI6383, Efizonerimod, GSK3174998, BMS-986178, and MOXR0916, etc.), anti-HVEM antibodies, anti-CD27 antibodies (e.g., Varlilumab, etc.), anti-GITR antibody / GITR fusion proteins (e.g., Efaprinermin, etc.), alfa, Efgivanermin alfa, MK-4166, INCAGN01876, GWN323, and TRX-518, etc.), anti-CD28 antibodies, anti-CCR4 antibodies (e.g., Mogamulizumab, etc.), anti-B7-H3 antibodies (e.g., Enoblituzumab, Mirzotamab, Mirzotamab clezutoclax, and Omburtamab, etc.), anti-ICOS agonist antibodies (e.g., Vopratelimab and GSK3359609, etc.), anti-CD4 antibodies (e.g., Zanolimumab and IT1208, etc.), anti-DEC-205 antibody / NY-ESO-1 fusion protein (e.g., CDX-1401, etc.), anti-SLAMF7 antibodies (e.g., Azintuxizumab, Azintuxizumab vedotin and Elotuzumab, etc.), anti-CD73 antibodies (e.g., Oleclumab and BMS-986179, etc.), PEGylated IL-2 (e.g., Bempegaldesleukin),Anti-CD40 agonist antibodies (e.g., ABBV-428, APX005M, and RO7009789, etc.), IDO inhibitors (e.g., Epacadostat, Indoximod, and Linrodostat, etc.), TLR agonists (e.g., Motolimod, CMP-001, G100, Tilsotolimod, SD-101, and MEDI9197, etc.), adenosine A2A receptor antagonists (e.g., Preladenant, AZD4635, T aminadenant and ciforadenant, etc.), anti-NKG2A antibodies (e.g., monalizumab, etc.), anti-CSF-1 antibodies (e.g., PD0360324, etc.), immunopotentiators (e.g., PV-10, etc.), IL-15 superagonists (e.g., ALT-803), soluble LAG3 (e.g., IMP321, etc.), anti-CD47 antibodies / CD47 antagonists (e.g., ALX148, etc.), and IL-12 antagonists (e.g., M9241, etc.).

[0079] Furthermore, other antibody drugs include, for example, anti-IL-1β antibodies (eg, canakinumab, etc.) and anti-CCR2 antibodies (eg, plozalizumab, etc.).

[0080] The present invention will be described in more detail by way of the following examples, but the scope of the present invention is not limited thereto. Various changes and modifications may be made by those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention. [Example]

[0081] 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 a single-chain diabody (hereinafter abbreviated as "scDb") composed of the VH and VL of an anti-mouse CD4 antibody and the VH and VL of an anti-mouse PD-1 antibody. Figure 1 shows a schematic representation of its structure. The scDb was constructed by linking DNA encoding the variable regions of each antibody and a peptide linker in a predetermined order. The resulting DNA was inserted into an expression vector and expressed in animal cells (FreeStyle 293F cells). The scDb produced in the culture supernatant was collected and purified by Ni-NTA affinity chromatography and size-exclusion chromatography. Nine types of PD-1 / CD4 scDbs were prepared in this example, one of which was designated "Clone-1." The cDNAs encoding the variable regions (VH and VL) of the anti-mouse CD4 antibody and anti-mouse PD-1 antibody that constitute the scDb in this example were obtained according to a method similar to a known gene cloning method. In this example, as an example, J43 was used as the anti-mouse PD-1 antibody, and YTA3.1.2, YTS191, or GK1.5 was used as the anti-mouse CD4 antibody.

[0082] Example 2: Preparation of PD-1 x CD4 bispecific protein (PD-1 / CD4 scDb-Fc) The PD-1 / CD4 scDb-Fc in this example is a heterodimer formed by the association of two proteins: a polypeptide (hereinafter abbreviated as "polypeptide A") in which a bispecific scDb composed of the VH and VL of an anti-mouse CD4 antibody and the VH and VL of an anti-mouse PD-1 antibody is fused to a human IgG1 constant region from the hinge to the CH3 region (IgG1 constant region (hinge / CH3)), and a polypeptide (hereinafter abbreviated as "polypeptide B") consisting only of the IgG1 constant region (hinge / CH3). This heterodimer contains a mutation that eliminates Fcγ receptor binding. A schematic diagram of its structure is shown in Figure 2.

[0083] DNA encoding polypeptide A was prepared by linking DNA encoding the VH and VL of the anti-mouse CD4 antibody and anti-mouse PD-1 antibody obtained in Example 1 together with DNA encoding a peptide linker in a predetermined order to prepare the PD-1 / CD4 scDb. The cDNA encoding the IgG1 constant region (hinge / CH3), which constitutes part of polypeptides A and B, was obtained by PCR based on known genetic information. Expression vectors containing DNA sequences encoding polypeptides A and B were transfected into FreeStyle 293F cells for expression and production in the culture supernatant. The culture supernatant was collected and purified by Protein A affinity chromatography and size exclusion chromatography to prepare the PD-1 / CD4 scDb-Fc. Nine types of PD-1 / CD4 scDb-Fc were prepared in this example, one of which was designated "Clone-2."

[0084] Example 3: Binding evaluation of PD-1 / CD4 bispecific proteins The binding of the PD-1 / CD4 bispecific protein to mouse PD-1 was assessed by flow cytometry using mouse PD-1-expressing CHO-S cells. Because the PD-1 / CD4 scDb contains a 6xHis tag at its C-terminus, Alexa Fluor 488-conjugated anti-His tag antibody (MBL, product number D291-A48) was used to detect each recombinant protein bound to the cells. The results are shown in Figure 3. On the other hand, because the PD-1 / CD4 scDb-Fc contains the human IgG1 constant region, a PE-conjugated anti-human IgG-Fc antibody (Thermo Fisher Scientific, product number H10104) was used to detect binding to mouse PD-1. The results are shown in Figure 4.

[0085] Both PD-1 / CD4 bispecific proteins were confirmed to bind to mouse PD-1-expressing CHO-S cells. In contrast, no binding was observed to control CHO-S cells, confirming that the binding was specific to the expressed PD-1. Subsequently, using a similar method, specific binding of the PD-1 / CD4 bispecific proteins to mouse CD4 was confirmed using mouse CD4-expressing CHO-K1 cells. The results are shown in Figures 5 and 6.

[0086] Example 4: In vivo effects of PD-1 / CD4 bispecific proteins in a mouse model of experimental allergic encephalomyelitis (EAE) The in vivo effects of the PD-1 / CD4 bispecific protein were evaluated in an EAE model using C57BL / 6 mice. Killed Mycobacterium tuberculosis H37Ra (BD Biosciences, product number 231141) was mixed with incomplete Freund's adjuvant (BD Biosciences, product number 263910) to prepare 4 mg / mL of killed Mycobacterium tuberculosis H37Ra in complete Freund's adjuvant (CFA). An emulsion was prepared by mixing 1 mg / mL of MOG peptide (ANASPEC, product number AS-60130) with an equal volume of CFA to serve as an EAE inducer. C57BL / 6 mice were injected subcutaneously with 200 μL of the inducer into the base of their tails, and on the day of immunization and the second day, 200 μL of 1 μg / mL pertussis toxin (Sigma-Aldrich, product number P7208) was administered intravenously into the tail vein. Next, the C57BL / 6 mice were intraperitoneally administered Clone-1 or Clone-2 once daily for 5 days, from days 6 to 10 after immunization. From the day of immunization onward, neurological symptoms were evaluated according to the method of Onuki et al. (Onuki M, et al., Microsc Res Tech 2001;52:731-9). The severity of neurological symptoms was scored (normal: score 0; tail flaccid: score 1; partial hindlimb paralysis: score 2; hindlimb paralysis: score 3; forelimb paralysis: score 4 and moribund or dead: score 5). If multiple neurological symptoms were observed, the highest score was used as the neurological symptom score on the day of evaluation. The cumulative neurological symptom scores for the observation period from the day of immunization to 30 days after immunization were calculated. The evaluation results are shown in Figure 7.

[0087] Clone-1 and Clone-2 suppressed neurological symptoms in the EAE model.

[0088] Example 5: Effect of PD-1 / CD4 bispecific proteins on blood cytokine levels in mice in vivo Mice were administered the PD-1 / CD4 bispecific proteins of the present invention, and changes in plasma cytokine concentrations were evaluated in vivo. C57BL / 6 mice were administered 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. Additionally, the PD-1 / CD3 bispecific scDb (J43×2C11_scDb) disclosed in Patent Document 1 was intraperitoneally administered at a dose of 0.5 mg / kg. Three hours after each administration, blood was collected from the tail vein of the mice, and plasma was separated. Plasma interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) concentrations were measured using a flow cytometer (BD Biosciences, BD FACSCantoII) with the BD Cytometric Bead Array Mouse Soluble Protein Flex Set (BD Biosciences, product numbers 558296, 558299, and 558297). The results are shown in Figure 8. Plasma IFN-γ, TNF-α, and IL-2 concentrations (pg / mL) in the figure are shown as mean ± standard error (N = 5).

[0089] J43×2C11_scDb increased plasma IFN-γ, TNF-α, and IL-2 concentrations in mice. In contrast, none of the PD-1 / CD4 bispecific proteins of the present invention increased plasma IFN-γ, TNF-α, or IL-2 concentrations. When the target molecule was CD4, induction of cytokine production or release was improved upon administration.

[0090] Example 6: Cytostatic effect of PD-1 / CD4 bispecific proteins on B-cell lymphoma cell lines Pan-T cells (STEMCELL, model number ST-70024) (human T cells) derived from peripheral blood of healthy donors were used to evaluate the cytostatic activity against B-cell lymphoma cell lines. Human T cells were seeded in a U-bottom 96-well plate with B-cell lymphoma cell lines EB-1 or SU-DHL-4 expressing human PD-1, labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887), and co-cultured for 48 hours with PD-1 / CD4 bispecific proteins. Cells were harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive viable cells was counted using a flow cytometer. The percentage reduction in DiD-positive viable cells in the PD-1 / CD4 bispecific protein-treated sample was calculated as the cytostatic rate, with the number of DiD-positive viable cells in the sample without the PD-1 / CD4 bispecific protein defined as 100%.

[0091] Example 7: Cytostatic effect of PD-1 / CD4 bispecific proteins on multiple myeloma cell lines Pan-T cells (STEMCELL, model number ST-70024) (human T cells) derived from peripheral blood of healthy donors were used to evaluate the cytostatic effect on multiple myeloma cell lines. Human T cells were seeded in a U-bottom 96-well plate with the multiple myeloma cell line RPMI8226 labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887), and co-cultured with PD-1 / CD4 bispecific recombinant protein for 48 hours. Cells were harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive viable cells was counted using a flow cytometer. The number of DiD-positive viable cells in the sample without PD-1 / CD4 bispecific protein was defined as 100%, and the percentage reduction in DiD-positive viable cells in the sample with PD-1 / CD4 bispecific protein was calculated as the cytostatic rate.

[0092] Example 8: Cytostatic effect of PD-1 / CD4 bispecific proteins on adult T-cell lymphoma cell lines Pan-T cells (STEMCELL, model number ST-70024) (human T cells) derived from peripheral blood of healthy donors were used to evaluate the cytostatic activity against adult T-cell lymphoma cell lines. Human T cells and the adult T-cell lymphoma cell line ILT-Mat labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded into a U-bottom 96-well plate, and co-cultured with PD-1 / CD4 bispecific recombinant protein for 48 hours. Cells were harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive viable cells was counted using a flow cytometer. The number of DiD-positive viable cells in the sample without PD-1 / CD4 bispecific protein was defined as 100%, and the percentage reduction in DiD-positive viable cells in the sample with PD-1 / CD4 bispecific protein was calculated as the cytostatic rate.

[0093] Example 9: Cytostatic effect of PD-1 / CD4 bispecific proteins on acute T-cell leukemia cell lines Pan-T cells (STEMCELL, model number ST-70024) (human T cells) derived from peripheral blood of healthy donors were used to evaluate the cytostatic activity against acute T-cell leukemia cell lines. Human T cells and Jurkat cells (acute T-cell leukemia cell line) overexpressing human PD-1 labeled with Vybrant DiD Cell-Labeling Solution (Thermo Fisher, model number V22887) were seeded into a U-bottom 96-well plate, and co-cultured for 48 hours with the addition of PD-1 / CD4 bispecific protein. Cells were harvested from each well, stained with Cell Viability Solution, and the number of DiD-positive viable cells was counted using a flow cytometer. The percentage reduction in DiD-positive viable cells in the sample with PD-1 / CD4 bispecific protein was calculated as the cytostatic rate, with the number of DiD-positive viable cells in the sample without PD-1 / CD4 bispecific protein defined as 100%. [Industrial Applicability]

[0094] The PD-1 / CD4 bispecific proteins of the present invention are useful for preventing, inhibiting the progression of symptoms, inhibiting recurrence, and / or treating autoimmune diseases, graft-versus-host disease (GVHD), blood cancers, and the like.

Claims

1. 1. A PD-1 / CD4 bispecific antibody having a first arm that specifically binds to PD-1 and a second arm that specifically binds to CD4, (a) the first arm comprises a VH and VL of an anti-PD-1 antibody, and the second arm comprises a VH and VL of an anti-CD4 antibody; (b)(i) having a structure in which, from the N-terminus, the VL of an anti-PD-1 antibody, the VH of an anti-CD4 antibody, the VL of an anti-CD4 antibody, and the VH of an anti-PD-1 antibody are linked via a peptide linker in this order; (ii) having a structure in which, from the N-terminus, the VL of an anti-PD-1 antibody, the VH of an anti-PD-1 antibody, the VH of an anti-CD4 antibody, and the VL of an anti-CD4 antibody are linked via a peptide linker, in this order; or (iii) The antibody has a structure in which, from the N-terminus, the VL of an anti-CD4 antibody, the VH of an anti-PD-1 antibody, the VL of an anti-PD-1 antibody, and the VH of an anti-CD4 antibody are linked via a peptide linker in this order.

2. The PD-1 / CD4 bispecific antibody of claim 1, wherein the N-terminus of a polypeptide comprising a human IgG constant region from the hinge region to the CH3 region is further linked, via a peptide linker, to the C-terminus of the VH of the anti-PD-1 antibody of claim 1(b)(i), the VL of the anti-CD4 antibody of claim 1(b)(ii), or the VH of the anti-CD4 antibody of claim 1(b)(iii).

3. The PD-1 / CD4 bispecific antibody of claim 2, further comprising a polypeptide having a human IgG constant region from the hinge region to the CH3 region.

4. 4. The PD-1 / CD4 bispecific antibody of claim 1 , wherein the peptide linker is Gly-Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser (provided that when multiple peptide linkers are present, they may be the same or different).

5. The IgG constant region of the polypeptide having the human IgG constant region from the hinge region to the CH3 region is １ The PD-1 / CD4 bispecific antibody of claim 2 or 3, which is derived from an antibody.

6. The PD-1 / CD4 bispecific antibody of any one of claims 1 to 5, wherein PD-1 and CD4 are human PD-1 and human CD4, respectively.

7. Anti-PD-1 antibodies include Nivolumab, Cemiplimab, Pembrolizumab, Spartalizumab, Tislelizumab, Dostarlimab, Toripalimab, Camrelizumab, Genolimzumab, Sintilimab, Lodap olimab, Retifanlimab, Balstilimab, Serplulimab, Budigalimab, Prolgolimab, Sasanlimab, Cetrelimab, Zimberelimab, Penpulimab, MEDI0680, STI-A1110, ENUM 388D4, ENUM 244C8, GLS010, CS1003, BAT-1306, AK103, BI 754091, LZM009, CMAB819, Sym021, SSI-361, JY034, HX008, ISU106, or CX-188.

8. 8. The PD-1 / CD4 bispecific antibody of any one of claims 1 to 7, wherein the anti-CD4 antibody is MTRX-1011A, TRX-1, Ibalizumab, BT-061, huB-F5, Zanolimumab, 4162W94, Clenoliximab, Keliximab, AD-519, PRO-542, Cedelizumab, or IT1208.

9. The PD-1 / CD4 bispecific antibody of any one of claims 1 to 8, which sufficiently reduces cytokine production during administration or within 24 hours after administration.

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