Therapeutic agent for T-cell tumor

Abstract

The present invention addresses the problem of providing a novel method for treating T-cell tumors, which is capable of inducing cytotoxicity in tumor cells and has a low possibility of causing immunodeficiency. According to the present invention, provided is a therapeutic agent for T cell tumors, which comprises a bispecific antigen-binding molecule, the bispecific antigen-binding molecule comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed in a T-cell tumor cell, and (2) at least one moiety that specifically binds to an antigen having a subtype, which is a normal T-cell side target antigen; wherein the target tumor antigen expressed in the T cell tumor cell is not present in the normal T cell, or even if the target tumor antigen is present, the normal T cell is substantially not activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in the normal T cell. Normal T cells are activated by binding a bispecific antigen binding molecule to a normal T cell side target antigen, and there is a sufficient proportion of subtypes of the normal T cell side target antigen in order to provide a sufficient number of activated T cells required for the treatment of T cell tumors.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority of Japanese Patent Application No. 2022 - 167027 filed on October 18, 2022, the entire disclosure of which is incorporated herein by reference in its entirety.

[0003] The present invention relates to a therapeutic agent for treating T - cell tumors, which is a therapeutic agent containing a bispecific antigen - binding molecule. The bispecific antigen - binding molecule contains at least one part that specifically binds to a target tumor antigen expressed in T - cell tumor cells and at least one part that specifically binds to an antigen having subtypes as a target antigen on the normal T - cell side. The present invention relates to the bispecific antigen - binding molecule and its use, and a method for treating T - cell tumors including administering the bispecific antigen - binding molecule to a subject. Background Art

[0004] Bispecific antibodies that can induce T - cell activity in target cells and selectively kill target cells in vivo have been developed. A bispecific antibody (Blinatumomab) that can simultaneously recognize human CD19 antigen and CD3 antigen has been approved for sale as a therapeutic agent for relapsed / refractory B - cell acute lymphoblastic leukemia (ALL) (Goebeler, M.E. and R. Bargou, Blinatumomab: a CD19 / CD3 bispecific T - cell engager (BiTE) with unique anti - tumor efficacy. Leuk Lymphoma, 2016. 57(5): p. 1021 - 32.; the entire disclosure of which is incorporated herein by reference in its entirety). Blinatumomab is a bispecific T - cell engaging antibody (BiTE TM ) and has a structure of a single - chain antibody obtained by connecting two single - chain variable region fragments (scFv) with a short linker. In addition to Blinatumomab, many bispecific antibodies targeting CD3 antigen and tumor cell antigens have been developed in recent years.

[0005] Lymphoid malignancies are roughly divided into B-cell malignancies and T-cell malignancies, and most bispecific antibodies have been developed for the treatment of B-cell malignancies. Acute myeloid leukemia cells and normal bone marrow progenitor cells have the same cell surface antigens. Therefore, bispecific antibodies against the tumor cell antigens of acute myeloid leukemia attack not only leukemia cells but also normal bone marrow progenitor cells, causing neutropenia and leading to the onset of febrile neutropenia, which is difficult to treat. In immunotherapy for B-cell malignancies, the antigens targeted are also expressed in normal B cells. Bispecific antibodies against the tumor cell antigens of B-cell malignancies attack not only tumor cells but also normal B cell progenitor cells, causing B-cell immunodeficiency and thus the inability to produce antibodies. However, by regularly administering immunoglobulin preparations, B-cell immunodeficiency can be prevented (Maciocia PM, et al., Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies. Nat Med. 2017 Dec;23(12):1416-1423.; the entire disclosure of which is hereby incorporated by reference in its entirety).

[0006] On the other hand, immunotherapy for T-cell malignancies is limited. Since CD3 is expressed equally in both normal T cells and tumor T cells, bispecific antibodies targeting CD3 and tumor antigens bind simultaneously to the CD3 antigen expressed in T-cell malignancies and the target antigen expressed in T-cell malignancies, connecting malignant tumor cells to each other ( Figure 1A ), so it is very likely that the therapeutic effect of the bispecific antibody cannot be fully obtained. Further, there is also the problem that the target antigens that distinguish normal T cells and malignant cells are unknown. The formation defect of T cells caused by targeting pan-T cell antigens may lead to severe and unacceptable immunodeficiency (Maciocia PM, et al. cited above).

[0007] In addition to the CD3 antigen, there are also bispecific antibodies that use TRGV9 expressed in γδ T cells as the target on the effector cell (T cell) side (WO2021 / 173896A1; the entire disclosure of which is hereby incorporated by reference in its entirety). However, γδ T cells account for less than a few percent of the total T cells, so there is a problem that a sufficient number of T cells cannot be mobilized to kill tumor cells to obtain a therapeutic effect. Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] There is a need for a new treatment method for T cell tumors that has a low possibility of causing immunodeficiency problems even when used for the treatment of T cell tumors and can achieve high treatment effects.

[0010] Therefore, an object of the present invention is to provide a new treatment method for T cell tumors that can induce cytotoxicity of tumor cells and has a low possibility of causing immunodeficiency.

[0011] Means for Solving the Problem

[0012] The present inventors repeatedly conducted in-depth research to solve the above problems, and as a result, found that a bispecific antigen-binding molecule that specifically binds to a target tumor antigen expressed in T cell tumor cells and a target antigen with a subtype on the normal T cell side mobilizes effector cells (normal T cells) and induces cytotoxicity of tumor cells. Further, the present inventors found that by using an antigen that expresses different subtypes between T cell tumor cells and normal T cells among antigens with subtypes as the target antigen on the normal T cell side to which the bispecific antigen-binding molecule binds, tumor cells of T cell malignancies can be killed while retaining sufficient normal T cells. The present invention was completed based on the above insights.

[0013] According to the present invention, the following inventions are provided.

[0014] [1] A therapeutic agent for T cell tumors, which comprises a bispecific antigen-binding molecule, wherein

[0015] The bispecific antigen-binding molecule comprises:

[0016] (1) at least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0017] (2) at least one part that specifically binds to an antigen with a subtype as the target antigen on the normal T cell side;

[0018] wherein the target tumor antigen expressed in T cell tumor cells does not exist in normal T cells, or even if it exists, when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in normal T cells, normal T cells are not substantially activated,

[0019] By binding of the bispecific antigen-binding molecule to the target antigen on the normal T cell side, normal T cells are activated, and

[0020] there is a sufficient proportion of subtypes of the target antigen on the normal T cell side to provide a sufficient number of activated T cells for the treatment of T cell tumors.

[0021] [2] The therapeutic agent according to [1], wherein the subtype of the target antigen on the normal T cell side is any one of the antigen subtypes that are not the antigen subtypes expressed in T cell tumor cells.

[0022] [3] The therapeutic agent according to [1] or [2], wherein the antigen having a subtype as the target antigen on the normal T cell side is TRBC (T cell receptor β constant region),

[0023] The subtype expressed in T cell tumor cells is TRBC1, and the subtype of the target antigen on the normal T cell side is TRBC2.

[0024] [4] The therapeutic agent according to [1] or [2], wherein the antigen having a subtype as the target antigen on the normal T cell side is TRBC,

[0025] The subtype expressed in T cell tumor cells is TRBC2, and the subtype of the target antigen on the normal T cell side is TRBC1.

[0026] [5] The therapeutic agent according to [1] or [2], wherein the antigen having a subtype as the target antigen on the normal T cell side is TRBC,

[0027] T cell tumor cells are negative for TRBC1 and negative for TRBC2, and the subtype of the target antigen on the normal T cell side is TRBC1 or TRBC2.

[0028] [6] The therapeutic agent according to any one of [1]-[5], wherein the target tumor antigen expressed in T cell tumor cells is selected from any one of CCR1, CCR4, CCR7, CCR8, CCR10, CXCR4, CXCR7, TIGIT, CADM1, GPR15, CXCR5, CXCL13, SLAM, ICOS, CD134, CXCR3, anaplastic lymphoma kinase, CD30, ST2(L), CCR5, Notch1, CD38, CD1a, CCR9(CD199), CD47, IL-7Rα(CD127), and CD40L(CD154).

[0029] [7] The therapeutic agent according to any one of [1]-[6], wherein the subtype of the target antigen on the normal T cell side is TRBC1,

[0030] The bispecific antigen-binding molecule comprises at least 1 moiety that specifically binds to TRBC1 of normal T cells, and the at least 1 moiety comprises a VH domain and a VL domain,

[0031] The VH domain comprises:

[0032] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10,

[0033] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0034] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0035] The VL domain comprises:

[0036] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13,

[0037] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0038] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15.

[0039] [8] The therapeutic agent according to [7], wherein at least one part specifically binding to TRBC1 of normal T cells comprises:

[0040] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4, and

[0041] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5.

[0042] [9] The therapeutic agent according to any one of [1] - [6], wherein the subtype of the target antigen on the normal T cell side is TRBC2,

[0043] The bispecific antigen-binding molecule comprises at least one part specifically binding to TRBC2 of normal T cells, and the aforementioned at least one part comprises a VH domain and a VL domain,

[0044] The VH domain comprises:

[0045] the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16,

[0046] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0047] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0048] The VL domain comprises:

[0049] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19,

[0050] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0051] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21.

[0052]

[10] The therapeutic agent according to [9], wherein at least one moiety that specifically binds to TRBC2 of normal T cells comprises:

[0053] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6, and

[0054] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7.

[0055]

[11] The therapeutic agent according to any one of [1]-

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CCR4,

[0056] The bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR4, and the aforementioned at least one moiety comprises a VH domain and a VL domain,

[0057] The VH domain comprises:

[0058] the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22,

[0059] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and

[0060] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24;

[0061] The VL domain comprises:

[0062] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25,

[0063] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and

[0064] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27.

[0065]

[12] The therapeutic agent according to

[11] , wherein at least one moiety that specifically binds to CCR4 comprises:

[0066] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8, and

[0067] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9.

[0068]

[13] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CD1a,

[0069] The bispecific antigen-binding molecule comprises at least 1 moiety that specifically binds to CD1a, and the aforementioned at least 1 moiety comprises a VH domain and a VL domain,

[0070] The VH domain comprises:

[0071] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:34, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:35, and

[0072] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:36;

[0073] The VL domain comprises:

[0074] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:37, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and

[0075] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:39.

[0076]

[14] The therapeutic agent according to

[13] , wherein the at least 1 moiety that specifically binds to CD1a comprises:

[0077] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:40, and

[0078] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:41.

[0079]

[15] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CCR9,

[0080] The bispecific antigen-binding molecule comprises at least 1 moiety that specifically binds to CCR9, and the aforementioned at least 1 moiety comprises a VH domain and a VL domain,

[0081] The VH domain comprises:

[0082] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 101, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102, and

[0083] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 103;

[0084] The VL domain comprises:

[0085] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105, and

[0086] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106.

[0087]

[16] The therapeutic agent according to

[15] , wherein at least one moiety that specifically binds to CCR9 comprises:

[0088] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 131, and

[0089] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 132.

[0090]

[17] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CXCR4,

[0091] the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CXCR4, and the foregoing at least one moiety comprises a VH domain and a VL domain,

[0092] The VH domain comprises:

[0093] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107,

[0094] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and

[0095] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;

[0096] The VL domain comprises:

[0097] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110,

[0098] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and

[0099] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0100]

[18] The therapeutic agent according to

[17] , wherein at least one moiety specifically binding to CXCR4 comprises:

[0101] a heavy chain variable region VH having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 133, and

[0102] a light chain variable region VL having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 134.

[0103]

[19] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is TIGIT,

[0104] the bispecific antigen-binding molecule comprises at least one moiety specifically binding to TIGIT, and the aforementioned at least one moiety comprises a VH domain and a VL domain,

[0105] the VH domain comprises:

[0106] the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89,

[0107] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and

[0108] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91;

[0109] the VL domain comprises:

[0110] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92,

[0111] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and

[0112] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94.

[0113]

[20] The therapeutic agent according to

[19] , wherein at least one moiety specifically binding to TIGIT comprises:

[0114] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 135, and

[0115] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 136.

[0116]

[21] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CCR8,

[0117] The bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR8, and the at least one moiety comprises a VH domain and a VL domain,

[0118] The VH domain comprises:

[0119] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, and

[0120] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97;

[0121] The VL domain comprises:

[0122] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99, and

[0123] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0124]

[22] The therapeutic agent according to

[21] , wherein the at least one moiety that specifically binds to CCR8 comprises:

[0125] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 137, and

[0126] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 138.

[0127]

[23] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CD30,

[0128] The bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD30, and the at least one moiety comprises a VH domain and a VL domain,

[0129] The VH domain comprises:

[0130] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and

[0131] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115;

[0132] The VL domain comprises:

[0133] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116,

[0134] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and

[0135] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0136]

[24] The therapeutic agent according to

[23] , wherein at least one moiety that specifically binds to CD30 comprises:

[0137] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 139, and

[0138] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 140.

[0139]

[25] The therapeutic agent according to any one of [1] -

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CD40L (CD154),

[0140] The bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD40L (CD154), and the aforementioned at least one moiety comprises a VH domain and a VL domain,

[0141] The VH domain comprises:

[0142] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119,

[0143] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120, and

[0144] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121;

[0145] The VL domain comprises:

[0146] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122,

[0147] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and

[0148] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124.

[0149]

[26] The therapeutic agent according to

[25] , wherein at least one moiety that specifically binds to CD40L (CD154) comprises:

[0150] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 141, and

[0151] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 142.

[0152]

[27] The therapeutic agent according to any one of [1]-

[26] , wherein the T cell tumor is T cell acute lymphoblastic leukemia / lymphoblastic lymphoma or mature T cell tumor, preferably T cell acute lymphoblastic leukemia / lymphoblastic lymphoma or mature T cell tumor, preferably any one selected from adult T cell leukemia (ATL), peripheral T cell lymphoma (PTCL), and T cell acute lymphoblastic leukemia (TALL).

[0153]

[28] A therapeutic agent, which is the therapeutic agent according to any one of [1]-

[27] for a method of treating a T cell tumor in a subject, wherein the aforementioned treatment method comprises: determining the subtype expressed in the T cell tumor cells of the subject, and administering the therapeutic agent to the subject; the therapeutic agent comprises a bispecific antigen-binding molecule, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to a target antigen on the normal T cell side, and the subtype of the target antigen on the normal T cell side is different from the subtype determined to be expressed in the T cell tumor cells of the subject.

[0154]

[29] A therapeutic agent, which is a therapeutic agent for T cell tumor comprising a bispecific antigen-binding molecule, wherein,

[0155] the aforementioned bispecific antigen-binding molecule comprises:

[0156] at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0157] at least one moiety that specifically binds to any one of TRBC1 or TRBC2 expressed in normal T cells.

[0158]

[30] The therapeutic agent according to

[29] , wherein when the T cell tumor cells are positive for TRBC1, the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC2 expressed in normal T cells, and when the T cell tumor cells are positive for TRBC2, the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC1 expressed in normal T cells.

[0159]

[31] The therapeutic agent according to

[29] or

[30] , wherein the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC1 expressed in normal T cells, and the at least one moiety comprises a VH domain and a VL domain.

[0160] The VH domain comprises:

[0161] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10,

[0162] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0163] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0164] The VL domain comprises:

[0165] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13,

[0166] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0167] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15.

[0168]

[32] The therapeutic agent according to

[31] , wherein the at least one moiety that specifically binds to TRBC1 expressed in normal T cells comprises:

[0169] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4, and

[0170] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5.

[0171]

[33] The therapeutic agent according to

[29] or

[30] , wherein the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC2 expressed in normal T cells, and the at least one moiety comprises a VH domain and a VL domain,

[0172] The VH domain comprises:

[0173] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16,

[0174] Heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and

[0175] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18;

[0176] The VL domain comprises:

[0177] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19,

[0178] Light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and

[0179] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0180]

[34] The therapeutic agent according to

[33] , wherein the at least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises:

[0181] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6, and

[0182] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0183]

[35] The therapeutic agent according to any one of

[29] -

[34] , wherein the at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is an scFv or Fab fragment.

[0184]

[36] The therapeutic agent according to any one of

[29] -

[35] , wherein the target tumor antigen expressed in T cell tumor cells is any one selected from CCR1, CCR4, CCR7, CCR8, CCR10, CXCR4, CXCR7, TIGIT, CADM1, GPR15, CXCR5, CXCL13, SLAM, ICOS, CD134, CXCR3, anaplastic lymphoma kinase, CD30, ST2(L), CCR5, Notch1, CD38, CD1a, CCR9 (CD199), CD47, IL-7Rα (CD127), and CD40L (CD154).

[0185]

[37] The therapeutic agent according to any one of

[29] -

[36] , wherein the bispecific antigen-binding molecule comprises at least 1 moiety that specifically binds to CCR4, and the at least 1 moiety comprises a VH domain and a VL domain.

[0186] The VH domain comprises:

[0187] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and

[0188] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24;

[0189] The VL domain comprises:

[0190] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and

[0191] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27.

[0192]

[38] The therapeutic agent according to

[37] , wherein the at least 1 moiety that specifically binds to CCR4 comprises:

[0193] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8, and

[0194] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9.

[0195]

[39] The therapeutic agent according to any one of

[29] -

[36] , wherein the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD1a, and the at least one moiety comprises a VH domain and a VL domain,

[0196] The VH domain comprises:

[0197] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and

[0198] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36;

[0199] The VL domain comprises:

[0200] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37, light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, and

[0201] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0202]

[40] The therapeutic agent according to

[39] , wherein the at least one moiety that specifically binds to CD1a comprises:

[0203] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 40, and

[0204] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 41.

[0205]

[41] The therapeutic agent according to any one of

[29] -

[36] , wherein the target tumor antigen expressed in T cell tumor cells is CCR9,

[0206] The bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR9, and the at least one moiety comprises a VH domain and a VL domain,

[0207] The VH domain comprises:

[0208] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 101, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102, and

[0209] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 103;

[0210] The VL domain comprises:

[0211] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105, and

[0212] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106.

[0213]

[42] The therapeutic agent according to

[41] , wherein at least one moiety that specifically binds to CCR9 comprises:

[0214] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 131, and

[0215] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 132.

[0216]

[43] The therapeutic agent according to any one of

[29] -

[36] , wherein the target tumor antigen expressed in T cell tumor cells is CXCR4,

[0217] the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CXCR4, and the aforementioned at least one moiety comprises a VH domain and a VL domain,

[0218] The VH domain comprises:

[0219] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and

[0220] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;

[0221] The VL domain comprises:

[0222] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and

[0223] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0224]

[44] The therapeutic agent according to

[43] , wherein at least one moiety that specifically binds to CXCR4 comprises:

[0225] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 133, and

[0226] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 134.

[0227]

[45] The therapeutic agent according to any one of

[29] -

[36] , wherein the target tumor antigen expressed in T cell tumor cells is TIGIT,

[0228] The bispecific antigen-binding molecule comprises at least one portion that specifically binds to TIGIT, and the aforementioned at least one portion comprises a VH domain and a VL domain,

[0229] The VH domain comprises:

[0230] A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and

[0231] A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91;

[0232] The VL domain comprises:

[0233] A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and

[0234] A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94.

[0235]

[46] The therapeutic agent according to

[45] , wherein the at least one portion that specifically binds to TIGIT comprises:

[0236] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 135, and

[0237] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 136.

[0238]

[47] The therapeutic agent according to any one of

[29] -

[36] , wherein the target tumor antigen expressed in T cell tumor cells is CCR8,

[0239] The bispecific antigen-binding molecule comprises at least one portion that specifically binds to CCR8, and the aforementioned at least one portion comprises a VH domain and a VL domain,

[0240] The VH domain comprises:

[0241] a heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:95, a heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:96, and

[0242] a heavy-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:97;

[0243] The VL domain comprises:

[0244] a light-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:98,

[0245] a light-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:99, and

[0246] a light-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:100.

[0247]

[48] The therapeutic agent according to

[47] , wherein at least one moiety that specifically binds to CCR8 comprises:

[0248] a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:137, and

[0249] a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:138.

[0250]

[49] The therapeutic agent according to any one of

[29] -

[36] , wherein the target tumor antigen expressed in T cell tumor cells is CD30,

[0251] The bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD30, and the aforementioned at least one moiety comprises a VH domain and a VL domain,

[0252] The VH domain comprises:

[0253] a heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:113,

[0254] a heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:114, and

[0255] a heavy-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:115;

[0256] The VL domain comprises:

[0257] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116,

[0258] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and

[0259] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0260]

[50] The therapeutic agent according to

[49] , wherein at least one moiety specifically binding to CD30 comprises:

[0261] a heavy chain variable region VH having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 139, and

[0262] a light chain variable region VL having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 140.

[0263]

[51] The therapeutic agent according to any one of

[29] -

[36] , wherein the target tumor antigen expressed in T cell tumor cells is CD40L (CD154),

[0264] the bispecific antigen-binding molecule comprises at least one moiety specifically binding to CD40L (CD154), and the foregoing at least one moiety comprises a VH domain and a VL domain,

[0265] the VH domain comprises:

[0266] the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119,

[0267] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120, and

[0268] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121;

[0269] the VL domain comprises:

[0270] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122,

[0271] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and

[0272] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124.

[0273]

[52] The therapeutic agent according to

[51] , wherein at least one moiety that specifically binds to CD40L (CD154) comprises:

[0274] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 141, and

[0275] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 142.

[0276]

[53] The therapeutic agent according to any one of

[29] -

[52] , wherein at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a scFv or Fab fragment.

[0277]

[54] The therapeutic agent according to any one of

[29] -

[53] , wherein the T cell tumor is T cell acute lymphoblastic leukemia / lymphoblastic lymphoma or mature T cell tumor, preferably any one selected from adult T cell leukemia (ATL), peripheral T cell lymphoma (PTCL), and T cell acute lymphoblastic leukemia (TALL).

[0278]

[55] The therapeutic agent according to any one of

[29] -

[54] , wherein the bispecific antigen-binding molecule is administered in combination with a chemotherapeutic agent, radiation, and / or other agents used in cancer immunotherapy.

[0279]

[56] A bispecific antigen-binding molecule, which is a bispecific antigen-binding molecule comprising the following moieties:

[0280] (1) at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0281] (2) at least one moiety that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side; wherein,

[0282] at least one moiety that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side is at least one moiety that specifically binds to TRBC1 or TRBC2 (T cell receptor β constant region 1 or 2) of normal T cells.

[0283]

[57] The bispecific antigen-binding molecule according to

[56] , wherein at least one moiety that specifically binds to TRBC1 expressed in normal T cells comprises a VH domain and a VL domain,

[0284] the VH domain comprises:

[0285] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0286] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0287] The VL domain comprises:

[0288] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0289] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15.

[0290]

[58] The bispecific antigen-binding molecule according to

[57] , wherein at least one moiety that specifically binds to TRBC1 expressed in normal T cells comprises:

[0291] a heavy chain variable region VH comprising at least 90% identity to the amino acid sequence set forth in SEQ ID NO:4, and

[0292] a light chain variable region VL comprising at least 90% identity to the amino acid sequence set forth in SEQ ID NO:5.

[0293]

[59] The bispecific antigen-binding molecule according to

[56] , wherein at least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises a VH domain and a VL domain,

[0294] The VH domain comprises:

[0295] the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0296] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0297] The VL domain comprises:

[0298] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0299] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21.

[0300]

[60] The bispecific antigen-binding molecule according to

[59] , wherein at least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises:

[0301] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6, and

[0302] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0303]

[61] The bispecific antigen-binding molecule according to any one of

[56] -

[60] , wherein the target tumor antigen expressed in T cell tumor cells is selected from the group consisting of CCR1, CCR4, CCR7, CCR8, CCR10, CXCR4, CXCR7, TIGIT, CADM1, GPR15, CXCR5, CXCL13, SLAM, ICOS, CD134, CXCR3, anaplastic lymphoma kinase, CD30, ST2(L), CCR5, Notch1, CD38, CD1a, CCR9(CD199), CD47, IL-7Rα(CD127), and CD40L(CD154).

[0304]

[62] The bispecific antigen-binding molecule according to

[61] , wherein at least one moiety that specifically binds to CCR4 comprises a VH domain and a VL domain,

[0305] The VH domain comprises:

[0306] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22,

[0307] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and

[0308] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24;

[0309] The VL domain comprises:

[0310] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25,

[0311] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and

[0312] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0313]

[63] The bispecific antigen-binding molecule according to

[62] , wherein at least one moiety specifically binding to CCR4 comprises:

[0314] a heavy chain variable region VH having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:8, and

[0315] a light chain variable region VL having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0316]

[64] The bispecific antigen-binding molecule according to

[61] , wherein at least one moiety specifically binding to CD1a comprises a VH domain and a VL domain,

[0317] The VH domain comprises:

[0318] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:34,

[0319] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:35, and

[0320] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:36;

[0321] The VL domain comprises:

[0322] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:37,

[0323] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and

[0324] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:39.

[0325]

[65] The bispecific antigen-binding molecule according to

[64] , wherein at least one moiety specifically binding to CD1a comprises:

[0326] a heavy chain variable region VH having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:40, and

[0327] a light chain variable region VL having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:41.

[0328]

[66] The bispecific antigen-binding molecule according to

[61] , wherein the target tumor antigen expressed in T cell tumor cells is CCR9,

[0329] The bispecific antigen-binding molecule comprises at least one part that specifically binds to CCR9, and the at least one part comprises a VH domain and a VL domain.

[0330] The VH domain comprises:

[0331] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 101, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102, and

[0332] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 103;

[0333] The VL domain comprises:

[0334] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104, light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105, and

[0335] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106.

[0336]

[67] The bispecific antigen-binding molecule according to

[66] , wherein the at least one part that specifically binds to CCR9 comprises:

[0337] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 131, and

[0338] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 132.

[0339]

[68] The bispecific antigen-binding molecule according to

[61] , wherein the target tumor antigen expressed in T cell tumor cells is CXCR4,

[0340] The bispecific antigen-binding molecule comprises at least one part that specifically binds to CXCR4, and the at least one part comprises a VH domain and a VL domain,

[0341] The VH domain comprises:

[0342] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and

[0343] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;

[0344] The VL domain comprises:

[0345] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and

[0346] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0347]

[69] The bispecific antigen-binding molecule according to

[68] , wherein at least one part specifically binding to CXCR4 comprises:

[0348] a heavy chain variable region VH having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 133, and

[0349] a light chain variable region VL having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 134.

[0350]

[70] The bispecific antigen-binding molecule according to

[61] , wherein the target tumor antigen expressed in T cell tumor cells is TIGIT,

[0351] the bispecific antigen-binding molecule comprises at least one part specifically binding to TIGIT, and the foregoing at least one part comprises a VH domain and a VL domain,

[0352] the VH domain comprises:

[0353] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and

[0354] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91;

[0355] the VL domain comprises:

[0356] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 92, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 93, and

[0357] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94.

[0358]

[71] The bispecific antigen-binding molecule according to

[70] , wherein at least one part specifically binding to TIGIT comprises:

[0359] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 135, and

[0360] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 136.

[0361]

[72] The bispecific antigen-binding molecule according to

[61] , wherein the target tumor antigen expressed in T cell tumor cells is CCR8,

[0362] The bispecific antigen-binding molecule comprises at least 1 portion that specifically binds to CCR8, and the aforementioned at least 1 portion comprises a VH domain and a VL domain,

[0363] The VH domain comprises:

[0364] A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, and

[0365] A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97;

[0366] The VL domain comprises:

[0367] A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99, and

[0368] A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0369]

[73] The bispecific antigen-binding molecule according to

[72] , wherein the at least 1 portion that specifically binds to CCR8 comprises:

[0370] A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 137, and

[0371] A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 138.

[0372]

[74] The bispecific antigen-binding molecule according to

[61] , wherein the target tumor antigen expressed in T cell tumor cells is CD30,

[0373] The bispecific antigen-binding molecule comprises at least 1 portion that specifically binds to CD30, and the aforementioned at least 1 portion comprises a VH domain and a VL domain,

[0374] The VH domain comprises:

[0375] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:113, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:114, and

[0376] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:115;

[0377] The VL domain comprises:

[0378] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:116, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:117, and

[0379] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:118.

[0380]

[75] The bispecific antigen-binding molecule according to

[74] , wherein at least one moiety that specifically binds to CD30 comprises:

[0381] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:139, and

[0382] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:140.

[0383]

[76] The bispecific antigen-binding molecule according to

[61] , wherein the target tumor antigen expressed in T cell tumor cells is CD40L (CD154),

[0384] the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD40L (CD154), and the aforementioned at least one moiety comprises a VH domain and a VL domain,

[0385] The VH domain comprises:

[0386] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:119,

[0387] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:120, and

[0388] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:121;

[0389] The VL domain comprises:

[0390] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:122,

[0391] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:123, and

[0392] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:124.

[0393]

[77] The bispecific antigen-binding molecule according to

[76] , wherein at least one moiety that specifically binds to CD40L (CD154) comprises:

[0394] a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:141, and

[0395] a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:142.

[0396]

[78] The bispecific antigen-binding molecule according to any one of

[56] -

[77] , which is used in a method for treating T cell tumors.

[0397]

[79] A method for treating T cell tumors, which comprises administering to a subject having a T cell tumor the bispecific antigen-binding molecule according to any one of

[56] -

[77] .

[0398]

[80] A bispecific antigen-binding molecule, which is a bispecific antigen-binding molecule for use in a method for treating T cell tumors, and which comprises:

[0399] (1) at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0400] (2) at least one moiety that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side;

[0401] wherein the target tumor antigen expressed in T cell tumor cells is not present in normal T cells, or even if present, when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in normal T cells, normal T cells are not substantially activated,

[0402] by binding of the bispecific antigen-binding molecule to the target antigen on the normal T cell side, normal T cells are activated, and

[0403] there is a sufficient proportion of subtypes of the target antigen on the normal T cell side to provide a sufficient number of activated T cells for the treatment of T cell tumors.

[0404]

[81] A treatment method, which is a method for treating T cell tumors in a subject, the method comprising administering a bispecific antigen-binding molecule to a subject having a T cell tumor, wherein,

[0405] the bispecific antigen-binding molecule comprises:

[0406] (1) at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0407] (2) at least one portion that specifically binds to an antigen having a subtype as a target antigen on normal T cells;

[0408] wherein, the target tumor antigen expressed in T cell tumor cells does not exist in normal T cells, or even if it exists, when the bispecific antigen-binding molecule binds to the antigen identical to the target tumor antigen present in normal T cells, normal T cells are not substantially activated,

[0409] by binding of the bispecific antigen-binding molecule to the target antigen on normal T cells, normal T cells are activated, and

[0410] there is a sufficient proportion of subtypes of the target antigen on normal T cells to provide a sufficient number of activated T cells for the treatment of T cell tumors.

[0411]

[82] The treatment method according to

[81] , wherein the method further comprises determining the subtype expressed in the T cell tumor cells of the subject, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to a target antigen on normal T cells, and the subtype of the target antigen on normal T cells is different from the subtype determined to be expressed in the T cell tumor cells of the subject.

[0412]

[83] Use of a bispecific antigen-binding molecule in the manufacture of a medicament for T cell tumors, wherein the bispecific antigen-binding molecule comprises:

[0413] (1) at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0414] (2) at least one portion that specifically binds to an antigen having a subtype as a target antigen on normal T cells;

[0415] wherein, the target tumor antigen expressed in T cell tumor cells does not exist in normal T cells, or even if it exists, when the bispecific antigen-binding molecule binds to the antigen identical to the target tumor antigen present in normal T cells, normal T cells are not substantially activated,

[0416] by binding of the bispecific antigen-binding molecule to the target antigen on normal T cells, normal T cells are activated, and

[0417] There is a sufficient proportion of subtypes of the normal T cell side target antigen to provide a sufficient number of activated T cells for the treatment of T cell tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0418] Figure 1A : Figure 1A It shows a state where the bispecific antibody binds to CD3 expressed on tumor cells and the target antigen expressed on tumor cells simultaneously to bridge the tumor cells to each other.

[0419] Figure 1B : Figure 1B It is a diagram showing the concept of the present invention. The bispecific antigen-binding molecule of the present invention recognizes the target antigen expressed in (T cell) tumor cells and "the antigen having subtypes as the normal T cell side target antigen" (in the figure, the subtype expressed in T lymphocytes is TRBC1), and shows a state of bridging tumor cells and T lymphocytes. In the figure, the bispecific antigen-binding molecule binds to the TRBC1 antigen of T lymphocytes and the target antigen expressed on tumor cells simultaneously. The TRBC2 subtype is expressed not only in tumor cells but also in T lymphocytes. In the figure, the bispecific antigen-binding molecule does not recognize T lymphocytes expressing the TRBC2 subtype.

[0420] Figure 2A : Figure 2A It shows the structure of the bispecific antibody prepared in the examples. The Fab-scFv type has a structure in which the scFv binding to one antigen is connected to the Fab part binding to the other antigen by a linker. The Fab-scFv-Fc type has a structure in which two scFvs binding to one antigen are connected to the Fab part binding to the other antigen by a linker and further has an Fc structure. The Fab-Fc-scFv type has a structure in which an scFv binding to the other antigen is connected to the C-terminus of the IgG binding to one antigen. A: The part binding to one antigen, B: The part binding to the other antigen.

[0421] Figure 2B : Figure 2BThe amino acid sequences of the heavy and light chains of the anti-TRBC1 antibody, anti-TRBC2 antibody, and anti-CCR4 antibody used in the production of the bispecific antibody are shown. The underlined portions represent the respective CDRs. The amino acid sequences of the heavy and light chains of the anti-CCR4 antibody are the amino acid sequences described in SEQ ID NO:9 and 14 of Japanese Patent No. 4052515 (humanized CDR-grafted antibody and antibody fragment thereof). The amino acid sequences of the heavy and light chains of the anti-TRBC1 antibody are the amino acid sequences described in SEQ ID NO:15 and 16 of US10730942 (protein-based T-cell receptor knockdown). The amino acid sequences of the heavy and light chains of the anti-TRBC2 antibody are the sequence of the VH domain having three amino acid substitutions T28K, Y32F, and A100N (Table 1 (page 23) of WO2020 / 089644) in the VH domain of hJOV-1 of WO2020 / 089644 (SEQ ID NO:1 of WO2020 / 089644) and the sequence of the VL domain of hJOV-1 (SEQ ID NO:2 of WO2020 / 089644). The underlined CDRs are defined according to the Kabat numbering scheme.

[0422] Figure 3A -K: Figure 3A -K shows the amino acid sequences of the heavy and light chains constituting the bispecific antibody produced in Example 1.

[0423] Figure 4A : Figure 4A Shows the increase in the percentage of TRBC1-positive cells after addition of the anti-TRBC1 antibody. N = 3.

[0424] Figure 4B : Figure 4B Shows gating live cells stained with Fixable Viability Dye eFluor TM 780, spreading with FSC and SSC, gating the main cell population, and then spreading with CD3 and TRBC1 to calculate the ratio of TRBC1-positive cells in CD3-positive cells (T cells).

[0425] Figure 5A : Figure 5A Shows the increase in cytotoxic T cells caused by addition of the anti-TRBC1 antibody.

[0426] Figure 5B : Figure 5B Shows gating live cells stained with Fixable Viability Dye eFluor TMGating was set with 780 staining, expanded with FSC and SSC. After selecting the main cell population, gating was performed with TRBC1-positive cells, expanded with CD27 and CD45RA, and the ratios of each fraction in TRBC1-positive cells were calculated. *Effector memory (EM): Circulates mainly in secondary lymphoid tissues (lymph nodes) and peripheral tissues, and rapidly produces cytokines and mounts an immune response when re-exposed to the same antigen. Central memory (CM): Resides in secondary lymphoid tissues (lymph nodes) and shows antigen response. Naive: T cells that have not been exposed to antigens. EM T cells re-expressing CD45RA (EMRA): Effector memory cells that are terminally differentiated. N = 3.

[0427] Figure 6A : Figure 6A Shows the positive rate (%) of activation markers in TRBC1-positive cells. (N = 3, *For Perforin, only data for Day0, N = 2)

[0428] Figure 6B : Figure 6B It is a graph that, after gating TRBC1-positive cells, is expanded with CD25 and CD69, and calculates the positive ratios of each marker in TRBC1-positive cells.

[0429] Figure 6C : Figure 6C It is a graph that is expanded with TRBC1 and Granzyme B, and calculates the Granzyme B positive ratio in TRBC1-positive cells.

[0430] Figure 6D : Figure 6D It is a graph that is expanded with TRBC1 and Perforin, and calculates the Perforin positive ratio in TRBC1-positive cells. The data are presented as mean (SD).

[0431] Figure 7A : Figure 7A Shows the change in the ratio of PD-1-positive cells caused by adding anti-TRBC1 antibody. N = 3.

[0432] Figure 7B : Figure 7B Is a graph that gates live cells with Fixable Viability Dye eFluor TM 780 staining, expands with FSC and SSC, circles the main cell population, then expands with TRBC1 and PD-1, and calculates the ratio of PD-1-positive cells in TRBC1-positive cells.

[0433] Figure 8 : Figure 8 Shows the quantification of cytokine release of T-LAK stimulated with anti-TRBC1 antibody.

[0434] Figure 9 : Figure 9 Shows an increase in the number of cells including TRBC2-positive T cells after addition of anti-TRBC2 antibody.

[0435] Figure 10 : (a) is a graph showing an increase in cytotoxic T cells caused by addition of anti-TRBC2 antibody. (b) shows that after gating live cells stained with Fixable Viability Dye eFluor TM 780, expanding with FSC and SSC, after circling the main cell population, gating with CD3-positive and TRBC1-negative cells (gating with TRBC2-positive cells), expanding with CD27 and CD45RA, and calculating the ratio of each fraction in TRBC2-positive cells (N = 3). EM: effector memory, CM: central memory, EMRA: EM T cells re-expressing CD45RA.

[0436] Figure 11A -B: (a) shows the positive rate (%) of activation markers in TRBC2-positive cells (N = 3). (b) is a graph showing that after gating TRBC2-positive cells, expanding with CD25 and CD69, and calculating the positive ratio of each marker in TRBC2-positive cells. (c) is a graph showing that after expanding with TRBC1 and granzyme B, calculating the granzyme B positive ratio in TRBC2-positive cells. (d) is a graph showing that after expanding with TRBC1 and perforin, calculating the perforin positive ratio in TRBC2-positive cells. Data are represented as mean (SD).

[0437] Figure 12 : (a) shows the change in the ratio of PD-1-positive cells caused by addition of anti-TRBC2 antibody (N = 3). (b) is a graph showing that after gating live cells stained with Fixable Viability Dye eFluor TM 780, expanding with FSC and SSC, after circling the main cell population, expanding with TRBC1 and PD-1, and calculating the PD-1 positive cell ratio in TRBC2-positive cells.

[0438] Figure 13 : Figure 13 Shows the quantification of cytokine release of T-LAK stimulated with anti-TRBC2 antibody. The vertical axis of each graph is concentration.

[0439] Figure 14A -C: Figure 14A -C shows the cytotoxic activity of bispecific antibodies CCR0001 and CCR0004.

[0440] Figure 15 :Figure 15 Shows the cytotoxic activity of bispecific antibodies CD1a1001 and CD1a1004.

[0441] Figure 16 : Figure 16 Shows the cytotoxic activity of the anti-CCR4 / anti-TRBC2 bispecific antibody against the cell line MT-2 from ATL.

[0442] Figure 17 : Figure 17 Shows the cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody against the cell line JM (expressing CD1a and TRBC1) from TALL.

[0443] Figure 18 : Figure 18 Shows the cytotoxic activity of the anti-CCR9 / anti-TRBC1 bispecific antibody against the cell line CCRF-CEM9 (expressing CCR9 and TRBC2) from TALL.

[0444] Figure 19 : Figure 19 Shows the cytotoxic activity of the anti-CXCR4 / anti-TRBC1 bispecific antibody against the cell line CCRF-CEM9 (expressing CXCR4 and TRBC2) from TALL.

[0445] Figure 20 : Figure 20 Shows the cytotoxic activity of the anti-CCR8 / anti-TRBC1 bispecific antibody against the cell line MT-1 (expressing CCR8) from ATL.

[0446] Figure 21 : Figure 21 Shows the cytotoxic activity of the anti-TIGIT / anti-TRBC1 bispecific antibody against the cell line ILT-Mat (expressing TIGIT) from ATL.

[0447] Figure 22 : Figure 22 Shows the cytotoxic activity of the anti-CD30 / anti-TRBC1 bispecific antibody against the cell line DL40 (expressing CD30) from PTCL.

[0448] Figure 23 : Figure 23 Shows the cytotoxic activity of the anti-CD40L / anti-TRBC1 bispecific antibody against the cell line MOLT3 (expressing CD40L) from TALL.

[0449] Figure 24 : Figure 24Results of adding T-LAK or NK cells to MOLT4 cells with forced CCR4 expression and measuring the cytotoxic activity of the anti-CCR4 / anti-TRBC1 bispecific antibody CCR0001 or mogamulizumab.

[0450] Figure 25 : Figure 25 Results of adding T-LAK to the CD3-expressing TALL cell line HPB-ALL cells and CD3-knockout HPB-ALL CD3 KO cells and measuring the cytotoxic activity of the anti-CD1a / anti-CD3 bispecific antibody CD1a1016 (a) and the anti-CD1a antibody and the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005 (b).

[0451] Figure 26 : Figure 26 Results of adding anti-TRBC2 antibody-sensitized T-LAK to the TALL cell line CCRF-CEM9 cells expressing TRBC2 and the TALL cell line JM cells expressing TRBC1 and measuring the cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006.

[0452] Figure 27 : Figure 27 Graph of IVIS measurements for each group on days 14 and 28 of NOG mice transplanted with tumor cells.

[0453] Figure 28 : Figure 28 Tumor distribution images of NOG mice transplanted with tumor cells.

[0454] Figure 29 : Figure 29 Results of adding T-LAK to the T-ALL cell line CCRF-CEM9 cells and CCRF-CEM CD1aKO cells expressing CD1a and measuring the cytotoxic activity of the bispecific antibodies CD1a1002 and CD1a1005.

[0455] Figure 30 : Figure 30 Results of adding anti-TRBC2 antibody-sensitized T-LAK to the TALL cell line JM cells and JM CD1a KO cells expressing CD1a and measuring the cytotoxic activity of the bispecific antibodies CD1a1003 and CD1a1006.

[0456] Figure 31 : Figure 31These are the results of adding T cells from normal humans to CCRF-CEM-Luc cells that express luciferase in the cell line CCRF-CEM from TALL, and measuring the cytotoxic activities of the anti-CD1a / anti-TRBC1 bispecific antibody (CD1a1005) and the anti-CD1a / anti-TRBV5-5 bispecific antibodies (TRBV50003 and TRBV50004).

[0457] Figure 32 : Figure 32 These are the results of adding T cells from normal humans to JM-Luc-BFP cells that express luciferase in the cell line JM from TALL, and measuring the cytotoxic activities of the anti-CD1a / anti-TRBC2 bispecific antibody (CD1a1006) and the anti-CD1a / anti-TRBV5-5 bispecific antibodies (TRBV50003 and TRBV50004).

[0458] Figure 33 : Figure 33 These are the results of adding T-LAK cells stimulated with an anti-TRBV5-5 antibody (TRBV50001) to the cell line CCRF-CEM9 from TALL, and measuring the cytotoxic activities of the anti-CD1a / anti-TRBV5-5 bispecific antibodies (TRBV50003 and TRBV50004). Detailed implementation mode

[0459] The description of the present invention recorded below is sometimes based on representative embodiments and specific examples, but the present invention is not limited by such embodiments. It should be noted that the numerical range indicated by "~" in this specification means a range including the numerical values recorded before and after "~" as the lower limit value and the upper limit value.

[0460] The present invention relates to the treatment of T cell tumors. Hematological malignancies are roughly divided into lymphocytic tumors and myeloid tumors based on the origin or maturity of their tumor cells. Furthermore, lymphocytic tumors can be roughly divided into T cell tumors and B cell tumors. Lymphocytic tumors originate from lymphocyte precursor cells, and myeloid tumors originate from bone marrow precursor cells. Cells in which T lymphocytes are malignant are T cell tumor cells, and cells in which B lymphocytes are malignant are B cell tumor cells.

[0461] In this specification, "T cell tumor" includes T cell acute lymphoblastic leukemia / lymphoblastic lymphoma and mature T cell tumors, and is not limited thereto as long as it is a tumor of T cells or their precursor cells.

[0462] For acute lymphoblastic leukemia / lymphoblastic lymphoma (ALL / LBL), when lymphoblasts infiltrate (>25%) into the bone marrow and appear in the peripheral blood, it is acute lymphoblastic leukemia (ALL), and when it invades lymph nodes and extranodal organs, it is lymphoblastic lymphoma (LBL). Acute lymphoblastic leukemia / lymphoblastic lymphoma includes B-cell and T-cell types, which are respectively called "B-cell acute lymphoblastic leukemia / lymphoblastic lymphoma" and "T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma". T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma includes the following (Swerdlow SH, et al (Editors). WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2017; all of its records are hereby incorporated by reference in their entirety): T-lymphoblastic leukemia / lymphoma (Provisional entity: Early T-cell precursor lymphoblastic leukemia, Provisional entity).

[0463] Mature T-cell neoplasms include the following (Swerdlow SH, et al (Editors). WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2017). T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, systemic EBV-positive T-cell lymphoma of childhood, hydroa vacciniforme-like lymphoproliferative disorder, adult T-cell leukemia / lymphoma, extranodal T-cell lymphoma, nasal typenasal type), enteropathy-associated T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma, indolent T-cell lymphoproliferative disorder of the GI tract, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous CD30 positive T-cell lymphoproliferative disorders, lymphomatoid papulosis, primary cutaneous anaplastic large cell lymphoma, 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 T-cell lymphoproliferative disorder, peripheral T-cell lymphoma, not otherwise specifiedNOS), Angioimmunoblastic T-cell lymphoma, Follicular T-cell lymphoma, Nodal peripheral T-cell lymphoma with TFH phenotype, Anaplastic large cell lymphoma, ALK positive, Anaplastic large cell lymphoma, ALK negative, Breast implant-associated anaplastic large-cell lymphoma.,

[0464] In this specification, "antigen-binding molecule" refers to a molecule that specifically binds to an epitope (antigenic determinant), for example, an antibody or an antibody fragment. "Bispecific" means that the antigen-binding molecule specifically binds to two different antigenic determinants. A bispecific antigen-binding molecule comprises at least two antigen-binding portions that bind to different antigenic determinants.

[0465] An antibody exhibits a structure in which two heavy chains (H chains) and two light chains (L chains) are bound. The light chain and the heavy chain are linked by disulfide bonds (SS bonds) to form a heterodimer, and then two such heterodimers bind to form a Y-shaped heterotetramer. Typically, a heavy chain contains a heavy chain variable region VH, heavy chain constant regions CH1, CH2, CH3, and a hinge region located between CH1 and CH2, and a light chain contains a light chain variable region VL and a light chain constant region CL. The variable region contains complementarity-determining regions (CDRs) and framework regions (FRs). There are three CDRs each (heavy chain CDR1-3 and light chain CDR1-3) and four FRs each (heavy chain FR1-4 and light chain FR1-4) in the variable regions of the light chain and the heavy chain. Methods for identifying CDRs are well known. For example, the IMGT / V-QUEST search page (http: / / www.imgt.org / IMGT_vquest / input), Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008), and the reference: Giudicelli, V., Brochet, X., Lefranc, M.-P., Cold Spring Harb Protoc. 2011 Jun 1; 2011(6). pii: pdb.prot5633. doi: 10.1101 / pdb.prot5633. PMID: 21632778) can be used; the entire disclosures of the two documents are hereby incorporated by reference in their entirety. In addition, any other means well known in the art can also be used as long as the same results can be obtained (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342:877; the entire disclosures of the two documents are hereby incorporated by reference in their entirety).

[0466] In this specification, an antibody fragment is an antigen-binding antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, F(ab’) 2 , Fv, and scFv. Fab refers to an antibody fragment in which the VL-CL and VH-CH1 domains are linked by disulfide bonds. F(ab’) 2 refers to an antibody fragment in which two Fabs are linked by disulfide bonds in the hinge region. The Fv fragment contains VL and VH. ScFv is a fusion protein in which VH and VL are linked by a linker peptide (about 10-25 amino acids in length).

[0467] The first embodiment of the present invention relates to a therapeutic agent for T cell tumors comprising a bispecific antigen-binding molecule. The bispecific antigen-binding molecule constituting the therapeutic agent of the present invention comprises:

[0468] (1) at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0469] (2) at least one moiety that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side;

[0470] wherein the target tumor antigen expressed in T cell tumor cells is not present in normal T cells, or even if present, when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in normal T cells, normal T cells are not substantially activated,

[0471] by binding of the bispecific antigen-binding molecule to the target antigen on the normal T cell side, normal T cells are activated, and

[0472] there is a sufficient proportion of subtypes of the target antigen on the normal T cell side to provide a sufficient number of activated T cells for the treatment of T cell tumors.

[0473] In the present invention, the description of "a moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells" (hereinafter referred to as the tumor antigen-binding moiety) refers to a polypeptide molecule that specifically binds to a target tumor antigen expressed in T cell tumor cells. In one aspect, the tumor antigen-binding moiety can direct normal T cells to T cell tumor cells expressing the target tumor antigen. The tumor antigen-binding moiety comprises an antibody or a fragment thereof.

[0474] In the present specification, "specific binding" means selectively binding to a specific antigen and can be distinguished from non-specific interactions. The binding ability of an antigen-binding molecule to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR) technology, etc. In a specific embodiment, the antigen-binding molecule can bind with a dissociation constant (K -5 M (mol / L) or less, 5×10 -6 M or less, 1×10 -6 M or less, 5×10 -7 M or less, 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×10 -10 M or less)D ) binds to the target antigen.

[0475] Preferably, the target tumor antigen expressed in T cell tumor cells is absent in normal T cells, or even if present, when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in normal T cells, normal T cells are not substantially activated. The absence of the target tumor antigen expressed in T cell tumor cells in normal T cells can be determined by flow cytometry analysis using a fluorescent dye-labeled antibody that specifically binds to the target tumor antigen. The presence or absence of a certain antigen in normal T cells, or whether the expression of a certain antigen is positive or negative in normal T cells, can be judged based on the graph obtained from flow cytometry analysis. The specific content of flow cytometry analysis will be described later. In a preferred embodiment, the target tumor antigen expressed in T cell tumor cells is absent in normal T cells, whereby the bispecific antigen-binding molecule can appropriately bridge normal T cells and T cell tumor cells (e.g., Figure 1B ). In addition, "when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in normal T cells, normal T cells are not substantially activated" can be achieved by preparing a tumor antigen-binding portion based on an antibody that does not have agonist activity, because it is considered that the agonist activity of the antibody plays a major role in the activation of normal T cells caused by the binding of the bispecific antigen-binding molecule (e.g., antibody) to the antigen.

[0476] CD3 is expressed in both normal T cells and T cell tumor cells. A bispecific antibody targeting CD3 and a tumor antigen binds simultaneously to the CD3 antigen expressed in T cell malignancies and the target antigen expressed in T cell malignancies, thereby connecting malignant tumor cells to each other ( Figure 1A ), so it is likely that the therapeutic effect of the bispecific antibody cannot be fully obtained. In the examples described later, compared with the same activity against a tumor cell line that does not express CD3 (CD3 knockout tumor cell line), the cytotoxic activity of the bispecific antibody that binds CD3 and a tumor antigen against a tumor cell line that expresses CD3 is shown to be reduced. This result implies that CD3 expression in the tumor cell line weakens the cytotoxic activity. In bispecific antigen-binding molecules for T cell tumors (mostly CD3-positive), the target for effector T cell induction should be set to other molecules than CD3.

[0477] The target tumor antigen expressed in T cell tumor cells is a different antigen from the target antigen on the normal T cell side. The "(1) at least one portion that specifically binds to the target tumor antigen expressed in T cell tumor cells" and "(2) at least one portion that specifically binds to the antigen with a subtype as the target antigen on the normal T cell side" of the bispecific antigen-binding molecule are preferably configured to bind to different antigens and respectively bind to different types of antigens expressed in T cell tumor cells or normal T cells.

[0478] In a specific manner, preferably, even if the target tumor antigen expressed in T cell tumor cells is present in normal T cells, it is expressed in T cell tumor cells at a higher frequency than in normal T cells. For example, for the target tumor antigen, as long as the ratio of the number of molecules expressed per T cell tumor cell to the number of molecules expressed per normal T cell is 110% or more, for example, it is preferably 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% or more. The number of expressed molecules of the target tumor antigen per cell can be determined by flow cytometry analysis using a fluorescent dye-labeled antibody that specifically binds to the target tumor antigen. By expressing the target tumor antigen in T cell tumor cells at a higher frequency than in normal T cells, the bispecific antigen-binding molecule can bridge a sufficient number of normal T cells for treatment with T cell tumor cells, thereby obtaining a therapeutic effect. Specific examples of the target tumor antigen are described later.

[0479] <Antigen-binding portion on the normal T cell side>

[0480] In the present invention, the description of "a portion that specifically binds to the antigen with a subtype as the target antigen on the normal T cell side" (hereinafter, referred to as the antigen-binding portion on the normal T cell side) refers to a polypeptide molecule that specifically binds to the target antigen expressed in normal T cells. The antigen-binding portion on the normal T cell side includes an antibody or a fragment thereof. In one mode, the antigen-binding portion on the normal T cell side can transduce and activate the TCR signal (activation of T cells) via the target antigen on the normal T cell side. Further, it is preferably possible to induce cellular responses such as cytokine production or cytotoxic activity of T cells.

[0481] "Antigen with subtypes" refers to a substance in the substances constituting T cells (e.g., peptides, proteins, sugars, lipids, or other organic or inorganic substances) that has variants with the same or substantially the same function but different structures (e.g., the amino acid sequence of peptides or proteins, the three-dimensional structure, the constituent units of sugars, the types, the arrangement of constituent elements of lipids, the types). Each variant is called a subtype. In one mode, it is preferable that only one of the subtypes of the "antigen with subtypes" is expressed on each T cell. In one example, the "antigen with subtypes" can be an antigen with subtypes based on TCR diversity generated by somatic recombination (also called "V(D)J gene rearrangement"), but is not limited thereto. Most T cell receptors are composed of an α chain and a β chain. The TCR α chain and β chain include a variable region at the N-terminus and a constant region at the C-terminus. TCR diversity is generated by somatic recombination that occurs when each TCR chain selects the variable region (V), the diversity region (D), the joining region (J), and the constant region (C). The V, D, J gene segments are contained in the β chain, and the V, J gene segments are contained in the α chain. For example, one of the 30 TRBV polypeptides generated by V(D)J gene rearrangement and allele elimination is expressed on each T cell (WO2022 / 119955; the entire description is hereby incorporated by reference in its entirety). The TRBV polypeptide is an example of an antigen with 30 subtypes. However, the "antigen with subtypes" is an antigen expressed at any position on T cells and is not limited to the antigen present on the TCR.

[0482] As long as the number of subtypes of the "antigen with subtypes" is two or more, there is no particular limitation. When the number of subtypes is large, the number of normal T cells to which the normal T cell-side antigen-binding portion of the bispecific antigen-binding molecule can bind via the target antigen is small, so it may sometimes be impossible to provide a sufficient number of activated T cells required for the treatment of T cell tumors. In one mode, the number of subtypes of the "antigen with subtypes" can be, for example, 50 or less, preferably 40 or less, 30 or less, 20 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less, or 2.

[0483] The normal T cell population contains normal T cells expressing any one of the subtypes of the "antigen with subtypes that is a target antigen on the normal T cell side". For example, when the number of subtypes of the "antigen with subtypes" is three (subtypes A, B, and C), cells expressing subtype A, cells expressing subtype B, and cells expressing subtype C are mixed and present in the normal T cell population. Even in T cell tumor cells, usually any one of the above subtypes (for example, any one of subtypes A to C above) is expressed. The normal T cell side antigen-binding portion of the bispecific antigen-binding molecule is configured to specifically bind to a subtype different from the subtype expressed in the T cell tumor cells. For example, when the subtype expressed in the T cell tumor cells is subtype A, the normal T cell side antigen-binding portion of the bispecific antigen-binding molecule is configured to specifically bind to subtype B or is configured to specifically bind to subtype C. The tumor antigen-binding portion of the bispecific antigen-binding molecule is configured to specifically bind to an antigen that is different from the "antigen with subtypes that is a target antigen on the normal T cell side" and is highly expressed in T cell tumor cells. Thereby, the bispecific antigen-binding molecule can appropriately bridge normal T cells and T cell tumor cells.

[0484] In the present invention, it is preferable to activate normal T cells by binding a bispecific antigen-binding molecule to the target antigen on the normal T cell side. Activation of T cells can be evaluated, for example, by culturing T cells in the presence of IL-2 and the normal T cell side antigen-binding portion of the bispecific antigen-binding molecule as described in the reference example below, and observing or measuring cell phenotypes, T cell activation markers, cell proliferation, cell viability, and the amount of secreted cytokines.

[0485] In the present invention, in order to provide a sufficient number of activated T cells required for the treatment of T cell tumors, it is preferable that there is a sufficient proportion of the subtypes of the target antigen on the normal T cell side. The subtypes of the target antigen on the normal T cell side are preferably expressed in cells accounting for 25% or more of the total number of cells in the normal T cell population, more preferably in cells accounting for 30% or more, 40% or more, or 50% or more. When selecting the "antigen with subtypes", for the proportion of the subtypes of the target antigen on the normal T cell side, the average value of humans can be referred to, or the analysis value in the patient to be treated can be based on. In one mode, in order to provide a sufficient number of activated T cells required for the treatment of T cell tumors, the blood concentration of cells expressing the subtypes of the target antigen on the normal T cell side can be 50 cells / μL or more, preferably 100 cells / μL or more, 200 cells / μL or more, or 300 cells / μL or more. Further, the "antigen with subtypes" is not an antigen present in γδ T cells, but is preferably present in αβ T cells. This is because γδ T cells account for only a few percent or less of the total T cells, and thus a sufficient number of T cells cannot be mobilized to kill tumor cells to obtain a therapeutic effect.

[0486] In one mode, the subtype of the target antigen on the normal T cell side is any subtype of the antigen subtype that is not the antigen subtype expressed in T cell tumor cells. For example, a cell population of clonally proliferated T cell tumor cells has any subtype. Therefore, as the target antigen on the normal T cell side of the bispecific antigen-binding molecule, a subtype different from the subtype expressed in the cell population of T cell tumor cells can be selected.

[0487] In one mode, the situation where the target antigen on the normal T cell side is not expressed in tumor cells is the case where the subtype of the target antigen on the normal T cell side is any subtype of the antigen subtype that is not the antigen subtype expressed in T cell tumor cells.

[0488] In one mode, the antigen with subtypes as the target antigen on the normal T cell side is TRBC (T cell receptor β constant region), the subtype expressed in T cell tumor cells is TRBC1, and the subtype of the target antigen on the normal T cell side is TRBC2. That is, in a subject, if the subtype of TRBC expressed in T cell tumor cells is analyzed and it is TRBC1, the target antigen on the normal T cell side of the bispecific antigen-binding molecule can be set as TRBC2. Thereby, normal T cells can be mobilized as effector cells by the bispecific antigen-binding molecule.

[0489] In a further mode, the antigen with subtypes as the target antigen on the normal T cell side is TRBC, the subtype expressed in T cell tumor cells is TRBC2, and the subtype of the target antigen on the normal T cell side is TRBC1. That is, in a subject, if the subtype of TRBC expressed in T cell tumor cells is analyzed and it is TRBC2, the target antigen on the normal T cell side of the bispecific antigen-binding molecule can be set as TRBC1. Thereby, normal T cells can be mobilized as effector cells by the bispecific antigen-binding molecule.

[0490] In a further mode, the antigen with subtypes as the target antigen on the normal T cell side is TRBC, the T cell tumor cells are negative for TRBC1 and negative for TRBC2, and the subtype of the target antigen on the normal T cell side is TRBC1 or TRBC2. That is, in a subject, if the subtype of TRBC expressed in T cell tumor cells is analyzed and it is negative for TRBC1 and negative for TRBC2, the target antigen on the normal T cell side of the bispecific antigen-binding molecule can be either TRBC1 or TRBC2. At this time, neither TRBC1 nor TRBC2 is expressed in T cell tumor cells, but either one is expressed in normal T cells. Therefore, through the action of the bispecific antigen-binding molecule having a normal T cell side antigen-binding portion that binds to either TRBC1 or TRBC2, normal T cells can be mobilized as effector cells.

[0491] In this specification, TRBC1 and TRBC2 refer to two functionally identical proteins of the constant region of the TCRβ chain. The T cell receptor (TCR) is an antigen receptor molecule expressed on T cells that recognizes antigens bound to major histocompatibility gene complex (MHC) molecules. There are two functionally identical genes (TRBC1 (GeneID: 28639), TRBC2 (Gene ID: 28636)) at the locus of the constant region of the TCRβ chain (Chr7: q34). The mature proteins of TRBC1 and TRBC2 differ by only four amino acid residues. The amino acid sequences of TCRs with TRBC1 and TRBC2 (TRBC1-TCR and TRBC2-TCR) are substantially the same, yet they can still be distinguished by antibodies (Maciocia PM, et al., cited above). In addition, in the peripheral blood T cells of normal humans, there are approximately 35% TRBC1-positive cells and 65% TRBC2-positive cells mixed, and monoclonality of TRBC has been confirmed in many types of T cell malignancies by flow cytometry and immunohistochemistry (IHC) methods (Maciocia PM, et al., cited above).

[0492] It has been reported that in the normal T cell population, cells expressing TRBC1 and cells expressing TRBC2 coexist, but in the overall cell population of T cell tumor cells, either TRBC1 or TRBC2 is exclusively expressed (Maciocia PM, et al., cited above). Thus, it has been proposed to use TRBC1 or TRBC2 as a target antigen on the tumor cell side (Japanese Patent No. 6767872 (Japanese Patent Application No. 2016-554603; the entire description thereof is hereby incorporated by reference in its entirety)). However, prior to this application, it was not known to use a TRBC subtype different from the subtype of TRBC expressed in tumor cells as a target antigen on the normal T cell side or effector cell side.

[0493] In a specific embodiment, the therapeutic agent for T cell tumors of the present invention comprises a bispecific antigen-binding molecule, the bispecific antigen-binding molecule comprising:

[0494] at least one moiety that specifically binds to a target tumor antigen expressed on T cell tumor cells, and

[0495] at least one moiety that specifically binds to either TRBC1 or TRBC2 expressed on normal T cells.

[0496] In the present invention, the description of "a moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells" (hereinafter referred to as the TRBC1 or TRBC2 binding moiety) refers to a polypeptide molecule that specifically binds to TRBC1 or TRBC2 expressed in normal T cells. The TRBC1 or TRBC2 binding moiety includes an antibody or a fragment thereof. In one mode, the TRBC1 or TRBC2 binding moiety can transduce and activate TCR signals (activation of T cells) via TRBC1 or TRBC2. Further, it is preferred to induce cellular responses such as cytokine production or cytotoxic activity of T cells.

[0497] The bispecific antigen-binding molecule preferably binds simultaneously to TRBC1 or TRBC2 expressed in normal T cells and a target tumor antigen expressed in T cell tumor cells.

[0498] In one mode, when the T cell tumor cell is positive for TRBC1, the aforementioned bispecific antigen-binding molecule includes at least one moiety that specifically binds to TRBC2 expressed in normal T cells. When the T cell tumor cell is positive for TRBC2, the aforementioned bispecific antigen-binding molecule includes at least one moiety that specifically binds to TRBC1 expressed in normal T cells. By using a TRBC of a different subtype from the TRBC expressed in the T cell tumor cell as the target on the effector cell (T lymphocyte) side (when the tumor cell expresses TRBC1, the target on the effector cell side is set as TRBC2, and when the tumor cell expresses TRBC2, the target on the effector cell side is set as TRBC1), it is impossible to cause T cell immunodeficiency, and a high therapeutic effect can be achieved.

[0499] In the following examples, compared with the cytotoxic activity against a TRBC2-positive tumor cell line, the bispecific antibody in which the normal T cell-side antigen-binding moiety binds to TRBC2 showed higher cytotoxic activity against a TRBC1-positive tumor cell line (Example 15). Compared with the TRBC2-positive tumor cell line, the bispecific antibody in which the normal T cell-side antigen-binding moiety binds to TRBC1 showed high cytotoxic activity (Example 4). From these, it was confirmed that in bispecific antibodies for treating tumors, by recognizing a TRBC of a different subtype from the TRBC expressed in T cell tumor cells with the normal T cell-side antigen-binding moiety, high cytotoxic activity can be obtained.

[0500] In the present invention, "TRBC1 positive" of T cell tumor cells can be understood to mean that the T cell tumor cells are "TRBC2 negative". In addition, in the present invention, "TRBC2 positive" of T cell tumor cells can be understood to mean that the T cell tumor cells are "TRBC1 negative". It has been reported that cells expressing TRBC1 and cells expressing TRBC2 are mixedly present in a normal T cell population, but in the whole cell population of T cell tumor cells, either TRBC1 or TRBC2 is exclusively expressed (Maciocia PM, et al., cited above).

[0501] In one aspect, there may be a case where T cell tumor cells are negative for both TRBC1 and TRBC2. When the T cell tumor cells are negative for both TRBC1 and TRBC2, the normal T cell side target antigen of the aforementioned bispecific antigen-binding molecule can be either TRBC1 or TRBC2, that is, it can include either at least one part that specifically binds to TRBC1 expressed in normal T cells or at least one part that specifically binds to TRBC2 expressed in normal T cells.

[0502] In one aspect, the target tumor antigen is not either TRBC1 or TRBC2. That is, the at least one part that specifically binds to the target tumor antigen expressed in T cell tumor cells is designed to specifically bind to an antigen selected from antigens other than TRBC1 and TRBC2, and thus does not bind or substantially does not bind to either TRBC1 or TRBC2.

[0503] In the case of a T cell malignancy that is TRBC1 positive, TRBC1 is set as the target antigen of the tumor cells, and in the case of a malignancy that is TRBC2 positive, TRBC2 is set as the target antigen of the tumor cells. For example, a chimeric antigen receptor (CAR) containing an antigen-binding domain that selectively binds to either TRBC1 or TRBC2 has been proposed for the treatment of T cell lymphoma or leukemia (Maciocia PM, et al., cited above, Japanese Patent No. 6767872 (Japanese Patent Application No. 2016-554603)). However, CAR-T cells using a TRBC1 antibody kill not only TRBC1 positive cancer cells but also TRBC1 positive T lymphocytes. Approximately 50% of T lymphocytes may die, and thus T cell immunodeficiency may be caused. In the present invention, a bispecific antigen-binding molecule that sets an antigen other than TRBC expressed in T cell malignancy as the target on the tumor cell side and further sets a TRBC of a different subtype from the TRBC subtype expressed in T cell malignancy as the target on the effector cell (T lymphocyte) side is used to induce normal T cells to kill tumor cells, and thus the possibility of causing T cell immunodeficiency is low.

[0504] T cell tumor cells are reported to exclusively express either TRBC1 or TRBC2 in their entire cell population. However, it is also possible that T cell tumor cells do not express either TRBC1 or TRBC2. For example, in the case of malignant transformation of T cells that do not express TCR. The present invention can treat T cell tumors even when neither TRBC1 nor TRBC2 is expressed in T cell tumor cells. This is because normal T cells express either TRBC1 or TRBC2, so a bispecific antigen-binding molecule that targets either TRBC1 or TRBC2 on the T cell side and targets an antigen other than TRBC expressed in T cell tumor cells on the tumor cell side can induce approximately half of the normal T cell population to T cell tumor cells for killing.

[0505] In the following examples, with respect to tumor cell lines that do not express either TRBC1 or TRBC2, both the bispecific antibody with the normal T cell side antigen-binding portion binding to TRBC1 and the bispecific antibody with the normal T cell side antigen-binding portion binding to TRBC2 showed cytotoxic activity (Examples 3 and 5).

[0506] Whether a cell is positive or negative for TRBC1 can be determined by flow cytometry analysis using a fluorescent pigment-labeled antibody that specifically binds to TRBC1. Examples of antibodies that specifically bind to TRBC1 include the JOVI-1 clone (based on protein T-cell receptor knockdown SEQ ID No.15, SEQ ID No.16 in US10730942; the entire disclosure of US10730942 is hereby incorporated by reference in its entirety). Similarly, whether a T cell is positive or negative for TRBC2 can be determined by flow cytometry analysis using a fluorescent pigment-labeled antibody that specifically binds to TRBC2. Examples of antibodies that specifically bind to TRBC2 include various antibodies described in WO2020 / 089644 (the entire disclosure of which is hereby incorporated by reference in its entirety).

[0507] Whether the expression of a cellular antigen is positive or negative can be determined based on a graph obtained by flow cytometry analysis. The position that appears in the graph sometimes varies depending on the voltage setting, sensitivity setting, antibody clone used, staining conditions, pigment used, etc. of the machine, but those skilled in the art can appropriately draw a line in the obtained graph without cutting into the cell population observed as a group. In determining whether the expression of the target cell antigen is positive or negative, the case of using an isotype control antibody can be used as a negative control for determination. An isotype control antibody is an antibody that does not react with a specific antigen. Generally, in an experiment using an antibody, background may be generated due to non-specific binding to proteins other than the target and binding to Fc receptors on the cell surface. By comparing with a system using an antibody as a negative control, it can be clarified that the reaction of the primary antibody to the target antigen is specific. In addition, the influence of the background can be excluded, and the intensity of the signal can be correctly interpreted.

[0508] <TRBC1 binding portion>

[0509] The bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may include a TRBC1 binding portion and a tumor antigen binding portion. The TRBC1 binding portion may be an anti-TRBC1 antibody described in US Patent No. 10730942 (the entire description thereof is hereby incorporated by reference in its entirety), (US10730942 is based on protein T-cell receptor knockdown SEQ ID No. 15, SEQ ID No. 16).

[0510] In one embodiment, the bispecific antigen-binding molecule includes at least one portion that specifically binds to TRBC1 expressed in normal T cells, and the at least one portion includes a VH domain and a VL domain.

[0511] The VH domain includes:

[0512] Heavy chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 10,

[0513] Heavy chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 11, and

[0514] Heavy chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 12;

[0515] The VL domain includes:

[0516] Light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 13,

[0517] Light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 14, and

[0518] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15.

[0519] In a particular manner, at least one moiety that specifically binds to TRBC1 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5. In a further particular manner, at least one moiety that specifically binds to TRBC1 expressed in normal T cells comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:4, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:5. Amino acid mutations (deletions, insertions and substitutions) with respect to the amino acid sequences of SEQ ID NO:4 or 5 may be present in the complementarity determining regions (CDRs) or frameworks (FRs).

[0520] In the present specification, the "percent amino acid sequence identity" related to a reference polypeptide sequence is defined as: the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide when the sequences are aligned and gaps are introduced as needed to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved using various methods within the skill of those in the art, such as computer software that is publicly available, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithm required to achieve the maximum alignment for the full length of the sequences to be compared.

[0521] In a particular manner, in order to improve the binding affinity and / or other biological properties of the TRBC1 binding moiety, amino acid sequence variants of the bispecific antigen-binding molecule comprising the TRBC1 binding moiety are made. The amino acid sequence variants can be made by introducing suitable mutations (deletions, insertions and substitutions) into the nucleotide sequence encoding the anti-TRBC1 antibody or by peptide synthesis. The mutations can be introduced into the complementarity determining regions (CDRs) or frameworks (FRs) of the antibody according to the properties to be improved. For the amino acid sequence variants, screening is carried out for maintenance or improvement of binding affinity, maintenance or improvement of antigen specificity, reduction of immunogenicity, etc.

[0522] <TRBC2 binding moiety>

[0523] The bispecific antigen-binding molecule that constitutes the therapeutic agent of the first embodiment of the present invention may comprise a TRBC2-binding portion and a tumor antigen-binding portion. The TRBC2-binding portion may be various anti-TRBC2 antibodies described in WO2020 / 089644, and antigen-binding domains that bind to TRBC2. The TRBC2-binding portion may also comprise the VH domain of hJOVI-1 of WO2020 / 089644 (SEQ ID NO:1 of WO2020 / 089644) having three amino acid substitutions T28K, Y32F, and A100N (Table 1 (page 23) of WO2020 / 089644) and the VL domain of hJOVI-1 (SEQ ID NO:2 of WO2020 / 089644).

[0524] In one aspect, the bispecific antigen-binding molecule comprises at least one portion that specifically binds to TRBC2 expressed in normal T cells, and the at least one portion comprises a VH domain and a VL domain,

[0525] The VH domain comprises:

[0526] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16,

[0527] Heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0528] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0529] The VL domain comprises:

[0530] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19,

[0531] Light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0532] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21.

[0533] In a specific manner, at least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises: a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7. In a further specific manner, at least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:6, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:7. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequences of SEQ ID NO:6 or 7 may be present in the complementarity determining regions (CDRs) or frameworks (FRs).

[0534] In a specific manner, to improve the binding affinity and / or other biological properties of the TRBC2-binding moiety, amino acid sequence variants of the bispecific antigen-binding molecule comprising the TRBC2-binding moiety are made. The amino acid sequence variants can be made, for example, by introducing suitable mutations (deletions, insertions and substitutions) into the nucleotide sequence encoding the anti-TRBC2 antibody or by peptide synthesis. The mutations can be introduced into the complementarity determining regions (CDRs) or frameworks (FRs) of the antibody according to the properties to be improved. For the amino acid sequence variants, screening is carried out for maintenance or improvement of binding affinity, maintenance or improvement of antigen specificity, reduction of immunogenicity, etc.

[0535] In a specific manner, at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells can be an scFv or Fab fragment.

[0536] The CDR sequences of the TRBC1 or TRBC2 antigen-binding sites are set forth in the following table.

[0537] [Table 1]

[0538]

[0539] <Tumor antigen-binding moiety>

[0540] The bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may comprise a TRBC1 or TRBC2 binding portion and a tumor antigen binding portion. The tumor antigen that is the target of the present invention is an antigen expressed in T cell tumor cells. Antigens expressed in T cell tumor cells are mostly well-known in the art and can be set as target tumor antigens in the present invention. Examples of target tumor antigens are described in other parts of this application. The tumor antigen binding portion can be produced based on well-known antibodies. For T cell tumor antigens, most antibodies are well-known in the art and can be used in the present invention. A person skilled in the art can select appropriate light chain variable regions and heavy chain variable regions from the amino acid sequence information of well-known antibodies and construct bispecific antibodies by genetic engineering methods. In genetic engineering methods, the gene sequence encoding the antibody can be inserted into an expression vector, which is then transformed into a host cell, and then the host cell is cultured to produce the antibody. Examples of Fc domains, peptide linkers, and antibody forms that can be used in the constructed bispecific antibodies are described in other parts of this application.

[0541] When the monoclonal antibody specifically binding to the target tumor antigen is unknown, the hybridoma method, phage display method, etc. can be used to produce and use in the bispecific antigen-binding molecule. In the hybridoma method, hybridomas are produced by fusing B cells collected from the spleen or lymph nodes of animals immunized with the peptide of the target tumor antigen, especially rats or mice, with myeloma cells, and hybridomas that produce antibodies reactive with the antigen are selected. Using the selected hybridomas, monoclonal antibodies can be produced. The phage display method is a technique that uses a library that functionally presents antibody variable regions on phages to select antibodies having affinity for the target molecule. The gene of the antibody contained in the phage can be sequenced, and monoclonal antibodies can be produced based on the sequence information.

[0542] In one mode, the tumor antigen that is the target of the present invention is not a pan-T cell antigen such as CD2, CD3, CD5, CD7, etc. This is because they can cause T cell exhaustion and there may be a clinically unacceptable level of immunodeficiency. In one mode, when a TRBC1-binding portion is used as the normal T cell-side antigen-binding portion of the bispecific antibody of the present invention, the tumor antigen that is the target of the present invention is not TRBC1, or when a TRBC2-binding portion is used as the normal T cell-side antigen-binding portion of the bispecific antibody of the present invention, the tumor antigen that is the target of the present invention is not TRBC2. This is because they can cause normal T cell exhaustion and there may be a clinically unacceptable level of immunodeficiency. For example, WO2022 / 177889A1 (the entire disclosure of which is hereby incorporated by reference in its entirety) discloses that normal TRBC1-positive T cells can be made to commit fratricide and die by a bispecific antibody that binds to normal TRBC1-positive T cells and TRBC1-positive cancer cells.

[0543] (Target tumor antigen)

[0544] In the present invention, examples of target tumor antigens expressed in T cell tumor cells include, but are not limited to, the following T cell antigens: CD4, CD8, CD13, CD16, CD17, CD18, CD19, CD20, CD21, CD23, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32b, CD35, CD37, CD38, CD39, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD62L, CD63, CD68, CD69, CD70, CD71, CD73, CD74, CD75S, CD80, CD81, CD82, CD84, CD85A, CD85J, CD86, CD87, CD92, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD107a, CD107b, CD108, CD109, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD124, CD126, CD127, CD128, CD129, CD130, CD132, CD134, CD137, CD146, CD147, CD148, CD150, CD152, CD153, CD40L (CD154), CD156b, CD158a, CD158b1, CD158b2, CD158e1 / e2, CD158f, CD158g, CD158h, CD158h, CD158i, CD158j, CD158k, CD159a, CD160, CD161, CD162, CD164, CD172g, CD178, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198, CDw199, CD205, CD210a, CDw210b, CD212, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD229, CD230, CD231, CD244, CD245, CD246, CD247, CD253,CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD267, CD268, CD270, CD272, CD273, CD274, CD275, CD277, CD278, CD279, CD283, CD288, CD289, CD290, CD294, CD295, CD296, CD298, CD300a, CD300c, CD300e, CD305, CD306, CD307c, CD314, CD316, CD317, CD319, CD321, CD328, CD351, CD352, CD352, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362, CD363。

[0545] In one embodiment, the target tumor antigens expressed in T cell tumor cells can be selected from CCR1, CCR4, CCR7, CCR8, CCR10, CXCR4, CXCR7, TIGIT, CADM1, GPR15, CXCR5, CXCL13, SLAM, ICOS, CD134, CXCR3, anaplastic lymphoma kinase, CD30, ST2(L), CCR5, Notch1, CD38, CD1a, CCR9 (CD199), CD47, IL-7Rα (CD127) and CD40L (CD154), but are not limited thereto.

[0546] CCR1 (C-C chemokine receptor 1), CCR4, CCR5, CCR7, CCR8, CCR9 (CD199), CCR10, CXCR (C-X-C chemokine receptor) 3, CXCR4, CXCR5 and CXCR7 are chemokine receptors. CCR1 is also known as CD191.

[0547] CXCL13 (C-X-C chemokine ligand 13) is a chemokine ligand.

[0548] TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an immune checkpoint receptor present in NK cells, cytotoxic T cells, memory T cells and regulatory T cells (Tregs).

[0549] CADM1 (Cell adhesion molecule 1) is an intercellular adhesion molecule belonging to the immunoglobulin superfamily of cell adhesion molecules (IgCAM).

[0550] GPR15 (G protein-coupled receptor 15) is a class A orphan G protein-coupled receptor, which can be found in epithelial cells, synovial macrophages, endothelial cells, lymphocytes, especially T cells.

[0551] SLAM (signaling lymphocytic activation molecule) is the signaling lymphocytic activation molecule (SLAM) family.

[0552] ICOS (inducible T cell co-stimulator) is a 55-60 kDa disulfide-linked homodimeric T cell surface glycoprotein, also known as CD278.

[0553] CD134 is a member 4 of the tumor necrosis factor receptor (TNFR) superfamily.

[0554] Anaplastic lymphomakinase (ALK) is a 220 kDa transmembrane glycoprotein mainly expressed in the developing nervous system, also known as CD246.

[0555] CD30 is a membrane-bound glycoprotein with a molecular weight of 105-120 kD belonging to the TNF receptor superfamily, and is strongly expressed in the mononuclear Hodgkin cells and multinucleated Reed-Sternberg cells of Hodgkin lymphoma, and the tumor cells of anaplastic large cell lymphoma (ALCL).

[0556] ST2(L) is the ST2 gene product, which has a secreted form (ST2) and a transmembrane receptor form (ST2L). The ST2 gene was cloned as a gene specifically expressed during the onset of cell proliferation.

[0557] Notch1 is a member of the Notch family.

[0558] CD38 is also known as cyclic ADP ribose hydrolase

[0559] CD1a is a CD1 isoform. There are 4 CD1 isoforms in humans (CD1a, CD1b, CD1c, CD1d). CD1a is a lipid-presenting molecule, and its expression is essentially limited to cortical / thymic T cell acute lymphoblastic leukemia (cortical T-ALL) and Langerhans cell (LC) histiocytosis, and is substantially absent in human tissues other than developing cortical thymocytes and LCs (Blood. 2019; 133(21):2291-2304; the entire description is hereby incorporated by reference in its entirety).

[0560] CD47 is also known as integrin-associated protein (IAP).

[0561] IL-7Rα (CD127) is mainly expressed in thymocytes, dendritic cells, monocytes, mature T cells, etc. IL-7R is a heterodimer of the α chain (CD127) of the type I cytokine receptor and the common γ chain (γc chain).

[0562] CD40L (CD40 ligand, CD154) is a ligand protein belonging to the TNF superfamily and is mainly expressed in activated CD4 + T cells. The CD40 / CD40L system is implicated in tumor formation.

[0563] CCR1, CCR4, CCR7, CCR8, CCR10, CXCR4, CXCR7, TIGIT, CADM1, and GPR15 can be target antigens for the treatment of adult T-cell leukemia (ATL) because they are expressed in ATL cells (Leukemia & Lymphoma, 2006; 47(10): 2163-2173; the entire disclosure thereof is hereby incorporated by reference in its entirety).

[0564] CXCR5, CXCL13, SLAM, ICOS, CD134, CXCR3 are expressed in angioimmunoblastic T-cell lymphoma (AITL) cells and can therefore be target antigens for the treatment of AITL (Blood. 2004; 103: 236-241; the entire disclosure thereof is hereby incorporated by reference in its entirety).

[0565] Anaplastic lymphoma kinase, CD30, ST2(L), CCR5 can be used as target antigens for the treatment of peripheral T-cell lymphoma (PTCL) including anaplastic large cell lymphoma because they are expressed in anaplastic large cell lymphoma cells (Blood. 2004; 103: 236-241).

[0566] Notch1, CXCR4, CD38, CD1a, CCR9 (CD199), CD47, IL-7Rα (CD127), and CD40L (CD154) are expressed in T-cell acute lymphoblastic leukemia (TALL) cells and thus can be target antigens for TALL treatment (for CD1a, refer to Blood. 2019; 133(21): 2291-2304; for IL-7Rα, refer to Blood 2021; 138(12): 1040-1052; for CCR9 (CD199), refer to Blood. 2022; 140(1): 25-37; for CD38 and CD47, refer to Blood 2022; 140(1): 45-57; for Notch1, CXCR4, and CD38, refer to Blood Cancer Discov 2021; 2: 19-31. The entire disclosures of these documents are hereby incorporated by reference in their entirety).

[0567] (CCR4 binding portion)

[0568] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be CCR4. Thus, the bispecific antigen-binding molecule may comprise a CCR4 binding portion and a TRBC1 binding portion, or may comprise a CCR4 binding portion and a TRBC2 binding portion. The CCR4 binding portion may be mogamulizumab, an anti-CCR4 antibody described in Japanese Patent No. 4052515 (the entire disclosure of which is hereby incorporated by reference in its entirety).

[0569] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule containing at least one portion that specifically binds to CCR4, wherein the at least one portion comprises a VH domain and a VL domain.

[0570] The VH domain comprises:

[0571] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, and

[0572] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24;

[0573] The VL domain comprises:

[0574] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and

[0575] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27.

[0576] In a specific manner, at least one moiety that specifically binds to CCR4 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9. In a further specific manner, at least one moiety that specifically binds to CCR4 comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:8, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:9. Amino acid mutations (deletions, insertions and substitutions) with respect to the amino acid sequences of SEQ ID NO:8 or 9 may be present in the complementarity determining regions (CDRs) or frameworks (FRs).

[0577] In a specific manner, at least one moiety that specifically binds to CCR4 may be an scFv or Fab fragment.

[0578] In a specific manner, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0579] At least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0580] (a) Comprising a VH domain and a VL domain,

[0581] The VH domain comprises:

[0582] A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0583] A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0584] The VL domain comprises:

[0585] A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0586] A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0587] or

[0588] (b) comprises a VH domain and a VL domain,

[0589] The VH domain comprises:

[0590] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0591] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0592] The VL domain comprises:

[0593] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0594] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0595] and

[0596] at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises the VH domain and the VL domain,

[0597] The VH domain comprises:

[0598] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and

[0599] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24;

[0600] The VL domain comprises:

[0601] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and

[0602] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27.

[0603] In a particular manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0604] at least one portion that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0605] (a) comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5, or

[0606] (b) comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7,

[0607] and

[0608] at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9.

[0609] (CD1a binding portion)

[0610] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be CD1a. Thus, the bispecific antigen-binding molecule may comprise a CD1a binding portion and a TRBC1 binding portion, or may comprise a CD1a binding portion and a TRBC2 binding portion. The CD1a binding portion may be the anti-CD1a antibody SC02-113 described in WO2005 / 063819 (the entire disclosure of which is hereby incorporated by reference in its entirety).

[0611] In one mode, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule containing at least one portion that specifically binds to CD1a, wherein the at least one portion comprises a VH domain and a VL domain,

[0612] The VH domain comprises:

[0613] a heavy chain CDR1 containing the amino acid sequence set forth in SEQ ID NO:34,

[0614] a heavy chain CDR2 containing the amino acid sequence set forth in SEQ ID NO:35, and

[0615] a heavy chain CDR3 containing the amino acid sequence set forth in SEQ ID NO:36;

[0616] The VL domain comprises:

[0617] a light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO:37,

[0618] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and

[0619] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:39.

[0620] In a specific manner, at least one moiety that specifically binds to CD1a comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:40 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:41. In a further specific manner, at least one moiety that specifically binds to CD1a comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:40, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:41. Amino acid mutations (deletions, insertions and substitutions) with respect to the amino acid sequences of SEQ ID NO:40 or 41 may be present in the complementarity determining regions (CDRs) or the framework (FR).

[0621] In a specific manner, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0622] at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0623] (a) comprising a VH domain and a VL domain,

[0624] wherein the VH domain comprises:

[0625] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0626] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0627] wherein the VL domain comprises:

[0628] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0629] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0630] or

[0631] (b) Comprising a VH domain and a VL domain,

[0632] The VH domain comprises:

[0633] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0634] The heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0635] The VL domain comprises:

[0636] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0637] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0638] and

[0639] At least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a VH domain and a VL domain,

[0640] The VH domain comprises:

[0641] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:34, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:35, and

[0642] The heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:36;

[0643] The VL domain comprises:

[0644] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:37, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and

[0645] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:39.

[0646] In a specific manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0647] At least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0648] (a) At least one moiety that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5, or

[0649] (b) At least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, and

[0650] At least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:41 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:42.

[0651] (CCR9 binding moiety)

[0652] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be CCR9. Thus, the bispecific antigen-binding molecule may comprise a CCR9 binding moiety and a TRBC1 binding moiety, or may comprise a CCR9 binding moiety and a TRBC2 binding moiety. The CCR9 binding moiety may be the anti-CCR9 antibody 9G7 described in WO2023 / 037125 (the entire description of which is hereby incorporated by reference in its entirety).

[0653] In one embodiment, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule containing at least one moiety that specifically binds to CCR9, wherein the at least one moiety comprises a VH domain and a VL domain,

[0654] The VH domain comprises:

[0655] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:101, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:102, and heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:103;

[0656] The VL domain comprises:

[0657] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104,

[0658] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105, and

[0659] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106.

[0660] In a specific manner, at least one moiety that specifically binds to CCR9 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 131 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 132. In a further specific manner, at least one moiety that specifically binds to CCR9 comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 131, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 132. Amino acid mutations (deletions, insertions and substitutions) with respect to the amino acid sequences of SEQ ID NO: 131 or 132 may be present in the complementarity determining regions (CDRs) or the framework (FR).

[0661] In a specific manner, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0662] at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0663] (a) comprising a VH domain and a VL domain,

[0664] wherein the VH domain comprises:

[0665] the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10,

[0666] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and

[0667] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12;

[0668] the VL domain comprises:

[0669] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,

[0670] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0671] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0672] or

[0673] (b) comprising a VH domain and a VL domain,

[0674] wherein the VH domain comprises:

[0675] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16,

[0676] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0677] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0678] wherein the VL domain comprises:

[0679] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0680] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0681] and

[0682] at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises the VH domain and the VL domain,

[0683] wherein the VH domain comprises:

[0684] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:101, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:102, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:103;

[0685] wherein the VL domain comprises:

[0686] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:104, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:105, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:106.

[0687] In a specific manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0688] At least one part that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0689] (a) At least one part that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5, or

[0690] (b) At least one part that specifically binds to TRBC2 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7, and

[0691] At least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 131 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 132.

[0692] (CXCR4 binding part)

[0693] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be CXCR4. Thus, the bispecific antigen-binding molecule may comprise a CXCR4 binding part and a TRBC1 binding part, or may comprise a CXCR4 binding part and a TRBC2 binding part. The CXCR4 binding part may be the anti-CXCR4 antibody (hz515H7 VH1D76N-VL2) described in WO2010 / 125162 (the entire description of which is hereby incorporated by reference in its entirety).

[0694] In one mode, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule containing at least one part that specifically binds to CXCR4, wherein the at least one part comprises a VH domain and a VL domain,

[0695] The VH domain comprises:

[0696] A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and

[0697] The heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;

[0698] The VL domain comprises:

[0699] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and

[0700] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0701] In certain embodiments, at least one moiety that specifically binds to CXCR4 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 133 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 134. In further particular embodiments, at least one moiety that specifically binds to CXCR4 comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 133, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 134. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequences of SEQ ID NO: 133 or 134 may be present in the complementarity determining regions (CDRs) or framework (FR).

[0702] In certain embodiments, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0703] At least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0704] (a) Comprising a VH domain and a VL domain,

[0705] The VH domain comprises:

[0706] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and

[0707] The heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12;

[0708] The VL domain comprises:

[0709] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0710] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0711] Or

[0712] (b) Comprising a VH domain and a VL domain,

[0713] The VH domain comprises:

[0714] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0715] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0716] The VL domain comprises:

[0717] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0718] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0719] And

[0720] At least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a VH domain and a VL domain,

[0721] The VH domain comprises:

[0722] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:107, the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:108, and the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:109;

[0723] The VL domain comprises:

[0724] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:110, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:111, and the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:112.

[0725] In a specific manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0726] At least one part that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0727] (a) At least one part that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4 and a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5, or

[0728] (b) At least one part that specifically binds to TRBC2 expressed in normal T cells comprises a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6 and a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7, and

[0729] At least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 133 and a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 134.

[0730] (TIGIT binding part)

[0731] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be TIGIT. Thus, the bispecific antigen-binding molecule may comprise a TIGIT binding part and a TRBC1 binding part, or may comprise a TIGIT binding part and a TRBC2 binding part. The TIGIT binding part may be the anti-TIGIT antibody Vibostolimab described in WO2016 / 028656 (the entire description of which is hereby incorporated by reference in its entirety).

[0732] In one mode, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule containing at least one part that specifically binds to TIGIT, wherein the at least one part comprises a VH domain and a VL domain,

[0733] The VH domain comprises:

[0734] A heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 89, a heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 90, and

[0735] The heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91;

[0736] The VL domain comprises:

[0737] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:92, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93, and

[0738] The light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:94.

[0739] In certain embodiments, at least one moiety that specifically binds to TIGIT comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:135 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:136. In further certain embodiments, at least one moiety that specifically binds to TIGIT comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:135, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:136. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequences of SEQ ID NO:135 or 136 may be present in the complementarity determining regions (CDRs) or frameworks (FRs).

[0740] In certain embodiments, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0741] At least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0742] (a) Comprising a VH domain and a VL domain,

[0743] The VH domain comprises:

[0744] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10,

[0745] The heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0746] The heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0747] The VL domain comprises:

[0748] A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13,

[0749] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0750] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0751] or

[0752] (b) comprising a VH domain and a VL domain,

[0753] wherein the VH domain comprises:

[0754] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16,

[0755] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0756] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0757] wherein the VL domain comprises:

[0758] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19,

[0759] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0760] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0761] and

[0762] at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises the VH domain and the VL domain,

[0763] wherein the VH domain comprises:

[0764] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:89, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:90, and

[0765] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91;

[0766] wherein the VL domain comprises:

[0767] The light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:92, the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93, and

[0768] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:94.

[0769] In a specific manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0770] at least one part that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0771] (a) At least one part that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5, or

[0772] (b) At least one part that specifically binds to TRBC2 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, and

[0773] at least one part that specifically binds to the target tumor antigen expressed in T cell tumor cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:135 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:136.

[0774] (CCR8 binding part)

[0775] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be CCR8. Thus, the bispecific antigen-binding molecule may comprise a CCR8 binding part and a TRBC1 binding part, or may comprise a CCR8 binding part and a TRBC2 binding part. The CCR8 binding part may be the anti-CCR8 antibody ABBV-514 described in WO2023 / 010054 (the entire description of which is hereby incorporated by reference in its entirety).

[0776] In one mode, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule containing at least one part that specifically binds to CCR8, wherein the at least one part comprises a VH domain and a VL domain,

[0777] The VH domain comprises:

[0778] The heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95,

[0779] the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, and

[0780] the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97;

[0781] The VL domain comprises:

[0782] the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98,

[0783] the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99, and

[0784] the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0785] In certain embodiments, at least one moiety that specifically binds to CCR8 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 137 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 138. In further certain embodiments, at least one moiety that specifically binds to CCR8 comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 137, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 138. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequences of SEQ ID NO: 137 or 138 may be present in the complementarity determining regions (CDRs) or the frameworks (FRs).

[0786] In certain embodiments, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0787] at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0788] (a) comprising a VH domain and a VL domain,

[0789] The VH domain comprises:

[0790] The heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10,

[0791] the heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0792] the heavy-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0793] The VL domain comprises:

[0794] the light-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, the light-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0795] the light-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0796] Or

[0797] (b) Comprising a VH domain and a VL domain,

[0798] The VH domain comprises:

[0799] the heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, the heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0800] the heavy-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0801] The VL domain comprises:

[0802] the light-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, the light-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0803] the light-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0804] And

[0805] At least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a VH domain and a VL domain,

[0806] The VH domain comprises:

[0807] the heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:95, the heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:96, and

[0808] A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:97;

[0809] The VL domain comprises:

[0810] A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:98, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:99, and

[0811] A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:100.

[0812] In a specific manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0813] At least one part that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0814] (a) At least one part that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5, or

[0815] (b) At least one part that specifically binds to TRBC2 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, and

[0816] At least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:137 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:138.

[0817] (CD30 binding part)

[0818] The target tumor antigen of the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may be CD30. Thus, the bispecific antigen-binding molecule may comprise a CD30 binding part and a TRBC1 binding part, or may comprise a CD30 binding part and a TRBC2 binding part. The CD30 binding part may be the anti-CD30 antibody AC10 described in Japanese Patent No. 4303964 (the entire description of which is hereby incorporated by reference in its entirety).

[0819] In one embodiment, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one moiety that specifically binds to CD30, wherein said at least one moiety comprises a VH domain and a VL domain.

[0820] The VH domain comprises:

[0821] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113,

[0822] Heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and

[0823] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115;

[0824] The VL domain comprises:

[0825] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116,

[0826] Light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and

[0827] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0828] In certain embodiments, the at least one moiety that specifically binds to CD30 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 139 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 140. In further certain embodiments, the at least one moiety that specifically binds to CD30 comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 139, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 140. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequences of SEQ ID NO: 139 or 140 may be present in the complementarity determining regions (CDRs) or the framework (FR).

[0829] In certain embodiments, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0830] The at least one moiety that specifically binds to TRBC1 or TRBC2 expressed on normal T cells is:

[0831] (a) comprises a VH domain and a VL domain,

[0832] The VH domain comprises:

[0833] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and

[0834] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12;

[0835] The VL domain comprises:

[0836] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and

[0837] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15;

[0838] Or

[0839] (b) comprises a VH domain and a VL domain,

[0840] The VH domain comprises:

[0841] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0842] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0843] The VL domain comprises:

[0844] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0845] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0846] And

[0847] at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a VH domain and a VL domain,

[0848] The VH domain comprises:

[0849] The heavy-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113, the heavy-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114, and

[0850] the heavy-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115;

[0851] The VL domain comprises:

[0852] the light-chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, the light-chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and

[0853] the light-chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0854] In a particular embodiment, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0855] at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0856] (a) at least one moiety that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4 and a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5, or

[0857] (b) at least one moiety that specifically binds to TRBC2 expressed in normal T cells comprises a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6 and a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7, and

[0858] at least one moiety that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy-chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 139 and a light-chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 140.

[0859] (CD40L binding moiety)

[0860] The target tumor antigen of the bispecific antigen-binding molecule that constitutes the therapeutic agent of the first embodiment of the present invention may be CD40L (CD154). Thus, the bispecific antigen-binding molecule may comprise a CD40L-binding portion and a TRBC1-binding portion, or may comprise a CD40L-binding portion and a TRBC2-binding portion. The CD40L-binding portion may be the anti-CD40L (CD154) antibody ABI793 described in Japanese Patent Application Laid-Open No. 2003-526371 (WO01 / 068860), the entire disclosure of which is hereby incorporated by reference in its entirety.

[0861] In one embodiment, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD40L (CD154), wherein the at least one portion comprises a VH domain and a VL domain.

[0862] The VH domain comprises:

[0863] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119,

[0864] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121;

[0865] The VL domain comprises:

[0866] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122,

[0867] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124.

[0868] In a specific manner, at least one moiety that specifically binds to CD40L (CD154) comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 141 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 142. In a further specific manner, at least one moiety that specifically binds to CD40L (CD154) comprises: a heavy chain variable region VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 141, and a light chain variable region VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 142. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequences of SEQ ID NO: 141 or 142 may be present in the complementarity determining regions (CDRs) or frameworks (FRs).

[0869] In a specific manner, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention,

[0870] at least one moiety that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0871] (a) comprising a VH domain and a VL domain,

[0872] wherein the VH domain comprises:

[0873] a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10,

[0874] a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and

[0875] a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12;

[0876] wherein the VL domain comprises:

[0877] a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13,

[0878] a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and

[0879] a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15;

[0880] or

[0881] (b) It comprises a VH domain and a VL domain,

[0882] The VH domain comprises:

[0883] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and

[0884] Heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18;

[0885] The VL domain comprises:

[0886] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and

[0887] Light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21;

[0888] And

[0889] At least one part that specifically binds to the target tumor antigen expressed in T cell tumor cells comprises the VH domain or the VL domain,

[0890] The VH domain comprises:

[0891] Heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:119, heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:120, and heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:121;

[0892] The VL domain comprises:

[0893] Light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:122, light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:123, and light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:124.

[0894] In a specific manner, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention,

[0895] At least one part that specifically binds to TRBC1 or TRBC2 expressed in normal T cells is:

[0896] (a) At least one portion that specifically binds to TRBC1 expressed in normal T cells, comprising a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5, or

[0897] (b) At least one portion that specifically binds to TRBC2 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7, and

[0898] At least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:141 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:142.

[0899] In a further specific manner, in order to improve the binding affinity and / or other biological properties of the tumor antigen-binding portion, amino acid sequence variants of the bispecific antigen-binding molecule comprising the tumor antigen-binding portion are prepared. The amino acid sequence variants can be prepared, for example, by introducing suitable mutations (deletions, insertions, and substitutions) into the nucleotide sequence encoding the antibody or by peptide synthesis. The mutations can be introduced into the complementarity-determining regions (CDRs) or frameworks (FRs) of the antibody according to the properties to be improved. For the amino acid sequence variants, screening is carried out for the maintenance or improvement of binding affinity, the maintenance or improvement of antigen specificity, the reduction of immunogenicity, etc.

[0900] The CDR sequences of each tumor antigen-binding site are set forth in the following table.

[0901] [Table 2]

[0902]

[0903] (Configuration)

[0904] In a specific manner, in the bispecific antigen-binding molecule that constitutes the therapeutic agent of the present invention, at least one part that specifically binds to an antigen having a subtype expressed in normal T cells is a scFv, at least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells is a Fab fragment, and the scFv is linked to the C-terminus of the heavy chain of the Fab fragment via a peptide linker. In a further specific manner, in the bispecific antigen-binding molecule, at least one part that specifically binds to an antigen having a subtype expressed in normal T cells is a scFv, at least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells is a scFv, and the two scFVs are linked via a peptide linker.

[0905] The "peptide linker" is a peptide containing about 2 to 20 amino acids, such as the peptide linker of (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n , where "n" is generally an integer from 1 to 10. Typically, the peptide linker is selected from GGGGS (SEQ ID NO:51), GGGGSGGGGS (SEQ ID NO:52), GGGGSGGGGSGGGGS (SEQ ID NO:64), SGGGGSGGGG (SEQ ID NO:53), and GGGGSGGGGSGGGG (SEQ ID NO:54), and the sequence GSPGSSSSGS (SEQ ID NO:55), (G 4 S) 4 , GSGSGSGS (SEQ ID NO:56), GSGSGNGS (SEQ ID NO:57), GGSGSGSG (SEQ ID NO:58), GGSGSG (SEQ ID NO:59), GGSG (SEQ ID NO:60), GGSGNGSG (SEQ ID NO:61), GGNGSGSG (SEQ ID NO:62), and GGNGSG (SEQ ID NO:63) can be used.

[0906] In a specific manner, the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention further comprises an Fc domain. In a further specific manner, the Fc domain is the Fc domain of IgG1, IgG2, IgG3, or IgG4, and the Fc domain is preferably the IgG1 Fc domain. In a further specific manner, the polypeptide of the IgG1 Fc domain may have more than one amino acid mutation, particularly preferably an amino acid mutation that alters the binding ability to the Fc receptor, and more preferably an amino acid mutation that reduces the binding ability to the Fc receptor.

[0907] In a specific manner, the Fc domain is of human IgG1 isotype and may have the leucine at positions 234 and 235 mutated to alanine, i.e., L234A L235A (LALA mutation), or a corresponding mutation. These mutations can be made at equivalent positions in other isotypes and subtypes. The LALA mutation renders the binding to complement component (C1q) and Fcγ receptor (FcgR) ineffective, and thus can prevent in vitro FcgR-mediated co-activation of innate immune effector cells including natural killer (NK) cells, monocytes / macrophages, and neutrophils without altering the functional binding to FcRn (fetal Fc receptor) (Clin Cancer Res (2016) 22(13):3286 - 3297.; the entire disclosure of which is incorporated herein by reference in its entirety). Thus, the bispecific antigen-binding molecule having an Fc domain carrying the LALA mutation has no complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) activities, and the only immune effector cells involved can be T cells.

[0908] In a specific manner, the configuration of the bispecific antigen-binding molecule can be any of the structures / configurations described in ULRICH H. WEIDLE et al., “The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer,” Cancer Genomics & Proteomics January 2013, 10(1) 1 - 18 (the entire disclosure of which is incorporated herein by reference in its entirety). Examples include, but are not limited to, cross Mab, IgG-dssFv2, DVD, IgG-dsFv, IgG-scFab, scFab-dssFv, Fv2-Fc, Fab-scFv2, Fab-scFv, Fab-scFv-Fc, BiTE (scFv-scFv), diabody, DART, etc.

[0909] In a specific manner, the bispecific antigen-binding molecule is monovalent with respect to both the target antigen on the normal T cell side and the target tumor antigen expressed in T cell tumor cells.

[0910] In a specific manner, the bispecific antigen-binding molecule is bivalent with respect to both the target antigen on the normal T cell side and the target tumor antigen expressed in T cell tumor cells.

[0911] In a specific manner, the bispecific antigen-binding molecule is trivalent with respect to both the target antigen on the normal T cell side and the target tumor antigen expressed in T cell tumor cells.

[0912] In a specific manner, the bispecific antigen-binding molecule is monovalent with respect to the target antigen on the normal T cell side and bivalent with respect to the target tumor antigen expressed in T cell tumor cells.

[0913] In a specific manner, the bispecific antigen-binding molecule is bivalent with respect to the target antigen on the normal T cell side and monovalent with respect to the target tumor antigen expressed in T cell tumor cells.

[0914] In a specific manner, the bispecific antigen-binding molecule has a structure in which 2 scFvs that specifically bind to the target antigen on the normal T cell side (e.g., TRBC1 or TRBC2) and a Fab fragment that specifically binds to the target tumor antigen expressed in T cell tumor cells are linked by a peptide linker (i.e., bivalent + bivalent), and further has a structure with an Fc domain (Fab-scFv-Fc).

[0915] In a specific manner, the T cell tumor can be adult T cell leukemia (ATL), peripheral T cell lymphoma (PTCL), or T cell acute lymphoblastic leukemia (TALL). Peripheral T cell lymphoma (peripheral T-cell lymphoma: PTCL) Peripheral T cell lymphoma is a general term for lymphomas originating from T cells that differentiate and mature in the thymus and migrate to peripheral organs. PTCL accounts for approximately 10% of all lymphomas in Japan. PTCL is divided into peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), angioimmunoblastic T cell lymphoma (AITL), ALK-positive anaplastic large cell lymphoma (ALCL), and ALK-negative ALCL. PTCL-NOS is difficult to distinguish from ATL (adult T cell lymphoma) in histopathology, and screening for anti-HTLV-1 antibodies is required.

[0916] In a specific manner, the therapeutic agent of the present invention can be administered in combination with chemotherapeutic agents, radiation, and / or other agents used in cancer immunotherapy.

[0917] The dosage and interval of administration of the therapeutic agent of the present invention can be individually adjusted to provide a sufficient plasma level of the bispecific antigen-binding molecule of the present invention required to maintain the therapeutic effect. The dosage varies depending on the therapeutic effect as the target, the administration method, the treatment period, age, body weight, etc., and is about 0.1 to 50 mg / kg / day. A therapeutically effective plasma level can be achieved by administering multiple doses per day. The level in plasma can be measured by, for example, HPLC.

[0918] In combination therapy, the therapeutic agent of the present invention and one or more agents or therapies can be administered simultaneously or sequentially. When administered simultaneously or sequentially, all the agents or therapies can be administered by the same route, or they can also be administered by different routes.

[0919] In this specification, simultaneous administration means administering two or more agents or therapies at intervals of several minutes to less than several seconds. For example, two agents or therapies are administered at intervals of about 15 minutes to less than 1 minute. In this specification, sequential administration means administering at intervals of several minutes, several hours, several days, or several weeks. For example, two agents or therapies are administered after a time of more than 15 minutes, more than 30 minutes, more than 60 minutes, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 2 weeks, 3 weeks, or 4 weeks.

[0920] The therapeutic agent of the present invention can be a pharmaceutical composition. The pharmaceutical composition can further contain a pharmaceutically acceptable carrier. Preparations of pharmaceutical compositions having a pharmaceutically acceptable carrier are well known in the art (e.g., Remington: The Science and Practice of Pharmacy (23rd edition (2020))). As pharmaceutically acceptable carriers, there are buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto.

[0921] The production of the pharmaceutical composition of the present invention is preferably carried out under conditions (good manufacturing practice, GMP) suitable for the manufacturing control and quality control rules of pharmaceuticals and quasi-drugs.

[0922] In one mode, the pharmaceutical composition is a liquid such as a solution, suspension, emulsion, microemulsion, gel, etc. The pharmaceutical composition may be an aqueous preparation and may contain at least 50% (w / w) or more of water. In one mode, the dosage form of the pharmaceutical composition is a form suitable for injection. The injection is, for example, subcutaneous, intramuscular, intraperitoneal, intravitreal or intravenous.

[0923] In one mode, the pharmaceutical composition may be in a solid dosage form. The solid dosage form is, for example, in a freeze-dried or spray-dried form. For example, a solvent and / or diluent may be added before administration for administration.

[0924] The pharmaceutical composition may be provided by packaging such as a syringe, vial, or infusion bag. The syringe may contain the pharmaceutical composition in a freeze-dried form (which needs to be dissolved by, for example, water for injection before administration) or in an aqueous form. Additionally, as a solid dosage form, it may be a powder, granule, tablet, or capsule.

[0925] In a specific mode, the therapeutic agent of the present invention can be used in a method for treating a T cell tumor of a subject. The treatment method includes: determining the subtype expressed in the T cell tumor cells of the subject, and administering the therapeutic agent to the subject; the therapeutic agent contains a bispecific antigen-binding molecule, and the bispecific antigen-binding molecule contains at least one part that specifically binds to a target antigen on the normal T cell side, and the subtype of the target antigen on the normal T cell side is different from the subtype determined to be expressed in the T cell tumor cells of the subject. The determination of the subtype of the antigen expressed in the T cell tumor cells of the subject can be determined by flow cytometry analysis using a fluorescent dye-labeled antibody that specifically binds to the subtype. When the antigen is TRBC and the subtype is TRBC1, the JOVI-1 clone antibody can be used. When the subtype is TRBC2, various antibodies described in WO2020 / 089644 can be used.

[0926] The second embodiment of the present invention is a method for treating a T cell tumor of a subject, and the method includes administering a bispecific antigen-binding molecule to a subject having a T cell tumor.

[0927] In a specific mode, the treatment method further includes determining the subtype expressed in the T cell tumor cells of the subject for the target antigen on the normal T cell side, and the bispecific antigen-binding molecule contains at least one part that specifically binds to the target antigen on the normal T cell side, and the subtype of the target antigen on the normal T cell side is different from the subtype determined to be expressed in the T cell tumor cells of the subject.

[0928] The third embodiment of the present invention is a bispecific antigen-binding molecule for a method of treating T cell tumors.

[0929] The fourth embodiment of the present invention is the use of a bispecific antigen-binding molecule in the manufacture of a medicament for T cell tumors.

[0930] The bispecific antigen-binding molecules in the second, third, and fourth embodiments of the present invention are the same as the bispecific antigen-binding molecules constituting the therapeutic agent of the first embodiment in this specification.

[0931] The term "subject" as used in this specification means a mammal, such as a mouse, rat, primate, particularly a human.

[0932] <Bispecific antigen-binding molecule>

[0933] The fifth embodiment of the present invention relates to a bispecific antigen-binding molecule. According to the present invention, there is provided a bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment.

[0934] The bispecific antigen-binding molecule of the present invention comprises:

[0935] (1) at least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells, and

[0936] (2) at least one portion that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side;

[0937] At least one portion that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side is at least one portion that specifically binds to TRBC1 or TRBC2 (T cell receptor β constant region 1 or 2) of normal T cells (TRBC1-binding portion or TRBC2-binding portion).

[0938] The amino acid sequences of the TRBC1-binding portion or TRBC2-binding portion and the target tumor antigen-binding portion that the bispecific antigen-binding molecule of the present invention may have are the same as those described above for the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment. In addition, the configurations that the bispecific antigen-binding molecule of the present invention may adopt are the same as those described above in this specification for the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment.

[0939] Furthermore, according to the present invention, there is provided a nucleic acid molecule encoding the heavy chain and light chain of the bispecific antigen-binding molecule of the fifth embodiment. The nucleic acid molecule of the present invention can be RNA, DNA, or cDNA.

[0940] The nucleic acid molecule of the present invention may be in the form of a vector, may be present in a vector, and / or may be a part of a vector such as a plasmid, cosmid or YAC. The vector may particularly be an expression vector, or may be a vector that provides for the expression of the bispecific antigen-binding molecule of the present invention in a host cell, host organism and / or expression system. An expression vector typically comprises at least one nucleic acid of the present invention operably linked to more than one appropriate expression control element (e.g., promoter, enhancer, terminator, etc.). The selection of elements and their sequences for expression in a particular host is a common knowledge of those skilled in the art. As specific examples of regulatory elements and other elements useful or indispensable in the expression of the heavy and light chains of the bispecific antigen-binding molecule of the present invention, there may be mentioned a promoter, enhancer, terminator, integration element, selection marker, leader sequence, reporter gene, etc.

[0941] The nucleic acid molecule of the present invention can be prepared or obtained by known methods (e.g., by automated DNA synthesis and / or recombinant DNA techniques) based on the information related to the amino acid sequences of the heavy and light chains of the bispecific antigen-binding molecule of the present invention disclosed in this specification, and / or can be isolated from appropriate natural resources.

[0942] Furthermore, according to the present invention, there is provided one or more host cells that express or can express the heavy and light chains of the bispecific antigen-binding molecule of the fifth embodiment. The host cell of the present invention may contain a nucleic acid or a vector. Preferred host cells of the present invention are bacterial cells, fungal / yeast cells or mammalian cells.

[0943] Suitable bacterial cells include cells of Gram-negative strains (e.g., Escherichia coli, Proteus and Pseudomonas) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus and Lactococcus).

[0944] Suitable fungal / yeast cells include cells of species of Trichoderma, Neurospora and Aspergillus; or include Saccharomyces, e.g., Schizosaccharomyces, e.g., Schizosaccharomyces pombe, Pichia, e.g., Pichia pastoris and Pichia methanolica, and species of Hansenula. Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0945] However, amphibian cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins can also be used in the present invention.

[0946] As described above, the heavy and light chains of the bispecific antigen-binding molecule of the present invention can be produced intracellularly, then isolated from the host cell, and optionally further purified. Alternatively, it can be produced extracellularly (e.g., in the culture medium of the host cell), then isolated from the medium, and optionally further purified.

[0947] Methods and reagents for the recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are well known in the art. Similarly, protein separation and purification techniques suitable for the methods of making the bispecific antigen-binding molecule of the present invention are well known to those skilled in the art.

[0948] However, the bispecific antigen-binding molecule of the present invention can also be obtained by other protein production methods known in the art, such as chemical synthesis including solid-phase or liquid-phase synthesis.

[0949] (Cytotoxic activity)

[0950] The bispecific antigen-binding molecule of the present invention having a TRBC1-binding portion or a TRBC2-binding portion can mobilize TRBC1-positive T cells or TRBC2-positive T cells, inducing cytotoxicity and / or cell death of tumor cells. The cytotoxic activity of the bispecific antigen-binding molecule can be determined by culturing target tumor cells together with the bispecific antigen-binding molecule in the presence of effector cells in vitro and measuring the survival rate of the target tumor cells. As effector cells, for example, activated T cells (e.g., T-LAK) can be used. The mixing ratio of effector cells to target tumor cells can be from 10:1 to 1:10. In one embodiment, the bispecific antigen-binding molecule of the present invention can induce cytotoxicity of tumor cells with an EC50 of less than about 500 pM in vitro, and in a preferred embodiment, can induce cytotoxicity of tumor cells with an EC50 of less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, or less than about 50 pM.

[0951] The anti-TRBC1 antibody for the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention can increase the percentage of TRBC1-positive cells in human peripheral blood mononuclear cells (PBMCs). Specifically, as shown in Reference Example 1 described later, when 10 nM of the anti-TRBC1 antibody was added to human PBMCs and cultured in the presence of IL-2, the proportion of TRBC1-positive cells, which was 30% on Day 0, increased to more than 80% on Day 15. This implies that the addition of the anti-TRBC1 antibody selectively proliferates TRBC1-positive cells. It is considered that the addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody also selectively proliferates TRBC1-positive cells.

[0952] The anti-TRBC1 antibody for the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention can increase cytotoxic T cells in the presence of IL-2. Specifically, as shown in Reference Example 1 described later, on Day 15, in the presence of the anti-TRBC1 antibody and IL-2, the proportion of effector memory T cells increased significantly from approximately 20% to approximately 70%. From this result, it is considered that the addition of the anti-TRBC1 antibody can increase T cells that cause cytotoxic effects. It is considered that the addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody also increases T cells that cause cytotoxic effects.

[0953] The anti-TRBC1 antibody for the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention can activate T cells. Specifically, as shown in Reference Example 1 described later, it is implied that the stimulation with the anti-TRBC1 antibody increases the ratios of CD69, granzyme B, and perforin-positive cells, and the TRBC1-positive cells are activated by the anti-TRBC1 antibody. The bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody used in this example is also expected to have a T cell activation effect.

[0954] As shown in Reference Example 1 described later, the stimulation with the anti-TRBC1 antibody for the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention does not cause a significant change in the ratio of PD-1-positive cells. This implies that even when T cells are activated with the anti-TRBC1 antibody, the T cells are not easily exhausted. It is considered that the bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody used in this example also does not easily exhaust T cells.

[0955] As shown in Reference Example 1 described below, the anti-TRBC1 antibody for the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention can induce T cell activation and release cytokines, but the amount of IL-6 released as an inflammatory cytokine is extremely small. This indicates the possibility of a low risk of cytokine release syndrome caused by the anti-TRBC1 antibody. It is implied that the risk of cytokine release syndrome is also low for bispecific antibodies having the same antigen-binding portion as the anti-TRBC1 antibody.

[0956] The anti-TRBC2 antibody for the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention can increase the percentage of TRBC2-positive cells in PBMC. Specifically, as shown in Reference Example 2 described below, after removing cells (TRBC1-positive T cells) that bind to the TRBC1 antibody from human PBMC using beads, 10 nM of the anti-TRBC2 antibody was added, and when cultured in the presence of IL-2, the cell number was about 5×10 6 ~7×10 6 cells at Day 0, and increased to about 18×10 6 ~42×10 6 cells at Day 15. This implies that the addition of the anti-TRBC2 antibody selectively proliferates TRBC2-positive cells. It is considered that even the addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC2 antibody will similarly selectively proliferate TRBC2-positive cells.

[0957] The anti-TRBC2 antibody for the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention can increase cytotoxic T cells in the presence of IL-2. Specifically, as shown in Reference Example 2 described below, on Day 15, in the presence of the anti-TRBC2 antibody and IL-2, the proportion of effector memory T cells increased significantly from about 27% to about 81%. From this result, it is considered that the stimulation by the anti-TRBC2 antibody can increase T cells that cause cytotoxic effects. It is considered that even the stimulation by a bispecific antibody having the same antigen-binding portion as the anti-TRBC2 antibody will similarly increase T cells that cause cytotoxic effects.

[0958] The TRBC2 antibody for the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention can activate T cells. Specifically, as shown in Reference Example 2 described below, it is implied that the stimulation by the anti-TRBC2 antibody increases the ratio of CD69, granzyme B, and perforin-positive cells, and TRBC2-positive cells are activated by the anti-TRBC2 antibody. The same T cell activation effect can also be expected for bispecific antibodies having the same antigen-binding portion as the anti-TRBC2 antibody.

[0959] As shown in the reference examples described below, the anti-TRBC2 antibody of the TRBC2-binding portion of the bispecific antigen-binding molecule for the present invention does not cause a significant change in the ratio of PD-1 positive cells upon stimulation. This suggests that even when T cells are activated with an anti-TRBC2 antibody, the T cells are not easily exhausted. The same is considered to be true for bispecific antibodies having the same antigen-binding portion as the anti-TRBC2 antibody.

[0960] As shown in Reference Example 2 described below, the anti-TRBC2 antibody of the TRBC2-binding portion of the bispecific antigen-binding molecule for the present invention can induce the activation of T cells and the release of cytokines, but the amount of IL-6 released as an inflammatory cytokine is extremely small. This indicates the possibility of a low risk of cytokine release syndrome caused by the anti-TRBC2 antibody. It is suggested that the risk of cytokine release syndrome is also low for bispecific antibodies having the same antigen-binding portion as the anti-TRBC2 antibody.

[0961] The bispecific antigen-binding molecule of the present invention having a TRBC1-binding portion or a TRBC2-binding portion can be used in the following methods of use:

[0962] (i) for inducing the cytotoxicity of tumor cells,

[0963] (ii) for increasing the TRBC1 positive cell rate or increasing the TRBC2 positive cell rate,

[0964] (iii) for increasing cytotoxic T cells,

[0965] (iv) for activating T cells,

[0966] (v) for stimulating or enhancing the cellular response without exhausting T cells,

[0967] (vi) for stimulating or enhancing the cellular response without increasing the risk of cytokine release syndrome,

[0968] (vii) for treating T cell tumors,

[0969] (viii) for delaying the progression of T cell tumors, or

[0970] (ix) for prolonging the survival of a subject suffering from a T cell tumor.

[0971] The bispecific antigen-binding molecule of the present invention can have an anti-tumor effect. The anti-tumor effect can be measured in vivo, for example. For example, immunodeficient mice NSG mice (Jackson Laboratory, Japan) were treated with human PBMC 1x10 7Cells / only intravenously transplanted, and 7 to 10 days later, a T cell tumor line stably expressing luciferase was intravenously transplanted at 1x10 7 cells / animal. Then, the bispecific antigen-binding molecule of the present invention was administered at a dose of 0.5 mg / kg at a frequency of 2 times / week. 2 to 3 weeks after the start of administration, luciferin was administered to the peritoneal cavity of the mice at 150 mg / kg, and the in vivo luciferase activity was quantified using an IVIS imaging system (Revvity) etc., and thus the T cell tumor amount was measured. By comparing the T cell tumor amounts of the control group and the group administered with the bispecific antigen-binding molecule of the present invention, the anti-tumor effect can be evaluated.

[0972] Example

[0973] The present invention will be more specifically described based on the following examples, but the present invention is not limited by these examples.

[0974] <Example 1> Production of Bispecific Antibody

[0975] 1.1 Bispecific Antibody

[0976] In this example, a bispecific antibody that binds to TRBC1 or TRBC2 and a target tumor antigen was produced.

[0977] TRBC1 or TRBC2 binding portion

[0978] The TRBC1-binding portion was produced based on the sequence information of the anti-TRBC1 antibody JOVI-1 clone described in the specification of US Patent No. 10730942 (US10730942 is based on T-cell receptor knockdown of proteins SEQ ID No. 15, SEQ ID No. 16).

[0979] The TRBC2-binding portion was produced based on the VH domain of hJOVI-1 of WO2020 / 089644 (binding domain) having three amino acid substitutions T28K, Y32F, and A100N (Table 1 (page 23) of WO2020 / 089644) and the VL domain of hJOVI-1 (SEQ ID NO: 2 of WO2020 / 089644) (the heavy and light chain amino acid sequences described in SEQ ID NO: 6 and 7 of the present application).

[0980] The portion that binds to the target tumor antigen expressed in T cell tumor cells was produced based on the sequence information disclosed in the documents described in the following table.

[0981] [Table 3]

[0982]

[0983] The sequences of the heavy and light chains of the bispecific antibody produced in this example are shown below.

[0984] [Table 4]

[0985]

[0986] Figure 2A Schematic diagrams of the Fab-scFv type, Fab-scFv-Fc type, and Fab-Fc-scFv type are shown.

[0987] CCR0010 was obtained by introducing T255K, Y259F, and A327N mutations into the heavy chain of CCR0001 and has a Fab-scFv structure. The previously developed anti-TRBC-2 antibody has a background obtained by introducing mutations into the anti-TRBC-1 antibody (JOVI-1) (for example, WO2015 / 132598A1, WO2020 / 089644A1), and the amino acid sequences of the CDRs of the two have high homology.

[0988] 1.2 Production and purification of recombinant antibodies

[0989] The bispecific antibodies described in Table 4 were constructed by separately expressing the heavy and light chains described in Table 4 using expression vectors. The detailed compositions of the heavy and light chains of each bispecific antibody are as Figure 3A described in each of the figures of -K.

[0990] Specifically, a DNA encoding a bispecific antibody and an expression vector (pcDNA3.4, Thermo Fisher Scientific) were used to construct an expression vector for the bispecific antibody (heavy and light chains). The combinations of the heavy and light chains of the bispecific antibody are as described in Table 4. The above expression vectors were mixed at a ratio of 1:1, and the DNA amount was 0.8 μg per 1 mL of the culture medium. ExpiFectamine CHO Reagent (Thermo Fisher Scientific) was used to introduce them into CHO cells. ExpiCHO Feed and ExpiFectamine CHO Enhancer were added on the day after transfection and 5 days later, and the cells were cultured at 32 °C for 10 - 12 days. Cells were removed from the culture medium by centrifugation and filtration, and the culture supernatant was recovered. The purification of the antibody was carried out by a combination of affinity chromatography using nickel-binding agarose and gel filtration chromatography or a combination of protein A affinity chromatography and gel filtration chromatography.

[0991] <Example 2> Preparation of cells

[0992] Preparation of the cells used in the experiments described in this example.

[0993] Use the following reagents.

[0994] RPMI1640 medium (Nacalai Tesque, 30264-56)

[0995] FBS (fetal bovine serum) (Nichirei Biosciences, 175012): Incubate in a water bath at 56 °C for 30 minutes for protein inactivation.

[0996] Penicillin-streptomycin mixed solution (Nacalai Tesque, 2625384)

[0997] MEM non-essential amino acid solution (100X) (Gibco, 11140-050)

[0998] Sodium pyruvate solution 100 mM (Gibco, 11360-070)

[0999] Anti-TRBC1 antibody (prepared and purified by the method described in Example 1. The heavy and light chain sequences of the anti-TRBC1 antibody used are SEQ ID NO: 4 and 5.)

[1000] Imunace injection 35 (IL-2, interleukin 2) (Shionogi & Co., Ltd.): Dissolve in PBS to 250 IU / μL and store at -25 °C.

[1001] PBS (phosphate-buffered saline) (Gibco, 10010-023)

[1002] Human PBMC (peripheral blood mononuclear cells) (CTL, CTL-UP1)

[1003] Various cells are prepared as described below.

[1004] T-LAK is a population of T cells that express lymphokine-activated killer (LAK) function. The preparation of T-LAK is based on Ochoa AC et al. (Lymphokine-activated killer activity in long-term cultures with anti-CD3 plus interleukin 2: identification and isolation of effector subsets. Cancer Res. 1989 Feb 15; 49(4): 963-8.;

[1005] All of its descriptions are specifically incorporated herein by reference, and the experimental system was optimized for implementation. Specifically, human PBMCs were dissolved in a 37°C water bath and washed once with culture medium. After culturing for 2 days in RPMI 1640 medium containing 10% FBS supplemented with 10 nM anti-TRBC1 antibody and 100 IU / mL of IL-2, further culture (up to 17 days) was carried out in RPMI 1640 medium containing IL-2 and 10% FBS to induce T-LAK. Passage was performed every 2 - 3 days, and 100 IU / mL of IL-2 was newly added when the culture medium was changed. Using an automatic cell counter (BIO RAD, TC20), the cell numbers on the culture start day (Day0) and the 15th day (Day15) were measured.

[1006] The MT-2 cells used in Example 3 are a human leukocyte cell line established by co-culture with human ATL (adult T-cell leukemia) cells. They were purchased from the JCRB cell bank (National Institute of Biomedical Innovation, Health and Nutrition, Japan) (cell number JCRB1210). The MT-2 cells are CCR4 positive (Haematologica 2018 Volume 103(1):126 - 135; all of its descriptions are specifically incorporated herein by reference). Using RPMI medium containing 10% FBS, non-essential amino acids, and sodium pyruvate, they were cultured in a 5% CO 2 , 37°C incubator and passaged 2 - 3 times a week.

[1007] The MOLT-4 cells used in Example 3 were purchased from the National Institute of Biomedical Innovation, Health and Nutrition, Japan. Using RPMI medium containing 10% FBS, they were cultured in a 5% CO 2 , 37°C incubator and passaged 2 - 3 times a week.

[1008] CCRF-CEM9 used in the following examples expresses CD1a and TRBC2. CCRF-CEM9 was obtained by monoclonalization of the cell line CCRF-CEM from TALL using the limiting dilution method. Specifically, the cell suspension of CCRF-CEM was serially diluted starting from 1x10 5 cells / mL until a concentration of 2.5 cells / ml was reached. The 2.5 cells / mL dilution was added to 2 96-well plates at 100 μL per well and cultured at 37°C. The wells (clones) where each cell proliferated were amplified and cultured, and the expression analysis of TRBC1 and CD1a was performed. As a result, CCRF-CEM9 in which the expression of both antigens was confirmed was obtained. Using RPMI medium containing 10% FBS, it was cultured in a 5% CO 2 , 37°C incubator and passaged 2 - 3 times a week.

[1009] <Reference Example 1>Verification of the effector activity of anti-TRBC1 antibody and quantification of cytokine release

[1010] Reagents used

[1011] Flow cytometry staining buffer (eBioscience, 00-4222-26)

[1012] Clear Back (MBL, MT-001)

[1013] Fixable viability dye eFluor780 (invitrogen, 65-0865-14)

[1014] Transcription factor buffer set (BD, 562574)

[1015] TF Fix / Perm buffer (4×) (51-9008100): Dilute 4-fold with TF dilution buffer before use.

[1016] TF dilution buffer (51-9008101)

[1017] TF Perm / Wash buffer (5×) (51-9008102): Dilute 5-fold with distilled water before use.

[1018] MILLIPLEX Human Cytokine / Chemokine / Growth Factor Panel A (Millipore, HCYTA-60K)

[1019] The staining antibodies are shown in the table below.

[1020] [Table 5]

[1021]

[1022] ※BV421, BV711: Abbreviations for BrilliantViolet421 (registered trademark), BrilliantViolet711 (registered trademark)

[1023] After the anti-TRBC1 antibody was prepared and purified by the method described in Example 1, it was labeled with a pigment using the HiLyte Fluor647 Labeling Kit-NH2 (Dojindo, LK15). The heavy and light chain sequences of the anti-TRBC1 antibody are SEQ ID NO: 4 and 5.

[1024] As an isotype control, human IgG (ChromPure Human IgG, whole molecule, Jackson ImmunoResearch, 009-000-003) was similarly labeled with a pigment.

[1025] Method

[1026] Conduct experiments with reference to WO2021 / 173896A1.

[1027] Method for cell surface staining

[1028] After centrifuging human PBMC or T-LAK (300 g × 5 minutes), remove the supernatant and add ClearBack. Incubate at room temperature for 5 minutes, then add various antibodies in flow cytometry staining buffer and react at 4 °C for 30 minutes. After the reaction, add flow cytometry staining buffer, centrifuge (500 g × 2 minutes), and discard the supernatant to wash the cells twice. Use BD LSRFortessa X-20 (BD Biosciences) to quantify the signals by flow cytometry analysis. The analysis software used is Flow Jo_v10.6.1.

[1029] Method for intracellular staining

[1030] After centrifuging human PBMC or T-LAK (300 g × 5 minutes), remove the supernatant and add ClearBack. Incubate at room temperature for 5 minutes, then add anti-CD3 antibody and anti-TRBC1 antibody in flow cytometry staining buffer and react at 4 °C for 30 minutes. After the reaction, add flow cytometry staining buffer, centrifuge (500 g × 2 minutes), and discard the supernatant to wash the cells twice. Add TF Fix / Perm buffer in the transcription factor buffer set and incubate on ice for 30 minutes for fixation and permeabilization treatment. After centrifuging (700 g × 2 minutes), add TF Perm / Wash buffer and centrifuge (700 g × 2 minutes), then discard the supernatant to wash the cells twice. Add anti-granzyme B antibody or anti-perforin antibody in TF Perm / Wash buffer and react on ice for 30 minutes. After the reaction, add TF Perm / Wash buffer, centrifuge (700 g × 2 minutes), and discard the supernatant to wash the cells twice. Use BD LSRFortessa X-20 (BD Biosciences) to quantify the signals by flow cytometry analysis. The analysis software used is Flow Jo_v10.6.1.

[1031] Method for quantifying cytokine release

[1032] After culturing T-LAK with anti-TRBC1 antibody for 24 hours, the amounts of various cytokines quantitatively released were measured using the MILLIPLEX Human Cytokine / Chemokine / Growth Factor Panel A. After washing T-LAK once with RPMI-1640 medium (culture medium) containing 10% FBS, an appropriate number of cells was counted and added to a 384-well plate. Anti-TRBC1 antibody was prepared in culture medium at final concentrations of 0, 0.1, 1.0, and 10 nM and added to the wells containing T-LAK. Incubation was carried out at 5% CO 2 , 37 °C for 24 hours. After centrifuging the 384-well plate (300 g, 5 minutes), the supernatant was recovered and IFN-γ, IL-2, IL-6, IL-10, and TNF-α were quantified using the MILLIPLEX Human Cytokine / Chemokine / Growth Factor Panel A. The quantification values were automatically calculated in the Luminex (registered trademark) 200xPONET (registered trademark) 3.1 system.

[1033] Results

[1034] TRBC1 positive cell rate

[1035] Figure 4A Figures A and B show an increase in the TRBC1 positive cell rate after addition of anti-TRBC1 antibody. Relative to human PBMC, when 10 nM of anti-TRBC1 antibody was added and cultured in the presence of IL-2, the proportion of TRBC1 positive cells, which was 30% on Day 0, increased to more than 80% on Day 15. It is suggested that addition of anti-TRBC1 antibody selectively proliferates TRBC1 positive cells. It is considered that even addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody used in this experiment will similarly selectively proliferate TRBC1 positive cells.

[1036] Increase in cytotoxic T cells

[1037] As Figure 5A shown in Figures A and B, using T-LAK on Day 0 and Day 15, staining with CD27 and CD45RA, the cell ratios of the effector memory, central memory, Naive, and EMRA fraction components were calculated. As a result, on Day 15, the proportion of effector memory increased significantly from approximately 20% to approximately 70%, and the proportions of the other fractions decreased. From this result, it is considered that stimulation with anti-TRBC1 antibody can increase cytotoxic T cells. It is considered that even stimulation by a bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody used in this experiment will similarly increase cytotoxic T cells.

[1038] Evaluation of T cell activation markers

[1039] CD69 is a cell surface glycoprotein that is expressed at a very early stage after the activation of T cells or B cells, but not in resting lymphocytes. Cytotoxic T cells have intracellular granules containing cytotoxic proteins (granzyme B, perforin). Granzyme B is a serine protease that, once delivered into the target cell, cleaves various intracellular substrates containing caspases, inducing apoptosis in the target cell. Perforin disrupts the cell membrane of the target cell, facilitating the delivery of granzyme B into the target cell.

[1040] Figure 6 shows the results of analyzing the positive cell ratios of CD25, CD69, granzyme B, and perforin using T-LAK on Day 0 and Day 15.

[1041] In this experiment, the positive cell ratio of CD25 decreased, but the positive cell ratios of CD69, granzyme B, and perforin increased, suggesting that TRBC1-positive cells were activated by the anti-TRBC1 antibody. The same effect is also expected for bispecific antibodies having the same antigen-binding portion as the anti-TRBC1 antibody used in this experiment. In this experiment, it is speculated that the addition of IL-2 during culture caused internalization and downregulation of CD25, resulting in a decrease in CD25 expression.

[1042] Change in the ratio of PD-1-positive cells

[1043] Figure 7 shows the results of analyzing the ratio of PD-1-positive cells, one of the immune checkpoint molecules of T cells, using T-LAK on Day 0 and Day 15. In the effector memory (EM) cells of functional cytotoxic T cells, the expression of immune checkpoint molecules such as PD-1 and TIGIT was hardly observed, whereas high expression of the above immune checkpoint molecules was observed in exhausted T cells that do not function properly. According to the results of Figure 7, the ratio of PD-1-positive cells did not change significantly. This suggests that even when T cells are activated with an anti-TRBC1 antibody, the T cells are not easily exhausted. The same is also considered to be true for bispecific antibodies having the same antigen-binding portion as the anti-TRBC1 antibody used in this experiment, that is, they are not likely to cause T cell exhaustion.

[1044] Quantification of cytokine release

[1045] Figure 8Quantitative results of the release amounts of IFN-γ, IL-2, IL-6, IL-10, and TNFα after 24-hour stimulation with T-LAK on Day 15 using anti-TRBC1 antibodies at 0.1, 1.0, or 10 nM are shown. As a result, an increase in the release amount dependent on the antibody concentration was confirmed for any of the cytokines, and it was considered that T cells were induced to be activated and release cytokines by the anti-TRBC1 antibody. In this experimental system, the release amount of the inflammatory cytokine IL-6 was extremely small. These data suggest the possibility of a low risk of cytokine release syndrome caused by the anti-TRBC1 antibody. It is suggested that the risk of cytokine release syndrome is also equally low for bispecific antibodies having the same antigen-binding portion as the anti-TRBC1 antibody used in this example.

[1046] <Reference Example 2> Evaluation of the agonist activity of anti-TRBC2 antibody

[1047] The anti-TRBC2 antibody was prepared and purified by the method described in Example 1. The heavy-chain and light-chain sequences of the anti-TRBC2 antibody are SEQ ID NO: 6 and 7. The reagents and staining antibodies used in this reference example are the same as those described in Reference Example 1.

[1048] Results

[1049] Number of TRBC2-positive cells

[1050] Human PBMCs were dissolved and washed in a 37°C water bath. The washed human PBMCs were reacted with JOVI-1mIgG (SantaCruz) at 4°C for 30 minutes. The PBMCs reacted with the antibody were suspended in a medium after washing and reacted with Dynabeads M-280 anti-mouse IgG (Invitrogen) at 4°C for 30 minutes. Using DynaMag (Invitrogen), the bead-unwanted cell conjugates were aggregated on the tube wall, and the supernatant containing TRBC2-positive T cells was recovered. The recovered cells were cultured for 2 days in RPMI 1640 medium containing 10% FBS supplemented with 10 nM of the anti-TRBC2 antibody and 100 IU / mL of IL-2, and then further cultured (up to 17 days) in RPMI 1640 medium containing IL-2 and 10% FBS to be induced into T-LAK. Subculture was performed every 2 to 3 days, and 100 IU / mL of IL-2 was newly added when the culture medium was changed.

[1051] Using an automatic cell counter (BIO RAD, TC20), the cell numbers on the culture start day (Day 0) and the 15th day (Day 15) were measured.

[1052] [Table 6]

[1053]

[1054] On Day0, the number of cells, approximately 5×10 6 ~7×10 6 increased to approximately 18×10 6 ~42×10 6 cells( Figure 9 ). It is suggested that the addition of anti-TRBC2 antibody selectively proliferates TRBC2-positive cells. It is considered that the addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC2 antibody used in this experiment also selectively proliferates TRBC2-positive cells.

[1055] Increase in cytotoxic T cells

[1056] As Figure 10 shown, T-LAK sensitized with anti-TRBC2 antibody on Day0 and Day15 was stained with CD27 and CD45RA, and the cell ratios of the fractional components of effector memory (EM), central memory (CM), Naive, and EMRA were calculated. As a result, at Day15, the proportion of effector memory increased significantly from approximately 27% to approximately 81%, and the proportions of other fractions decreased. From this result, it is considered that the addition of anti-TRBC2 antibody can increase cytotoxic T cells. It is considered that the addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC2 antibody used in this experiment also increases cytotoxic T cells. It should be noted that the cell population that is CD3-positive and TRBC1-negative is defined as TRBC2-positive cells.

[1057] Evaluation of T cell activation markers

[1058] Figure 11 shows the results of analyzing the positive cell ratios of CD25, CD69, granzyme B, and perforin, respectively, in T-LAK sensitized with anti-TRBC2 antibody on Day0 and Day15. In this experiment, the CD25-positive cell ratio decreased, but the CD69, granzyme B, and perforin-positive cell ratios increased, suggesting that TRBC2-positive cells were activated by anti-TRBC2 antibody. The same effect can also be expected for a bispecific antibody having the same antigen-binding portion as the anti-TRBC2 antibody used in this experiment. In this experiment, it is speculated that due to the addition of IL-2 during culture, internalization and downregulation of CD25 occurred, resulting in a decrease in CD25 expression. It should be noted that the cell population that is CD3-positive and TRBC1-negative is defined as TRBC2-positive cells.

[1059] Change in the ratio of PD-1-positive cells

[1060] Figure 12T-LAKs were sensitized with anti-TRBC2 antibodies on Day0 and Day15, and the results of analyzing the ratio of PD-1 positive cells, which is one of the immunosuppressive checkpoint molecules of T cells, were obtained. In the effector memory (EM) cells of functional cytotoxic T cells, the expression of immunosuppressive checkpoint molecules such as PD-1 and TIGIT was hardly observed. In contrast, high expression of the above immunosuppressive checkpoint molecules was observed in exhausted T cells that do not function properly. According to Figure 12 the results, the ratio of PD-1 positive cells did not change significantly. This suggests that even when T cells are activated with anti-TRBC2 antibodies in the same way as anti-TRBC1 antibodies, T cells are not easily exhausted. The same is thought to be true for bispecific antibodies having the same antigen-binding portion as the anti-TRBC2 antibody used in this experiment.

[1061] Quantification of cytokine release

[1062] Figure 13 The quantitative results of the release amounts of IFN-γ, IL-2, IL-6, IL-10, and TNFα after 24-hour stimulation with 0.1, 1.0, or 10 nM anti-TRBC2 antibody using T-LAKs on Day15 are shown. As a result, an increase in the release amount dependent on the antibody concentration was confirmed for any of the cytokines, and it was considered that T cells were induced to be activated and release cytokines by the anti-TRBC2 antibody. In this experimental system, the release amount of the inflammatory cytokine IL-6 was extremely small. These data suggest the possibility of a low risk of cytokine release syndrome caused by the anti-TRBC2 antibody. It is suggested that the risk of cytokine release syndrome is also low for bispecific antibodies having the same antigen-binding portion as the anti-TRBC2 antibody used in this experiment.

[1063] <Example 3> Cytotoxic Activity of Anti-CCR4 / anti-TRBC1 Bispecific Antibody

[1064] In this example, for the in vitro efficacy evaluation of the bispecific antibody prepared in Example 1, after culturing target cells with T-LAKs (effector cells) in the presence of specific antibodies, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index, and the effector cell-dependent cytotoxic activity was evaluated. The experiment was carried out with reference to Neri S et al. (Clin Diagn Lab Immunol. 2001 Nov; 8(6): 1131-5; the entire description is hereby incorporated by reference in its entirety).

[1065] The following reagents were used.

[1066] HBSS (Hank’s Balanced Salt Solution) without phenol red (Gibco, 14025092)

[1067] RPMI 1640 Medium, without phenol red (Gibco, 11835-030)

[1068] Probenecid water-soluble (Invitrogen, P36400)

[1069] Calcein-AM (invitrogen, C1430)

[1070] Lysis buffer 10X (Promega, G1821)

[1071] 3.1 Cytotoxic activity of bispecific antibody against MT-2 cells

[1072] Method

[1073] Using RPMI phenol-free medium containing 5 mM probenecid and 0.5% FBS (assay medium), the bispecific antibody was prepared at a final concentration of 100 nM to 100 fM and added to a 96-well round-bottom culture plate. As a control, wells containing only the assay medium were set up.

[1074] For the cytotoxic activity assay using a bispecific antibody with a CCR4-binding moiety, the required amount of MT-2 cells (target cells) was recovered from the culture medium and washed twice with HBSS. The target cells were labeled with calcein using 10 μM calcein-AM supplemented with 5 mM probenecid and incubated at 37 °C for 30 minutes. After incubation, the cells were washed three times with HBSS and, after the final wash, suspended in the assay medium. After counting the number of labeled target cells, they were added to a 96-well round-bottom culture plate pre-coated with the bispecific antibody. After recovering the required amount of effector cells from the culture medium, they were washed once with the assay medium, counted, and then added to the 96-well round-bottom culture plate pre-coated with the bispecific antibody. The ratio of effector cells to labeled target cells was added at 10:1. The plate containing the bispecific antibody, labeled target cells, and effector cells was reacted at 5% CO 2 , 37 °C for 2 hours. The 96-well round-bottom culture plate was centrifuged (300 g, 5 minutes), and the supernatant was recovered. The fluorescence of calcein released from the labeled target cells contained in the supernatant was measured using a microplate reader (Bio Tek, SYNERGY H1). The percentage of cytotoxicity of the target cells was calculated using the fluorescence value of calcein as an index, and the cytotoxic activity dependent on the bispecific antibody was evaluated.

[1075] The percentage of cytotoxicity was determined as follows.

[1076] The minimum fluorescence value of lysed target cells (lysis rate = 0%) is obtained from wells in which labeled target cells and effector cells were incubated in the absence of any bispecific antibody. The maximum fluorescence value of lysed target cells (lysis rate = 100%) is obtained by adding 10× lysis buffer to wells under the same conditions as for the minimum fluorescence value.

[1077] The percentage of cytotoxicity is calculated using the following formula:

[1078] Percentage of cytotoxicity = [(fluorescence value of lysed target cells in the presence of bispecific antibody) - (minimum fluorescence value of lysed target cells)] / [(maximum fluorescence value of lysed target cells) - (minimum fluorescence value of lysed target cells)].

[1079] The S-shaped dose-response curve is calculated using Prism software (GraphPad Software Inc.), and the EC50 value (50% effective concentration) and Emax value (maximum activity value) are calculated.

[1080] Result

[1081] Figure 14A And the following table shows the results of adding T-LAK to MT-2 cells, a cell line from ATL expressing CCR4, and measuring the cytotoxic activity of bispecific antibodies CCR0001 and CCR0004.

[1082] The EC50 value and Emax value (N = 3) are shown in the following table.

[1083] [Table 7]

[1084] Antibody name EC50 (pM) Standard deviation CCR0001 44.4 22.8 CCR0004 262.7 95.8

[1085] Antibody name Emax (%) Standard deviation CCR0001 106.9 12.4 CCR0004 106.5 13.3

[1086] It is considered that the bispecific antibodies CCR0001 and CCR0004 against CCR4 and TRBC1 show very strong cytotoxic activity against CCR4-expressing cells.

[1087] 3.2 Cytotoxic activity of negative control antibodies against MT-2 cells

[1088] An anti-CCR4 antibody, an anti-TRBC1 antibody, and their combination (anti-CCR4 antibody + anti-TRBC1 antibody) were used as negative control antibodies, and the cytotoxic activity was measured by the above method.

[1089] The anti-CCR4 antibody used in this example was mogamulizumab. The anti-TRBC1 antibody was prepared and purified by the method described in Example 1. The heavy chain and light chain sequences of the anti-TRBC1 antibody were SEQ ID NO: 4 and 5.

[1090] Figure 14B The results of adding T-LAK to the MT-2 cell line, a cell line derived from ATL that expresses CCR4, and measuring the cytotoxic activity of a negative control antibody are shown in the table below.

[1091] The EC50 values and Emax values (N = 3) are shown in the table below.

[1092] [Table 8]

[1093]

[1094]

[1095] ＊ For the anti-CCR4 antibody, the value could not be calculated in 1 out of 3 experiments, so the average value of the 2 experiments is shown. NC: Not calculated

[1096] T-LAK was added to the MT-2 cell line, a cell line derived from ATL that expresses CCR4, and then either anti-CCR4 antibody alone, anti-TRBC1 antibody alone, or both anti-CCR4 antibody and anti-TRBC1 antibody were added, and the cytotoxic activity was measured. As a result, the EC50 values and Emax were significantly lower than those when adding the anti-CCR4 / anti-TRBC1 bispecific antibodies CCR0001 and CCR0004. The anti-CCR4 antibody did not exhibit sufficient cytotoxic activity in this experimental system. The anti-TRBC1 antibody did not show sufficient cytotoxic activity.

[1097] 3.3 Cytotoxic activity of bispecific antibodies against negative control cells

[1098] Instead of MT-2 cells, the MOLT-4 cell line, a cell line derived from a T cell tumor that does not express CCR4, was used as the negative control cells, and the cytotoxic activity of the bispecific antibodies CCR0001 and CCR0004 was measured. The MOLT-4 cells were labeled with calcein-AM and suspended in the assay medium by the same method as the above MT-2 cells.

[1099] Figure 14C The results of adding T-LAK to the negative control cells, MOLT-4 cells, and measuring the cytotoxic activity of CCR0001 and CCR0004 are shown in the table below.

[1100] The EC50 values and Emax values (N = 3) are shown in the table below.

[1101] [Table 9]

[1102] Antibody name EC50 (pM) Standard deviation CCR0001 NC NC CCR0004 3348.4* NC

[1103] Antibody name Emax (%) Standard deviation CCR0001 17.2* NC CCR0004 12.5* NC

[1104] ＊ In one of the three experiments, the value could not be calculated, so the average value of the two experiments is recorded and the SD is not calculated. NC: Not calculated

[1105] For the T cell tumor line MOLT-4 cells that do not express CCR4, the EC50 value and Emax are both significantly lower in cytotoxic activity compared to CCR4-expressing cells. This result shows that the bispecific antibodies CCR0001 and CCR0004 are antibodies that specifically act on cells expressing CCR4.

[1106] <Example 4> Cytotoxic Activity of Anti-CD1a / Anti-TRBC1 Bispecific Antibody

[1107] In this example, for the in vitro efficacy evaluation of the anti-CD1a / anti-TRBC1 bispecific antibody prepared in Example 1, the target cells CCRF-CEM9 were cultured with T-LAK (effector cells) sensitized with anti-TRBC1 antibody in the presence of the specific antibody, and then the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate the effector cell-dependent cytotoxic activity.

[1108] The target cells CCRF-CEM9 express CD1a and TRBC2, and as described in Example 2, they were obtained by monoclonalization of the cell line CCRF-CEM from TALL.

[1109] The measurement of cytotoxic activity was carried out by the same method as described in Example 3.

[1110] Result

[1111] Figure 15 The following table shows the results of adding T-LAK to the cells of the TALL-derived cell line CCRF-CEM9 expressing CD1a and measuring the cytotoxic activity of the bispecific antibodies CD1a1001 and CD1a1004.

[1112] The EC50 value and Emax value (N = 3) are shown in the following table.

[1113] [Table 10]

[1114] Antibody name EC50 (pM) Standard deviation CD1a1001 2.9 1.2 CD1a1004 2.9 0.8

[1115] Antibody name Emax (%) Standard deviation CD1a1001 94.2 16.9 CD1a1004 77.0 18.7

[1116] T-LAK was added to CCRF-CEM9 cells, and the cytotoxic activities of CD1a1001 and CD1a1004 were measured. As a result, the EC50 values were both 2.9 pM, and the Emax values were 94.2% and 77.0%, respectively. Based on this result, it was considered that the bispecific antibody against CD1a and anti-TRBC1 showed very strong cytotoxic activity against cells expressing CD1a and TRBC2.

[1117] <Example 5> Cytotoxic Activity of Anti-CCR4 / Anti-TRBC2 Bispecific Antibody

[1118] In this example, for the in vitro efficacy evaluation of the anti-CCR4 / anti-TRBC2 bispecific antibody prepared in Example 1, the target cell MT-2 was cultured with anti-TRBC2 antibody-sensitized T-LAK cells (effector cells) in the presence of the bispecific antibody, and then the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index to evaluate the effector cell-dependent cytotoxic activity.

[1119] Results

[1120] Figure 16 These are the results of adding anti-TRBC2 antibody-sensitized T-LAK to the MT-2 cells, a cell line from ATL that expresses CCR4, and measuring the cytotoxic activity of CCR0010. The EC50 values and Emax values (N = 3) are shown in the following table.

[1121] [Table 11]

[1122] Antibody name EC50 (pM) Standard deviation CCR0010 1677.0 1063.6

[1123] Antibody name Emax (%) Standard deviation CCR0010 81.4 13.4

[1124] Anti-TRBC2 antibody-sensitized T-LAK was added to MT-2 cells, and the cytotoxic activity of CCR0010 was measured. As a result, the bispecific antibody CCR0010 that recognizes CCR4 and TRBC2 showed an activity of EC50 1677.0 pM and Emax 81.4% against CCR4-expressing cells. Therefore, it was considered that the bispecific antibody that recognizes CCR4 and TRBC2 showed cytotoxic activity against the ATL cell line expressing CCR4 in the same way as the bispecific antibody that recognizes CCR4 and TRBC1.

[1125] <Example 6> Cytotoxic Activity of Anti-CD1a / Anti-TRBC2 Bispecific Antibody

[1126] In this example, for the in vitro efficacy evaluation of the anti-CD1a / anti-TRBC2 bispecific antibody prepared in Example 1, the target cells JM were cultured with anti-TRBC2 antibody-sensitized T-LAK (effector cells, prepared by the method described in Reference Example 2) in the presence of the bispecific antibody, and then the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index to evaluate the effector cell-dependent cytotoxic activity. The measurement of the cytotoxic activity was carried out by the same method as described in Example 3. The target cells JM are a cell line derived from TALL that expresses CD1a and TRBC1. The JM cells were purchased from the RIKEN Bio Research Center (RCB0537). Using RPMI medium containing 10% FBS, they were cultured in a 5% CO 2 2, 37 °C incubator and passaged 2 - 3 times a week.

[1127] Results

[1128] Figure 17 These are the results of measuring the cytotoxic activities of the bispecific antibodies CD1a1003 and CD1a1006 after adding anti-TRBC2 antibody-sensitized T-LAK to the JM cells, which are a cell line derived from TALL that expresses CD1a.

[1129] The EC50 values and Emax values (N = 3) are shown in the following table.

[1130] [Table 12]

[1131] Antibody name EC50 (pM) Standard deviation CD1a1003 22.5 12.2 CD1a1006 12.9 7.6

[1132] Antibody name Emax (%) Standard deviation CD1a1003 90.1 9.6 CD1a1006 70.1 6.5

[1133] T-LAK was added to the JM cells and the cytotoxic activities of CD1a1003 and CD1a1006 were measured. As a result, the EC50 values were 22.5 pM and 12.9 pM respectively, and the Emax values were 90.1% and 70.1% respectively. Based on these results, it is considered that the bispecific antibody of anti-CD1a antibody and anti-TRBC2 shows very strong cytotoxic activity against cells expressing CD1a and TRBC1.

[1134] <Example 7> Cytotoxic Activity of Anti-CCR9 / anti-TRBC1 Bispecific Antibody

[1135] In this example, for the in vitro efficacy evaluation of the anti-CCR9 / anti-TRBC1 bispecific antibody prepared in Example 1, after culturing the target cells CCRF-CEM9 with T-LAK (effector cells) in the presence of specific antibodies, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index, and the cytotoxic activity dependent on effector cells was evaluated. The target cells CCRF-CEM9 express CCR9 and TRBC2, and as described in Example 2, they were obtained by monoclonalization of the cell line CCRF-CEM from TALL. The measurement of cytotoxic activity was carried out by the same method as described in Example 3.

[1136] Results

[1137] Figure 18 These are the results of adding T-LAK to CCRF-CEM9 cells from TALL that express CCR9 and measuring the cytotoxic activities of the bispecific antibodies CCR9-1002 and CCR9-1004.

[1138] The EC50 values and Emax values (N = 3) are shown in the following table.

[1139] [Table 13]

[1140] Antibody name EC50 (pM) Standard deviation CCR9-1002 29.1 9.3 CCR9-1004 6.6 3.1

[1141] Antibody name Emax (%) Standard deviation CCR9-1002 85.9 7.4 CCR9-1004 94.5 6.7

[1142] T-LAK was added to CCRF-CEM9 cells and the cytotoxic activities of CCR9-1002 and CCR9-1004 were measured. As a result, the EC50 values were 29.1 pM and 6.6 pM respectively, and the Emax values were 85.9% and 94.5% respectively. Based on these results, it is considered that the bispecific antibody against anti-CCR9 antibody and anti-TRBC1 shows very strong cytotoxic activity against cells expressing CCR9 and TRBC2.

[1143] <Example 8> Cytotoxic Activity of Anti-CXCR4 / anti-TRBC1 Bispecific Antibody

[1144] In this example, for the in vitro efficacy evaluation of the anti-CXCR4 / anti-TRBC1 bispecific antibody prepared in Example 1, the target cells CCRF-CEM9 were cultured with T-LAK (effector cells) in the presence of the specific antibody, and then, using a fluorescent dye (calcein activity) as an indicator, the cell viability of the target cells was measured to evaluate the effector cell-dependent cytotoxic activity. The target cells CCRF-CEM9 express CXCR4 and TRBC2, and as described in Example 2, they were obtained by monoclonalization of the cell line CCRF-CEM from TALL. The measurement of cytotoxic activity was carried out by the same method as described in Example 3.

[1145] Results

[1146] Figure 19 These are the results of adding T-LAK to the CCRF-CEM9 cells from TALL that express CXCR4 and measuring the cytotoxic activities of the bispecific antibodies CXCR4-1002 and CXCR4-1004.

[1147] The EC50 values and Emax values (N = 3) are shown in the following table.

[1148] [Table 14]

[1149] Antibody name EC50 (pM) Standard deviation CXCR4-1002 0.31 0.28 CXCR4-1004 0.73 0.46

[1150] Antibody name Emax (%) Standard deviation CXCR4-1002 88.3 7.8 CXCR4-1004 95.6 8.4

[1151] T-LAK was added to CCRF-CEM9 cells and the cytotoxic activities of CXCR4-1002 and CXCR4-1004 were measured. As a result, the EC50 values were 0.31 pM and 0.73 pM, respectively, and the Emax values were 88.3% and 95.6%, respectively. Based on these results, it is considered that the anti-CXCR4 / anti-TRBC1 bispecific antibody shows very strong cytotoxic activity against cells expressing CXCR4 and TRBC2.

[1152] <Example 9> Cytotoxic Activity of Anti-CCR8 / anti-TRBC1 Bispecific Antibody

[1153] In this example, for the in vitro efficacy evaluation of the anti-CCR8 / anti-TRBC1 bispecific antibody prepared in Example 1, after culturing target cells with T-LAK cells (effector cells) in the presence of a specific antibody, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator, and the effector cell-dependent cytotoxic activity was evaluated. The target cell MT-1 is a cell line derived from ATL that expresses CCR8. The cytotoxic activity was measured by the same method as described in Example 3. The MT-1 cells were purchased from the JCRB cell bank (National Institute of Biomedical Innovation, Health and Nutrition) (JCRB1209). Using RPMI medium containing 10% FBS, they were cultured in a 5% CO 2 2, 37 °C incubator and passaged 2 - 3 times a week.

[1154] Result

[1155] Figure 20 It shows the results of adding T-LAK to MT-1 cells, a cell line derived from ATL that expresses CCR8, and measuring the cytotoxic activities of CCR81001 and CCR81002. The EC50 values and Emax values (N = 3) are shown in the following table.

[1156] [Table 15]

[1157] Antibody name EC50 (pM) Standard deviation CCR81001 23.5 2.2 CCR81002 31.4 5.0

[1158] Antibody name Emax (%) Standard deviation CCR81001 89.9 0.6 CCR81002 97.1 1.7

[1159] T-LAK was added to MT-1 cells, and the cytotoxic activities of CCR81001 and CCR81002 were measured. As a result, the EC50 values were 23.5 pM and 31.4 pM respectively, and the Emax values were 89.9% and 97.1% respectively. Based on this result, it is considered that the bispecific antibodies CCR81001 and CCR81002 that recognize CCR8 and TRBC1 show very strong cytotoxic activity against CCR8-expressing cells.

[1160] <Example 10> Cytotoxic Activity of Anti-TIGIT / anti-TRBC1 Bispecific Antibody

[1161] In this example, for the in vitro efficacy evaluation of the anti-TIGIT / anti-TRBC1 bispecific antibody prepared in Example 1, after culturing target cells with T-LAK cells (effector cells) in the presence of specific antibodies, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator, and the effector cell-dependent cytotoxic activity was evaluated. The target cell ILT-Mat is a cell line derived from ATL that expresses TIGIT. The cytotoxic activity was measured by the same method as described in Example 3. The ILT-Mat cells were purchased from the RIKEN Bio Research Center (RCB0475). Using RPMI medium containing 320 IU / mL of IL-2 and 10% FBS, they were cultured in a 5% CO 2 2, 37 °C incubator and passaged 2 - 3 times a week.

[1162] Results

[1163] Figure 21 The results are those of adding T-LAK to the ILT-Mat cells, a cell line derived from ATL that expresses TIGIT, and measuring the cytotoxic activities of TIGIT2001 and TIGIT2002. The EC50 values and Emax values (N = 3) are shown in the following table.

[1164] [Table 16]

[1165] Antibody name EC50 (pM) Standard deviation TIGIT2001 3.4 0.5 TIGIT2002 5.6 0.3

[1166] Antibody name Emax (%) Standard deviation TIGIT2001 105.1 6.8 TIGIT2002 91.9 6.4

[1167] T-LAK was added to ILT-Mat cells and the cytotoxic activities of TIGIT2001 and TIGIT2002 were measured. As a result, the EC50 values were 3.4 pM and 5.6 pM respectively, and the Emax values were 105.1% and 91.9% respectively. Based on these results, it is considered that the bispecific antibodies TIGIT2001 and TIGIT2002 that recognize TIGIT and TRBC1 show very strong cytotoxic activity against TIGIT-expressing cells.

[1168] <Example 11> Cytotoxic Activity of Anti-CD30 / anti-TRBC1 Bispecific Antibody

[1169] In this example, for the in vitro efficacy evaluation of the anti-CD30 / anti-TRBC1 bispecific antibody prepared in Example 1, after culturing target cells with T-LAK cells (effector cells) in the presence of a specific antibody, using a fluorescent pigment (calcein activity) as an index, the cell viability of the target cells was measured, and the effector cell-dependent cytotoxic activity was evaluated. The target cell DL40 is a cell line derived from PTCL that expresses CD30. The measurement of cytotoxic activity was carried out by the same method as described in Example 3. The DL40 cells were purchased from the JCRB cell bank (National Institute of Biomedical Innovation, Health and Nutrition, Japan) (JCRB1337). Using RPMI medium containing 10% FBS, it was cultured in a 5% CO 2 2, 37 °C incubator, and passaged 2 - 3 times a week.

[1170] Result

[1171] Figure 22 The results show the cytotoxic activities of CD301001 and CD301003 by adding T-LAK to DL40 cells, which are cell lines derived from PTCL that express CD30. The EC50 values and Emax values (N = 3) are shown in the following table.

[1172] [Table 17]

[1173] Antibody name EC50 (pM) Standard deviation CD301001 151.1 95.4 CD301003 14.5 5.8

[1174] Antibody name Emax (%) Standard deviation CD301001 89.1 12.6 CD301003 95.7 2.2

[1175] T-LAK was added to DL40 cells, and the cytotoxic activities of CD301001 and CD301003 were measured. As a result, the EC50 values were 151.1 pM and 14.5 pM respectively, and the Emax values were 89.1% and 95.7% respectively. Based on this result, it is considered that the bispecific antibodies CD301001 and CD301003 of anti-CD30 antibody and anti-TRBC1 show very strong cytotoxic activity against CD30-expressing cells.

[1176] <Example 12> Cytotoxic Activity of Anti-CD40L / anti-TRBC1 Bispecific Antibody

[1177] In this example, for the in vitro efficacy evaluation of the anti-CD40L / anti-TRBC1 bispecific antibody prepared in Example 1, after culturing target cells with T-LAK cells (effector cells) in the presence of specific antibodies, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index, and the effector cell-dependent cytotoxic activity was evaluated. The target cell MOLT3 is a cell line from TALL that expresses CD40L (CD154). The measurement of cytotoxic activity was carried out by the same method as described in Example 3. The MOLT3 cells were purchased from the RIKEN Bio Research Center (RCB9048). Using RPMI medium containing 10% FBS, they were cultured in a 5% CO 2 , 37 °C incubator and passaged 2 - 3 times a week.

[1178] Result

[1179] Figure 23 are the results of adding T-LAK to MOLT3 cells, a cell line from TALL that expresses CD40L (CD154), and measuring the cytotoxic activities of CD40L1007 and CD40L1008. The EC50 values and Emax values (N = 3) are shown in the following table.

[1180] [Table 18]

[1181] Antibody Name EC50 (pM) Standard Deviation CD40L1007 125.0 8.2 CD40L1008 49.1 7.7

[1182] Antibody Name Emax (%) Standard Deviation CD40L1007 67.7 1.7 CD40L1008 72.7 2.2

[1183] T-LAK was added to MOLT3 cells and the cytotoxic activities of CD40L1007 and CD40L1008 were measured. As a result, the EC50 values were 125.0 pM and 49.1 pM respectively, and the Emax values were 67.7% and 72.7% respectively. Based on this result, it is considered that the bispecific antibodies CD40L1007 and CD40L1008 of anti-CD40L antibody and anti-TRBC1 show very strong cytotoxic activity against CD40L (CD154)-expressing cells.

[1184] <Example 13> Comparison of anti-CCR4 / anti-TRBC1 bispecific antibody with mogamulizumab

[1185] In this example, the in vitro efficacy of the anti-CCR4 / anti-TRBC1 bispecific antibody CCR0001 and the anti-CCR4 monoclonal antibody mogamulizumab prepared in Example 1 was evaluated using MOLT4 cells that forcibly express CCR4. The MOLT4 cells were purchased from the JCRB Cell Bank (National Institute of Biomedical Innovation, Health and Nutrition, Japan) (JCRB9031). They were cultured in RPMI1640 medium containing 10% FBS in a 5% CO 2 , 37 °C incubator, and passaged 2 - 3 times a week. The target cells, CCR4-expressing MOLT4 cells, were prepared by introducing a CCR4 expression plasmid into MOLT4 cells that do not express CCR4 using electroporation. Four types of cells with different expression levels were established and used for the evaluation.

[1186] Evaluation of anti-CCR4 / anti-TRBC1 bispecific antibody

[1187] After culturing the target cells, CCR4-expressing MOLT4 cells, with T-LAK (effector cells) in the presence of the bispecific antibody CCR0001, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator, and the effector cell-dependent cytotoxic activity was evaluated.

[1188] Evaluation of anti-CCR4 monoclonal antibody mogamulizumab

[1189] After culturing the target cells, CCR4-expressing MOLT4 cells, with NK cells (effector cells) isolated from normal human peripheral blood in the presence of mogamulizumab, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator, and the effector cell-dependent cytotoxic activity was evaluated.

[1190] Results

[1191] Figure 24 The results are those of adding T-LAK or NK cells to CCR4-expressing MOLT4 cells and measuring the cytotoxic activities of the anti-TRBC1 bispecific antibody CCR0001 and mogamulizumab. The cytotoxic activity of CCR0001 was higher than that of mogamulizumab for any of the four types of CCR4-expressing MOLT4 cells, suggesting that the anti-CCR4 and anti-TRBC1 bispecific antibody has higher efficacy compared to the existing drug mogamulizumab.

[1192] <Example 14> Evaluation of the cytotoxic activity of a bispecific antibody using an anti-CD3 antibody in T cell tumors

[1193] CD3 is expressed identically in both normal T cells and tumor T cells. Bispecific antibodies targeting CD3 and tumor antigens bind simultaneously to the CD3 antigen expressed in T cell malignancies and the target antigen expressed in T cell malignancies, linking malignant tumor cells to each other ( Figure 1A ), and thus it is likely that the therapeutic effect of the bispecific antibody cannot be fully obtained.

[1194] In this example, the cytotoxic activities of bispecific antibodies using anti-CD3 antibodies against tumor cell lines expressing CD3 and tumor cell lines with CD3 knocked out were evaluated. Further, the same experiment was conducted using a bispecific antibody using an anti-TRBC1 antibody.

[1195] By the method described in Example 1, the anti-CD1a / anti-CD3 bispecific antibody CD1a1016 and the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005 were prepared. The CD1a-binding portions of CD1a1016 and CD1a1005 contain the same amino acid sequence. The CD3-binding portion of CD1a1016 was designed based on the anti-CD3 antibody described in U.S. Patent No. 5,821,337 (the entire description of which is hereby incorporated by reference in its entirety).

[1196] [Table 19]

[1197]

[1198] After culturing the target cells HPB-ALL and HPB-ALL CD3 KO cells with T-LAK (effector cells) in the presence of each bispecific antibody, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index, and the effector cell-dependent cytotoxic activity was evaluated. The target cell HPB-ALL expresses CD1a, CD3, and TRBC2. The target cell HPB-ALL CD3KO cell line was obtained by knocking out the CD3 gene of the cell line HPB-ALL from TALL using the CRSPR-Cas9 method. The measurement of cytotoxic activity was carried out by the same method as described in Example 3. The target cell HPB-ALL was purchased from the RIKEN Bio Research Center (RCB1935). Using RPMI1640 medium containing 10% FBS, it was cultured in a 5% CO 2 , 37 °C incubator, and passaged 2 - 3 times per week.

[1199] Results

[1200] Figure 25The results are from adding T-LAK to the TALL-derived cell line HPB-ALL cells expressing CD3 and HPB-ALL CD3 KO cells with CD3 knocked out, and measuring the cytotoxic activities of the anti-CD1a / anti-CD3 bispecific antibody CD1a1016 and the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005.

[1201] The EC50 values and Emax values (N = 3) are shown in the following table.

[1202] [Table 20]

[1203] CD1a1016

[1204] Target Cell Name EC50 (pM) Standard Deviation HPB-ALL 9.7 3.9 HPB-ALL CD3 KO 1.0 0.5

[1205] Target Cell Name Emax (%) Standard Deviation HPB-ALL 71.9 13.3 HPB-ALL CD3 KO 85.3 13.1

[1206] CD1a1005

[1207] Target Cell Name EC50 (pM) Standard Deviation HPB-ALL 3.6 1.9 HPB-ALL CD3 KO 5.3 3.0

[1208] Target Cell Name Emax (%) Standard Deviation HPB-ALL 76.5 11.9 HPB-ALL CD3 KO 74.5 13.1

[1209] When CD3 is expressed in the target cells compared to when it is not expressed, the EC50 of the anti-CD1a / anti-CD3 bispecific antibody CD1a1016 is increased by about 10-fold and the Emax is also decreased. Based on this result, it is considered that when CD3 is expressed on the target cell side, the cytotoxic activity of the bispecific antibody using an anti-CD3 antibody on the effector cell side is weakened.

[1210] There is no significant difference in the EC50 and Emax of the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005 depending on the target cells.

[1211] <Example 15> Cytotoxic Activity of Bispecific Antibodies Using Anti-TRBC2 Antibodies against TRBC1-Expressing Tumor Cells and TRBC2-Expressing Tumor Cells

[1212] In this example, the cytotoxic activities of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 prepared in Example 1 against TRBC1-positive tumor cell lines and TRBC2-positive tumor cell lines were compared.

[1213] The target cells CCRF-CEM9 cells and JM cells were cultured with anti-TRBC2 antibody-sensitized T-LAK (effector cells) in the presence of bispecific antibody, and the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index to evaluate the effector cell-dependent cytotoxic activity. The target cell CCRF-CEM9 cells are a cell line derived from TALL that expresses CD1a and TRBC2. The target cell JM cells are a cell line derived from TALL that expresses CD1a and TRBC1. The measurement of cytotoxic activity was carried out by the same method as described in Example 3.

[1214] The cytotoxic activity of the anti-CD1a / anti-TRBC1 bispecific antibody against the TRBC2-positive tumor cell line CCRF-CEM9 was evaluated in Example 4. In addition, the cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 against the TRBC1-positive tumor cell line JM was also carried out in Example 6.

[1215] Results

[1216] Figure 26 These are the results of adding anti-TRBC2 antibody-sensitized T-LAK to the TALL-derived cell line CCRF-CEM9 cells expressing TRBC2 and the TALL-derived cell line JM cells expressing TRBC1, and measuring the cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006. The EC50 values and Emax values (N = 3) are shown in the following table.

[1217] [Table 21]

[1218] Target Cell Name EC50 (pM) Standard Deviation CCRF-CEM9 28.4 2.1 JM 18.0 1.9

[1219] Target Cell Name Emax (%) Standard Deviation CCRF-CEM9 33.0 4.1 JM 72.8 3.6

[1220] For the target cells CCRF-CEM9 and JM, the EC50 values were 28.4 pM and 18.0 pM, respectively, and the Emax values were 33.0 and 72.8%, respectively. These results indicate that the anti-CD1a / anti-TRBC2 bispecific antibody shows high cytotoxic activity against CD1a- and TRBC1-expressing cells, and low cytotoxic activity against CD1a- and TRBC2-expressing cells. In addition, as shown in Example 4, when the antibody on the effector side is TRBC1, very strong cytotoxic activity against the target cell CCRF-CEM9 is shown. These results indicate that it is important that the antibody on the effector side is an antibody that recognizes a subtype not expressed in tumor cells.

[1221] <Example 16> Evaluation of Antitumor Effect in Vivo

[1222] In this example, the in vivo anti-tumor effect of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 was studied.

[1223] Preparation of cells

[1224] The JM cell line is a cell line derived from TALL and expresses CD1a and TRBC1. It was purchased from the Cell Material Development Laboratory of the RIKEN (cell number RCB1164, product number: RCB0537). JM-Luc cells that stably express the luciferase gene were used. JM-Luc cells were cultured in RPMI1640 medium containing 10% FBS in a 5% CO 2 , 37 °C incubator, and the cultured cells were used for the in vivo anti-tumor effect test.

[1225] Test animals

[1226] Animal species: Mouse

[1227] System: NOD / Shi-scid,IL-2RγKO JiC(NOG)

[1228] Supplier: CLEA Japan, Inc.

[1229] Age: 6 weeks old (at the time of receipt)

[1230] Preparation of PBMC

[1231] PBMC was purchased from Hemacare Corporation (product number: PB009C-3, Lot No.: 22074722). The frozen PBMC (1×10 8 cells / vial) was thawed in a thermostatic bath at about 37 °C. The cell suspension was collected using a pipette and slowly added dropwise to a 50 mL centrifuge tube. The empty vial was rinsed with thawing media (Hanks' balanced salt solution (-) containing 10% FBS) and slowly added dropwise to the above centrifuge tube for mixing. After centrifugation (room temperature, 400×g, 5 minutes), the supernatant was aspirated off, and then washing solution (25 mL of D-PBS containing 0.005% FBS + 100 μL of 0.5 M EDTA) was added for resuspension. It was diluted 2-fold with trypan blue to calculate the number of viable cells. After centrifugation again (room temperature, 400×g, 5 minutes), the supernatant was aspirated off, and a cell suspension of 1×10 8 cells / mL was prepared using RPMI1640 medium.

[1232] Transplantation of PBMC

[1233] The prepared PBMC suspension was transferred to the breeding room on ice. Within 2 hours after preparation, for NOG mice, the tail was disinfected with an alcohol swab. Using a 1 mL disposable syringe and a 27G injection needle (Terumo Corporation), 0.1 mL (1×10 7 cells / mouse) was transplanted into the tail vein, and this was designated as "Day10".

[1234] Preparation of JM-Luc cells

[1235] On Day0, the cultured JM-Luc cells were harvested. Centrifugation was performed (room temperature, 400×g, 5 minutes). After aspirating and removing the supernatant, the cells were washed with PBS and then centrifuged again (room temperature, 400×g, 5 minutes). The supernatant was aspirated and removed, and the cells were appropriately diluted with PBS. After trypan blue staining, cell counting was performed, and the cells were prepared at a concentration of 1×10 7 cells / mL. The prepared cells were stored on ice from suspension until transplantation.

[1236] Transplantation of JM-Luc cells

[1237] The prepared JM-Luc cell suspension was transferred to the breeding room on ice. Within 2 hours after preparation, for NOG mice, the tail was disinfected with an alcohol swab. Using a 1 mL disposable syringe and a 27G injection needle (Terumo Corporation), 0.1 mL (1×10 6 cells / mouse) was transplanted into the tail vein, and this was designated as "Day0".

[1238] Preparation of antibodies

[1239] CD1a1006 was prepared by using PBS at dosages of 0.05 mg / kg (0.01 mg / mL), 0.15 mg / kg (0.03 mg / mL), 0.5 mg / kg (0.1 mg / mL), 1.5 mg / kg (0.3 mg / mL), and 5 mg / kg (1.0 mg / mL) for each antibody.

[1240] Administration of antibodies

[1241] On Day1, 7, 10, 14, 17, 21, and 25, the tail was disinfected with an alcohol swab. Using a 1 mL disposable syringe and a 27G injection needle (Terumo Corporation), the prepared antibody solution was transplanted into the tail vein at a dose of 0.1 mL per mouse.

[1242] Analysis method for anti-tumor effect

[1243] On Days 14 and 28, for all surviving individuals, the luminescence of JM-Luc cells was measured using an IVIS imaging system (Revvity). On each IVIS measurement day, luciferin was intraperitoneally administered at 150 mg / kg (10 mL / kg). After inhalation anesthesia with isoflurane, the animals were placed in the supine position in the IVIS chamber. Approximately 5 to 20 minutes after luciferin administration, the total flux (luminescence per minute) was measured with the whole body as the region of interest (ROI). For any measurement day and any individual, the maximum value of the luminescence per minute was set as the measurement value for that individual.

[1244] Results

[1245] The IVIS measurement values for each group on Days 14 and 28 are shown in Figure 27 , and the tumor distribution images are shown in Figure 28 . The IVIS measurement value for Group 1 on Day 14 was 56.34 ± 68.05×10 5 p / s. In contrast, the IVIS measurement values for Groups 2 - 6, which received CD1a1006 administration, were 11.74 ± 2.59×10 5 p / s, 7.17 ± 0.14×10 5 p / s, 7.10 ± 2.01×10 5 p / s, 8.54 ± 0.32×10 5 p / s, and 8.64 ± 1.05×10 5 p / s, respectively.

[1246] In addition, the IVIS measurement value for Group 1 on Day 28 was 3951.23 ± 5229.34×10 5 p / s. In contrast, the IVIS measurement values for Groups 2 - 6, which received CD1a1006 administration, were 7.43 ± 0.99×10 5 p / s, 7.81 ± 0.66×10 5 p / s, 6.43 ± 0.29×10 5 p / s, 7.66 ± 0.59×10 5 p / s, and 9.40 ± 0.20×10 5 p / s, respectively.

[1247] From these results, it can be seen that CD1a1006 has the efficacy of inhibiting the implantation and subsequent proliferation of JM-Luc cells.

[1248] <Example 17> Cytotoxic Activity Data of CD1a KO Cells 1

[1249] In this example, for the in vitro efficacy evaluation of the antigen specificity of the anti-CD1a / anti-TRBC1 bispecific antibody prepared in Example 1, the target cells CCRF-CEM9 and CCRF-CEM CD1aKO cells were cultured with T-LAK (effector cells) in the presence of the bispecific antibody, and then the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index to evaluate the effector cell-dependent cytotoxic activity.

[1250] The target cell CCRF-CEM9 expresses CD1a and TRBC2 and was obtained by monoclonalization of the cell line CCRF-CEM from T-ALL. The CCRF-CEM CD1a KO cell line was obtained by knocking out the CD1a gene in CCRF-CEM using the CRSPR-Cas9 method. The measurement of cytotoxic activity was carried out by the same method as described in Example 3.

[1251] Results

[1252] Figure 29 These are the results of measuring the cytotoxic activities of the bispecific antibodies CD1a1002 and CD1a1005 by adding T-LAK to the T-ALL-derived cell line CCRF-CEM9 cells and CCRF-CEM CD1a KO cells expressing CD1a.

[1253] The EC50 values and Emax values (N = 3) are shown in the following table.

[1254] [Table 22]

[1255] Target Cell Name Antibody EC50 (pM) Standard Deviation CCRF-CEM9 CD1a1002 3.7 0.5 CCRF-CEM9 CD1a1005 4.8 1.2 CCRF-CEM CD1a KO CD1a1002 208.2 NC CCRF-CEM CD1a KO CD1a1005 3737.0 NC

[1256] Target Cell Name Antibody Emax (%) Standard Deviation CCRF-CEM9 CD1a1002 103.4 8.5 CCRF-CEM9 CD1a1005 95.8 8.4 CCRF-CEM CD1a KO CD1a1002 2.7 1.1 CCRF-CEM CD1a KO CD1a1005 2.9 NC

[1257] For the EC50 of CCRF-CEM CD1a KO / CD1a1002, it could not be calculated in 1 out of 3 experiments, so the average value of 2 experiments is recorded.

[1258] For the EC50 of CCRF-CEM CD1a KO / CD1a1005, it could not be calculated in 2 out of 3 experiments, so the value of 1 experiment is recorded.

[1259] For the Emax of CCRF-CEM CD1a KO / CD1a1005, it could not be calculated in 1 out of 3 experiments, so the average value of 2 experiments is recorded.

[1260] T-LAK was added to CCRF-CEM9 cells and CCRF-CEM CD1a KO cells, and the cytotoxic activities of CD1a1002 and CD1a1005 were measured. As a result, Emax of 103.4% or 95.8% was shown in CCRF-CEM9 cells, while Emax of 2.7% or 2.9% was shown in CCRF-CEM CD1a KO cells, and no antibody concentration-dependent cytotoxic activity was observed. These results indicate that the CD1a / anti-TRBC1 bispecific antibody specifically acts on antigen-expressing cells.

[1261] <Example 18> Cytotoxic Activity Data 2 of CD1a KO Cells

[1262] In this example, for the in vitro efficacy evaluation of the antigen specificity of the anti-CD1a / anti-TRBC2 bispecific antibodies CD1a1003 and CD1a1006 prepared in Example 1, the target cells JM and JM CD1a KO cells were cultured with anti-TRBC2 antibody-sensitized T-LAK (effector cells) in the presence of the bispecific antibody, and then the cell viability of the target cells was measured using a fluorochrome (calcein activity) as an index to evaluate the effector cell-dependent cytotoxic activity.

[1263] The target cell JM is a cell line derived from TALL that expresses CD1a and TRBC1. The JM CD1a KO cell is a cell line obtained by knocking out the CD1a gene in the cell line JM derived from TALL using the CRISPR-Cas9 method. The measurement of cytotoxic activity was carried out by the same method as described in Example 3.

[1264] Results

[1265] Figure 30 These are the results of adding anti-TRBC2 antibody-sensitized T-LAK to JM cells, a cell line derived from TALL that expresses CD1a, and JM CD1a KO cells and measuring the cytotoxic activities of the bispecific antibodies CD1a1003 and CD1a1006.

[1266] The EC50 values and Emax values (N = 3) are shown in the following table.

[1267] [Table 23]

[1268] Target Cell Name Antibody EC50 (pM) Standard Deviation JM (Control) CD1a1003 15.7 8.2 JM (Control) CD1a1006 7.4 NC JM CD1a KO CD1a1003 92.3 NC JM CD1a KO CD1a1006 NC NC

[1269] Target Cell Name Antibody Emax (%) Standard Deviation JM (Control) CD1a1003 91.2 25.3 JM (Control) CD1a1006 66.1 NC JM CD1a KO CD1a1003 12.2 NC JM CD1a KO CD1a1006 6.9 NC

[1270] For the EC50 of JM CD1a KO / CD1a1003, it could not be calculated in 2 out of 3 experiments, so the value of 1 experiment is recorded.

[1271] Regarding the EC50 of JM CD1a KO / CD1a1006, it could not be calculated in 2 out of 2 experiments.

[1272] Regarding the Emax of JM CD1a KO / CD1a1003, it could not be calculated in 1 out of 3 experiments. Therefore, the average value of 2 experiments is recorded.

[1273] Anti-TRBC2 antibody-sensitized T-LAK was added to JM cells and JM CD1a KO cells, and the cytotoxic activities of CD1a1003 and CD1a1006 were measured. As a result, the Emax was 91.2% or 66.1% in the case of JM cells, while it was 12.2% or 6.9% in the case of JM CD1a KO cells. No antibody concentration-dependent cytotoxic activity was observed. This result indicates that the anti-CD1a / anti-TRBC2 bispecific antibody specifically acts on antigen-expressing cells.

[1274] <Example 19>Comparison 1 with the bispecific antibody using anti-TRBV5-5 antibody

[1275] The following reagents were used.

[1276] PBS (Nacalai Tesque, 14249-24)

[1277] Ficoll-PaqueTM PLUS (GE Healthcare, 17-1440-02)

[1278] Leucosep (Grener bio-one, 227290)

[1279] FBS (NICHIREI, 175012, Lot.19J00C)

[1280] 2% FBS-PBS (PBS 49 mL + FBS 1 mL)

[1281] Red blood cell lysis buffer (Roche, 11814389001)

[1282] CELLBUNKER2 (Takara, 11914)

[1283] Human pan T cell isolation kit (Miltenyi Biotec, 130-096-535)

[1284] 10% BSA (Thermo, 37525)

[1285] 0.5 mmol / L EDTA (Nacalai, 06894-14)

[1286] Steady-Glo (Promega, E2520)

[1287] RPMI1640 (Nacalai Tesque 30264 - 85)

[1288] 1M Hepes (Nacalai, 17557 - 94)

[1289] 100x Sodium Pyruvate (gibco, 11360 - 070)

[1290] 100x 2 - ME (Wako, 198 - 15781)

[1291] In this example, the cytotoxic activity of the anti - CD1a / anti - TRBC1 bispecific antibody of the present invention and the anti - CD1a / anti - TRBV5 - 5 bispecific antibody prepared based on WO2022 / 119955 was verified. WO2022 / 119955 discloses a bispecific antibody targeting CD3 and TRBV polypeptides for the treatment of T - cell tumors (patent claim). The TRBV polypeptide described in WO2022 / 119955 is an example of an antigen with 30 subtypes.

[1292] The anti - CD1a / anti - TRBV5 - 5 bispecific antibody was prepared by the method described in Example 1.

[1293] [Table 24]

[1294]

[1295] For the anti - CD1a / anti - TRBC1 bispecific antibody CD1a1005, those prepared in Example 14 were used. After culturing the target cells CCRF - CEM - Luc cells with T cells from normal humans (effector cells) in the presence of the bispecific antibody, the cell viability of the target cells was measured using the luminescence enzyme activity (luciferase activity) as an index, and the cytotoxic activity dependent on effector cells was evaluated.

[1296] The target cells CCRF - CEM - Luc cells are a cell line obtained by introducing the luciferase gene into the CCRF - CEM cell line from TALL through a lentiviral vector.

[1297] Method

[1298] Using RPMI1640 medium (TDCC medium) containing 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 0.1 mM 2 - ME, the bispecific antibody was prepared at a final concentration of 10 nM to 100 fM and added to a 384 - well flat - bottom culture plate. As a control, wells containing only TDCC medium were set.

[1299] For the cytotoxic activity assay using a bispecific antibody having antigen-binding sites against CD1a and TRBC1 or TRBV5-5, the required amount of CCRF-CEM-Luc cells (target cells) was recovered from the culture medium, suspended in TDCC medium, and the cell count was performed. Then, it was added to a 384-well flat-bottom culture plate prepared with the bispecific antibody. After recovering the required amount of effector cells, they were suspended in TDCC medium and the cell count was performed. Then, it was added to a 384-well flat-bottom culture plate prepared with the bispecific antibody. The ratio of effector cells to labeled target cells was added at 10:1. The plate containing the bispecific antibody, labeled target cells, and effector cells was incubated at 5% CO2 and 37 °C. After 72 hours, 25 μL of Steady-Glo was added to each well, and after 5 minutes, the luminescence of luciferase in the surviving cells was measured using a microplate reader (Bio Tek, Cytation5). The percentage of cytotoxicity of the target cells was calculated using the luminescence value of luciferase as an index, and the bispecific antibody-dependent cytotoxic activity was evaluated.

[1300] The percentage of cytotoxicity was determined as follows.

[1301] The maximum luminescence value of the labeled target cells (cytotoxicity percentage = 0%) was obtained from the wells in which the labeled target cells and effector cells were incubated in the absence of any bispecific antibody.

[1302] The percentage of cytotoxicity was calculated using the following formula:

[1303] Percentage of cytotoxicity = 100 - [100 × (luminescence value of target cells in the presence of bispecific antibody) / (maximum luminescence value)].

[1304] An S-shaped dose-response curve was calculated using Prism software (GraphPad Software Inc.), and the EC50 value (50% effective concentration) and Emax value (maximum activity value) were calculated.

[1305] Preparation of T cells from normal humans

[1306] Using blood collected from normal individuals, T cells were prepared as described below. The heparin-treated blood at the time of blood collection was diluted 3-fold with PBS and added to a Leucosep tube filled with Ficoll, and the volume was adjusted to 50 mL with PBS. Then, centrifugation was performed (1000×g, 10 min, 20 °C), the upper layer was removed, and the PBMC layer was transferred to another tube. After washing twice with PBS, it was suspended in 3 mL of 2% FBS-PBS, 6 mL of RBC buffer was added, and it was allowed to stand for 10 minutes. After washing with 10 mL of 2% FBS / PBS, it was suspended in CELLBUNKER2 and stored in liquid nitrogen until use. The stored human PBMC was dissolved in a 37 °C water bath and suspended in MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) at a concentration of 1x10 7 cells / 40 μL. Using a human pan-T cell isolation kit, T cells were fractionated from PBMCs and used for the assay.

[1307] Results

[1308] Figure 31 These are the results of adding T cells from normal individuals to CCRF-CEM-Luc cells that express luciferase in the cell line CCRF-CEM from TALL and measuring the cytotoxic activities of the anti-CD1a / anti-TRBC1 bispecific antibody (CD1a1005) and the anti-CD1a / anti-TRBV5-5 bispecific antibodies (TRBV50003 and TRBV50004).

[1309] The EC50 values and Emax values (N = 3) are shown in the following table.

[1310] [Table 25]

[1311] Target Cell Name Antibody EC50 (pM) Standard Deviation CCRF-CEM-Luc CD1a1005 1.2 0.8 CCRF-CEM-Luc TRBV50003 14.0 NC CCRF-CEM-Luc TRBV50004 0.5 NC

[1312] For the EC50 of CCRF-CEM-Luc / TRBV50003, it could not be calculated in 1 out of 3 runs, so the average value of 2 runs is recorded.

[1313] For the EC50 of CCRF-CEM-Luc / TRBV50004, it could not be calculated in 2 out of 3 runs, so the value of 1 run is recorded.

[1314] Target Cell Name Antibody Emax (%) Standard Deviation CCRF-CEM-Luc CD1a1005 79.6 13.3 CCRF-CEM-Luc TRBV50003 23.6 7.8 CCRF-CEM-Luc TRB50004 15.0 6.1

[1315] The Emax of the bispecific antibody of anti-CD1a antibody and anti-TRBC1 antibody showed good activity of 79.6%. On the other hand, the Emax of the anti-CD1a / anti-TRBV5-5 bispecific antibody was 23.6% or 15.4%, which was a result of significantly weaker activity compared to the bispecific antibody using anti-TRBC1.

[1316] It is known that there are 30 subtypes of TRBV, and only one isotype is randomly expressed by one T cell. Therefore, it is speculated that T cells expressing TRBV5-5 are only about 3% of the whole. On the other hand, there are 2 isotypes of TRBC, and one T cell only expresses either one of the isotypes of TRBC1 or TRBC2. Therefore, it is speculated that T cells expressing TRBC1 are about 50% of the whole, and the difference in the number of T cells (effector cells) that can bind to the bispecific antibody is considered to be the reason for the difference in the cytotoxic activity of this data. Based on this result, it is considered that a bispecific antibody using an anti-TRBC1 antibody that can make more T cells function in vivo can be expected to have higher efficacy.

[1317] It should be noted that as shown in Reference Example 3, when T cells expressing TRBV5-5 are sufficiently present, either of TRBV50003 and TRBV50004 shows an activity with Emax = about 60% or so. This is a result supporting that the difference in the number of T cells that can bind to the bispecific antibody will have a significant impact on the cytotoxic activity.

[1318] <Example 20> Comparison 2 with the bispecific antibody using anti-TRBV5-5 antibody

[1319] The same experiment as in Example 19 was performed on the target cell JM-Luc-BFP. The target cell JM-Luc-BFP is a cell line obtained by introducing the luciferase gene into the JM cell line derived from T-ALL through a lentiviral vector.

[1320] In this example, the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 prepared in Example 1 and the anti-CD1a / anti-TRBV5-5 bispecific antibodies TRBV50003 and TRBV50004 prepared in Example 19 were used. After culturing the target cell JM-Luc-BFP cells with T cells (effector cells) from normal humans in the presence of the bispecific antibody, the cell viability of the target cells was measured using the luminescence enzyme activity (luciferase activity) as an index, and the effector cell-dependent cytotoxic activity was evaluated.

[1321] Results

[1322] Figure 32This is the result of adding T cells from normal humans to JM-Luc-BFP cells that express luciferase in the JM cell line from TALL, and measuring the cytotoxic activities of the anti-CD1a / anti-TRBC2 bispecific antibody (CD1a1006) and the anti-CD1a / anti-TRBV5-5 bispecific antibodies (TRBV50003 and TRBV50004).

[1323] The EC50 values and Emax values (N = 2) are shown in the table.

[1324] [Table 26]

[1325] Target Cell Name Antibody EC50 (pM) Standard Deviation JM-Luc-BFP CD1a1006 6.1 NC JM-Luc-BFP TRBV50003 NC NC JM-Luc-BFP TRBV50004 660.7 NC

[1326] Target Cell Name Antibody Emax (%) Standard Deviation JM-Luc-BFP CD1a1006 51.1 NC JM-Luc-BFP TRBV50003 3.9 NC JM-Luc-BFP TRBV50004 3.8 NC

[1327] For the EC50 of JM-Luc-BFP / TRBV50003, it could not be calculated in 2 out of 2 runs, so it was recorded as NC. The EC50 of JM-Luc-BFP / TRBV50004 is the value from 1 run.

[1328] The Emax of the bispecific antibody of the anti-CD1a antibody and the anti-TRBC2 antibody showed good activity of 51.1%. On the other hand, the Emax of the anti-CD1a / anti-TRBV5-5 bispecific antibody was 3.9% or 3.8%, which was a result of significantly weaker activity compared to the bispecific antibody using anti-TRBC2. Based on this result, it is considered that the bispecific antibody using the anti-TRBC2 antibody that can enable more T cells to function in the body can be expected to have higher efficacy.

[1329] <Reference Example 3> Activity of the Bispecific Antibody Using the Anti-TRBV5-5 Antibody

[1330] This reference example was carried out to confirm that the bispecific antibodies TRBV50003 and TRBV5004 prepared in Examples 19 and 20 are antibodies with cytotoxic activity.

[1331] After culturing the target cells CCRF-CEM9 cells with T-LAK (effector cells) stimulated with the anti-TRBV5-5 antibody (TRBV50001) in the presence of the bispecific antibody, the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an index to evaluate the effector cell-dependent cytotoxic activity.

[1332] Preparation of T-LAK cells stimulated with the anti-TRBV5-5 antibody

[1333] Dissolve human PBMC in a 37°C water bath and wash once with culture medium. After culturing for 2 days in RPMI 1640 medium containing 10% FBS supplemented with 10 nM anti-TRBV5-5 antibody (TRBV50001) and 100 IU / mL of IL-2, further culture (up to 17 days) in RPMI 1640 medium containing IL-2 and 10% FBS to induce T-LAK. Transfer every 2 - 3 days and newly add 100 IU / mL of IL-2 when changing the culture medium.

[1334] Results

[1335] Figure 33 These are the results of measuring the cytotoxic activity of anti-CD1a / anti-TRBV5-5 bispecific antibodies (TRBV50003 and TRBV50004) by adding T-LAK cells stimulated with anti-TRBV5-5 antibody (TRBV50001) to the cell line CCRF-CEM9 cells from TALL.

[1336] The EC50 values and Emax values are shown in the following table.

[1337] [Table 27]

[1338] Target cell name Antibody EC50 (pM) CCRF-CEM9 TRBV50003 8.2 CCRF-CEM9 TRBV50004 8.0

[1339] Target cell name Antibody Emax (%) CCRF-CEM9 TRBV50003 57.5 CCRF-CEM9 TRBV50004 57.4

[1340] The Emax values of the anti-CD1a / anti-TRBV5-5 bispecific antibodies were 57.5% and 57.4% respectively, showing antibody concentration-dependent cytotoxic activity. It was confirmed that even for bispecific antibodies using anti-TRBV5-5 antibody on the effector side, cytotoxic activity was shown under conditions rich in activated T cells stimulated with anti-TRBV5-5 antibody and expressing TRBV5-5.

Claims

1. A therapeutic agent for treating T cell tumors, which is a therapeutic agent comprising a bispecific antigen-binding molecule, wherein, the bispecific antigen-binding molecule comprises: (1) at least one part that specifically binds to a target tumor antigen expressed in T cell tumor cells, and (2) at least one part that specifically binds to an antigen with subtypes as a target antigen on normal T cells; wherein the target tumor antigen expressed in T cell tumor cells does not exist in normal T cells, or even if it exists, when the bispecific antigen-binding molecule binds to the antigen identical to the target tumor antigen present in normal T cells, normal T cells are not substantially activated, by binding of the bispecific antigen-binding molecule to the target antigen on normal T cells, normal T cells are activated, and there is a sufficient proportion of subtypes of the target antigen on normal T cells to provide a sufficient number of activated T cells for the treatment of T cell tumors.

2. The therapeutic agent according to claim 1, wherein, the subtype of the target antigen on normal T cells is any subtype among the antigen subtypes that are not the antigen subtypes expressed in T cell tumor cells.

3. The therapeutic agent according to claim 1 or 2, wherein, the antigen with subtypes as the target antigen on normal T cells is TRBC (T cell receptor β constant region), the subtype expressed in T cell tumor cells is TRBC1, and the subtype of the target antigen on normal T cells is TRBC2.

4. The therapeutic agent according to claim 1 or 2, wherein, the antigen with subtypes as the target antigen on normal T cells is TRBC, the subtype expressed in T cell tumor cells is TRBC2, and the subtype of the target antigen on normal T cells is TRBC1.

5. The therapeutic agent according to claim 1 or 2, wherein, the antigen with subtypes as the target antigen on normal T cells is TRBC, T cell tumor cells are negative for TRBC1 and negative for TRBC2, and the subtype of the target antigen on normal T cells is TRBC1 or TRBC2.

6. The therapeutic agent according to claim 1 or 2, wherein, the subtype of the target antigen on normal T cells is TRBC1, the bispecific antigen-binding molecule comprises at least one part that specifically binds to TRBC1 of normal T cells, and the at least one part comprises a VH domain and a VL domain, the VH domain comprises: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12; the VL domain comprises: a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

15.

7. The therapeutic agent according to claim 6, wherein, the at least one part that specifically binds to TRBC1 of normal T cells comprises: A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4, and A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

5.

8. The therapeutic agent according to claim 1 or 2, wherein, The subtype of the target antigen on the normal T cell side is TRBC2, The bispecific antigen-binding molecule comprises at least one portion that specifically binds to TRBC2 of normal T cells, and the at least one portion comprises a VH domain and a VL domain, The VH domain comprises: A heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, A heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and A heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18; The VL domain comprises: A light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and A light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

21.

9. The therapeutic agent according to claim 8, wherein, At least one portion that specifically binds to TRBC2 of normal T cells comprises: A heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6, and A light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

7.

10. The therapeutic agent according to claim 1 or 2, wherein, The T cell tumor is T cell acute lymphoblastic leukemia / lymphoblastic lymphoma or mature T cell tumor.

11. A therapeutic agent, which is the therapeutic agent according to claim 1 or 2 for a method of treating a T cell tumor in a subject, wherein, The treatment method comprises: determining the subtype expressed in the T cell tumor cells of the subject, and administering the therapeutic agent to the subject; the therapeutic agent comprises a bispecific antigen-binding molecule, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to a target antigen on the normal T cell side, and the subtype of the target antigen on the normal T cell side is different from the subtype determined to be expressed in the T cell tumor cells of the subject.

12. A bispecific antigen-binding molecule, which comprises: (1) At least one portion that specifically binds to a target tumor antigen expressed in T cell tumor cells, and (2) At least one portion that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side; wherein, At least one portion that specifically binds to an antigen having a subtype as a target antigen on the normal T cell side is at least one portion that specifically binds to TRBC1 or TRBC2 (T cell receptor β constant region 1 or 2) of normal T cells.

13. The bispecific antigen-binding molecule according to claim 12, wherein, At least one portion that specifically binds to TRBC1 of normal T cells comprises a VH domain and a VL domain, The VH domain comprises: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:10, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:11, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:12; the VL domain comprises: a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

15.

14. The bispecific antigen-binding molecule according to claim 14, wherein at least one portion specifically binding to TRBC1 of normal T cells comprises: a heavy chain variable region VH having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:4, and a light chain variable region VL having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

5.

15. The bispecific antigen-binding molecule according to claim 12, wherein at least one portion specifically binding to TRBC2 of normal T cells comprises a VH domain and a VL domain, the VH domain comprises: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:16, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18; the VL domain comprises: a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:19, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:20, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

21.

16. The bispecific antigen-binding molecule according to claim 15, wherein at least one portion specifically binding to TRBC2 of normal T cells comprises: a heavy chain variable region VH having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:6, and a light chain variable region VL having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:7.

Images