Bispecific antibody that is effective for t-cell tumor patients with t-cell dysfunction

Abstract

The present invention addresses the problem of providing a novel treatment method for T-cell tumors that can induce cell damage in tumor cells and can be used in the treatment of a T-cell tumor of a subject with T-cell dysfunction. The present invention provides a therapeutic agent for a T-cell tumor of a subject with T-cell dysfunction, said therapeutic agent comprising a bispecific antigen-binding molecule, wherein the bispecific antigen-binding molecule includes (1) at least one section that specifically binds to a target tumor antigen expressed in T-cell tumor cells, and (2) at least one section that specifically binds to an antigen which is a normal T-cell-side target antigen and has a subtype, provided that the target tumor antigen expressed in T-cell tumor cells is not present in normal T-cells, or if present, the normal T-cells are substantially not activated when the bispecific antigen-binding molecule binds to the antigen that is present in the normal T-cells and is the same as the target tumor antigen, and a sufficient ratio of the subtype of the normal T-cell-side target antigen is present such that the normal T-cells are activated by the bispecific antigen-binding molecule binding to the normal T-cell-side target antigen and a sufficient number of activated T-cells for treatment of the T-cell tumor are provided.

Description

Bispecific antibodies effective in patients with T-cell dysfunction and T-cell malignancies

[0001] This application claims priority from Japanese Patent Application No. 2024-066502, filed April 17, 2024, the entire disclosure of which is expressly incorporated herein by reference.

[0002] The present invention relates to a therapeutic agent for T-cell tumors in a subject with T-cell dysfunction, comprising a bispecific antigen-binding molecule, the bispecific antigen-binding molecule comprising at least one moiety that specifically binds to a target tumor antigen expressed on T-cell tumor cells and at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype. The present invention also relates to the bispecific antigen-binding molecule and its use, as well as a method for treating T-cell tumors, comprising administering the bispecific antigen-binding molecule to a subject.

[0003] Bispecific T cell engager antibodies have been developed that can induce T cell activity in target cells and selectively kill target cells in vivo. A bispecific antibody (blinatumomab) that can simultaneously recognize human CD19 and CD3 antigens has been approved for sale as a treatment for relapsed / refractory B-cell acute lymphoblastic leukemia (ALL) (Goebeler, ME 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.). Blinatumomab is a bispecific T cell-inducing antibody (BiTE). ＴＭ ) and has a single-chain antibody structure consisting of two single-chain variable region fragments (scFv) linked by a short linker. In addition to blinatumomab, many bispecific antibodies targeting CD3 antigen and tumor cell antigen have been developed in recent years.

[0004] Lymphoid malignancies are broadly divided into B-cell malignancies and T-cell malignancies, and many bispecific antibodies have been developed for the treatment of B-cell malignancies. Acute myeloid leukemia cells share the same cell surface antigens as normal myeloid progenitor cells. Therefore, bispecific antibodies directed against tumor cell antigens of acute myeloid leukemia attack not only leukemia cells but also normal myeloid progenitor cells, causing neutropenia and resulting in febrile neutropenia, which is difficult to treat. In immunotherapy used for B-cell malignancies, the target antigens are also expressed on normal B cells. Bispecific antibodies directed against tumor cell antigens of B-cell malignancies attack not only tumor cells but also normal B-cell progenitor cells, resulting in B-cell immunodeficiency and the inability to produce antibodies. However, regular administration of immunoglobulin preparations can prevent B-cell immunodeficiency (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.).

[0005] On the other hand, immunotherapy for T-cell malignancies has been limited. Because CD3 is expressed similarly on both normal and tumor T cells, bispecific antibodies targeting both CD3 and tumor antigens likely bind to both the CD3 antigen expressed on T-cell malignancies and the target antigen expressed on T-cell malignancies simultaneously, linking malignant tumor cells to each other (Figure 1A), potentially limiting their therapeutic efficacy. Furthermore, there is a lack of known target antigens that distinguish between normal and malignant T cells. T-cell hypoplasia resulting from targeting pan-T-cell antigens can lead to severe and unacceptable immunodeficiency (Maciocia PM, et al., supra).

[0006] In addition to the CD3 antigen, a bispecific antibody that targets effector cells (T cells) utilizes TRGV9, which is expressed in γδ T cells (WO 2021 / 173896 A1). However, because γδ T cells account for only a few percent of all T cells, there is a problem in that a sufficient number of T cells cannot be mobilized to damage tumor cells and achieve a therapeutic effect.

[0007] Furthermore, some T-cell malignancies, such as adult T-cell leukemia (ATL), are known to cause T-cell dysfunction due to immune evasion by tumor cells. After infection with HTLV-1 (human T-cell leukemia virus type 1), many genetic mutations accumulate over time, ultimately leading to the development of ATL (Kataoka K, et al., Prognostic relevance of integrated genetic profiling in adult T-cell leukemia / lymphoma. Blood. 2018 Jan 11;131(2):215-225. doi: 10.1182 / blood-2017-01-761874. Epub 2017 Oct 30. PMID: 29084771; PMCID: PMC5757690.). Due to numerous genetic mutations, HTLV-1-infected cells have constantly activated TCR and NFκB signaling pathways, ultimately leading to clonally proliferating ATL cells (Benjy JY Tan, et al., HTLV-1 infection promotes excessive T cell activation and transformation into adult T cell leukemia / lymphoma. J Clin Invest. 2021;131(24):e150472. https: / / doi.org / 10.1172 / JCI150472.). ATL cells exhibit a Treg phenotype and highly express co-inhibitory molecules such as CTLA4 (cytotoxic T lymphocyte antigen 4) and LAG3 (lymphocyte activation gene 3), inhibiting the maturation of antigen-presenting cells (APCs). Furthermore, binding of CD80 / 86 on APCs to CTLA4 highly expressed on ATL cells inhibits the binding of CD28 on normal T cells to CD80 / 86 on APCs, resulting in T cell anergy in normal T cells. Meanwhile, ATL cells highly express HLA class II (human leukocyte antigen class II), and thus can also become antigen-presenting cells. However, because ATL cells do not express CD80 / 86, they induce T cell anergy in both naive and effector T cells (Benjy JY Tan, et al., supra).

[0008] Immunosuppressive checkpoint inhibitors are used as immunotherapies to counteract the immune evasion mechanisms of cancer cells. However, because ATL cells express high levels of PDL-1 (programmed cell death ligand 1), some cases have been reported in which ATL patients treated with the anti-PD-1 (programmed cell death 1) antibody nivolumab experienced rapid progression of the disease (Ratner L, Waldmann TA, Janakiram M, Brammer JE. Rapid Progression of Adult T-Cell Leukemia-Lymphoma after PD-1 Inhibitor Therapy. N Engl J Med. 2018 May 17;378(20):1947-1948. doi: 10.1056 / NEJMc1803181. PMID: 29768155.). Studies using mouse models have shown that loss of PD-1 induces changes in oncogenic signaling pathways in T cells, promoting the development of T-cell lymphoma (Wartewig T, Kurgyis Z, Keppler S et al. PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis. Nature 2017; 552: 121-125.). Thus, treatment with anti-PD-1 antibodies for T-cell malignancies, including ATL, is not always effective.

[0009] There is a need for new methods for treating T cell tumors that can provide high therapeutic efficacy even when used to treat T cell tumors in subjects with T cell dysfunction.

[0010] Therefore, an object of the present invention is to provide a new method for treating T cell tumors that can induce cytotoxicity in tumor cells and can be used to treat T cell tumors in subjects with T cell dysfunction.

[0011] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that a bispecific antigen-binding molecule that specifically binds to a target tumor antigen expressed on T cell tumor cells and a target antigen with a subtype on normal T cells mobilizes effector cells (normal T cells) and induces cytotoxicity of tumor cells. Furthermore, the present inventors have found that by using an antigen with subtypes that expresses different subtypes between T cell tumor cells and normal T cells as the target antigen on normal T cells to which the bispecific antigen-binding molecule binds, it is possible to damage tumor cells of T cell malignancies while sparing sufficient normal T cells. Furthermore, the present inventors have found that the bispecific antigen-binding molecule can induce cytotoxic activity even in patients with T cell tumors in a state of T cell dysfunction. The present invention was completed based on these findings.

[0012] The present invention provides the following: [1] A therapeutic agent for T cell tumors in a subject with T cell dysfunction, comprising a bispecific antigen-binding molecule, wherein the bispecific antigen-binding molecule comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T cell tumor cells, and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype, wherein the target tumor antigen expressed on T cell tumor cells is not present on normal T cells, or even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells, wherein the normal T cells are activated by the binding of the bispecific antigen-binding molecule to the target antigen on normal T cells, and wherein a sufficient proportion of subtypes of the target antigen on normal T cells is present to provide a sufficient number of activated T cells for the treatment of the T cell tumor. [2] The therapeutic agent according to [1], wherein the subtype of the target antigen on normal T cells is any one of the subtypes of antigens other than the subtype of the antigen expressed on T cell tumor cells. [3] The therapeutic agent according to [1] or [2], wherein the target antigen on normal T cells and having a subtype is TRBC (T cell receptor beta constant region), the subtype expressed on T cell tumor cells is TRBC1, and the subtype of the target antigen on normal T cells is TRBC2.

[0013] [4] The therapeutic agent according to [1] or [2], wherein the target antigen on normal T cells and having a subtype 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 [1] or [2], wherein the target antigen on normal T cells and having a subtype is TRBC, the T cell tumor cells are TRBC1-negative and TRBC2-negative, and the subtype of the target antigen on normal T cells is TRBC1 or TRBC2.

[0014] [6] The therapeutic agent according to any one of [1] to [5], wherein the target tumor antigen expressed in T-cell tumor cells is any one 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). [7] The therapeutic agent according to any one of [1] to [6], wherein the subtype of the target antigen on normal T cells is TRBC1, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC1 on normal T cells, said at least one moiety comprising: a VH domain comprising 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, and a VL domain comprising 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.

[0015] [8] The therapeutic agent according to [7], wherein at least one portion which 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, or comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149. [9] The therapeutic agent according to any one of [1] to [6], wherein the subtype of the target antigen on normal T cells is TRBC2, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC2 on normal T cells, said at least one moiety comprising: a VH domain comprising 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, and a VL domain comprising 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.

[0016]

[10] The therapeutic agent according to [9], wherein at least one moiety 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.

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

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CCR4, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR4, wherein the at least one moiety comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0017]

[12] The therapeutic agent according to

[11] , wherein at least one portion 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.

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

[10] , wherein the target tumor antigen expressed in T cell tumor cells is CD1a, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to CD1a, the at least one portion comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0018]

[14] The therapeutic agent according to

[13] , wherein at least one portion 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.

[0019]

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

[10] , wherein the target tumor antigen expressed in T-cell tumor cells is CCR9, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR9, the at least one moiety comprising: a VH domain comprising 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, and a VL domain comprising 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.

[16] The therapeutic agent according to

[15] , wherein the 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.

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

[10] , wherein the target tumor antigen expressed in T-cell tumor cells is CXCR4, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CXCR4, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[18] The therapeutic agent according to

[17] , wherein the 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.

[0020]

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

[10] , wherein the target tumor antigen expressed in T-cell tumor cells is TIGIT, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TIGIT, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94.

[20] The therapeutic agent according to

[19] , wherein the 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.

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

[10] , wherein the target tumor antigen expressed in T-cell tumor cells is CCR8, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR8, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[22] The therapeutic agent according to

[21] , wherein the 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.

[0021]

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

[10] , wherein the target tumor antigen expressed on T-cell tumor cells is CD30, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD30, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[24] The therapeutic agent according to

[23] , wherein 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.

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

[10] , wherein the target tumor antigen expressed in T-cell tumor cells is CD40L (CD154), and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD40L (CD154), wherein the at least one moiety comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119, 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, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, 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.

[26] The therapeutic agent according to

[25] , wherein at least one portion 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.

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

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

[28] The therapeutic agent according to any one of [1] to

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

[0022]

[29] A therapeutic agent for T-cell tumors in a subject with T-cell dysfunction, comprising a bispecific antigen-binding molecule, wherein the bispecific antigen-binding molecule comprises: at least one moiety that specifically binds to a target tumor antigen expressed on T-cell tumor cells; and at least one moiety that specifically binds to either TRBC1 or 2 expressed on normal T cells.

[30] The therapeutic agent according to

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

[0023]

[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 on normal T cells, and the at least one moiety comprises a VH domain comprising 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, and a VL domain comprising 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.

[32] The therapeutic agent according to

[31] , wherein at least one portion which 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, or comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149.

[0024]

[33] The therapeutic agent of

[29] or

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

[34] The therapeutic agent of

[33] , wherein the at least one moiety that specifically binds to TRBC2 expressed on 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.

[35] The therapeutic agent according to any one of

[29] to

[34] , wherein at least one moiety that specifically binds to TRBC1 or 2 expressed on normal T cells is an scFv or Fab fragment.

[0025]

[36] The therapeutic agent according to any one of

[29] to

[35] , wherein the target tumor antigen expressed in T-cell tumor cells is any one 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).

[0026]

[37] The therapeutic agent according to any one of

[29] to

[36] , wherein the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR4, the at least one moiety comprising a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[38] The therapeutic agent according to

[37] , wherein the 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.

[0027]

[39] The therapeutic agent according to any one of

[29] to

[36] , wherein the bispecific antigen-binding molecule comprises at least one portion that specifically binds to CD1a, and the at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0028]

[40] The therapeutic agent according to

[39] , wherein at least one portion 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.

[0029]

[41] The therapeutic agent according to any one of

[29] to

[36] , wherein the target tumor antigen expressed in T-cell tumor cells is CCR9, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR9, the at least one moiety comprising: a VH domain comprising 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, and a VL domain comprising 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.

[42] The therapeutic agent according to

[41] , wherein the 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.

[43] The therapeutic agent according to any one of

[29] to

[36] , wherein the target tumor antigen expressed in T-cell tumor cells is CXCR4, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CXCR4, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[44] The therapeutic agent according to

[43] , wherein the 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.

[0030]

[45] The therapeutic agent according to any one of

[29] to

[36] , wherein the target tumor antigen expressed in T-cell tumor cells is TIGIT, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TIGIT, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94.

[46] The therapeutic agent according to

[45] , wherein the 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.

[47] The therapeutic agent according to any one of

[29] to

[36] , wherein the target tumor antigen expressed in T-cell tumor cells is CCR8, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CCR8, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[48] The therapeutic agent according to

[47] , wherein the 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.

[0031]

[49] The therapeutic agent according to any one of

[29] to

[36] , wherein the target tumor antigen expressed in T-cell tumor cells is CD30, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD30, the at least one moiety comprising: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[50] The therapeutic agent according to

[49] , wherein 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.

[51] The therapeutic agent according to any one of

[29] to

[36] , wherein the target tumor antigen expressed in T-cell tumor cells is CD40L (CD154), and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD40L (CD154), wherein the at least one moiety comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119, 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, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, 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.

[52] The therapeutic agent according to

[51] , wherein at least one portion 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.

[0032]

[53] The therapeutic agent according to any one of

[29] to

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

[54] The therapeutic agent according to any one of

[29] to

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

[55] The therapeutic agent according to any one of

[29] to

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

[0033]

[56] A bispecific antigen-binding molecule for use in a method for treating a T-cell tumor in a subject with T-cell dysfunction, comprising: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T-cell tumor cells; and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype, wherein the at least one moiety that specifically binds to a target antigen on normal T cells having a subtype is at least one moiety that specifically binds to TRBC1 or 2 (T-cell receptor beta constant region 1 or 2) of normal T cells.

[57] The bispecific antigen-binding molecule of

[56] , wherein at least one portion which specifically binds to TRBC1 expressed in normal T cells comprises a VH domain comprising 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, and a VL domain comprising 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.

[58] The bispecific antigen-binding molecule of

[57] , wherein at least one portion which 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, or comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149.

[0034]

[59] The bispecific antigen-binding molecule of

[56] , wherein at least one portion which specifically binds to TRBC2 expressed on normal T cells comprises a VH domain comprising 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, and a VL domain comprising 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.

[60] The bispecific antigen-binding molecule of

[59] , wherein at least one portion which specifically binds to TRBC2 expressed on 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.

[0035]

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

[56] to

[60] , wherein the target tumor antigen expressed on T-cell tumor cells is any one 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).

[62] The bispecific antigen-binding molecule of

[61] , wherein at least one portion that specifically binds to CCR4 comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[63] The bispecific antigen-binding molecule of

[62] , wherein at least one portion 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.

[0036]

[64] The bispecific antigen-binding molecule of

[61] , wherein at least one portion that specifically binds to CD1a comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[65] The bispecific antigen-binding molecule of

[64] , wherein at least one portion 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.

[0037]

[66] The bispecific antigen-binding molecule of

[61] , wherein the target tumor antigen expressed in T-cell tumor cells is CCR9, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to CCR9, wherein the at least one portion comprises a VH domain comprising 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, and a VL domain comprising 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.

[67] The bispecific antigen-binding molecule of

[66] , wherein the at least one portion 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.

[0038]

[68] The bispecific antigen-binding molecule of

[61] , wherein the target tumor antigen expressed in T-cell tumor cells is CXCR4, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CXCR4, wherein the at least one moiety comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[69] The bispecific antigen-binding molecule of

[68] , wherein the 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.

[70] The bispecific antigen-binding molecule of

[61] , wherein the target tumor antigen expressed in T-cell tumor cells is TIGIT, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TIGIT, wherein the at least one moiety comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 91, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 94.

[71] The bispecific antigen-binding molecule of

[70] , wherein the 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.

[0039]

[72] The bispecific antigen-binding molecule of

[61] , wherein the target tumor antigen expressed in T-cell tumor cells is CCR8, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to CCR8, wherein the at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[73] The bispecific antigen-binding molecule of

[72] , wherein the at least one portion 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.

[74] The bispecific antigen-binding molecule of

[61] , wherein the target tumor antigen expressed on T-cell tumor cells is CD30, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to CD30, wherein the at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[75] The bispecific antigen-binding molecule of

[74] , wherein the at least one portion 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.

[0040]

[76] The bispecific antigen-binding molecule of

[61] , wherein the target tumor antigen expressed on T-cell tumor cells is CD40L (CD154), and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to CD40L (CD154), wherein the at least one portion comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119, 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, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, 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.

[77] The bispecific antigen-binding molecule of

[76] , wherein at least one portion 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.

[0041]

[78] A method for treating a T cell tumor in a subject with T cell dysfunction, comprising administering the bispecific antigen-binding molecule of any one of

[56] to

[77] to a subject having a T cell tumor.

[79] A bispecific antigen-binding molecule for use in a method for treating a T cell tumor in a subject with T cell dysfunction, comprising: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T cell tumor cells, and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype, wherein the target tumor antigen expressed on T cell tumor cells is not present on normal T cells, or even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells, and the normal T cells are activated by binding of the bispecific antigen-binding molecule to the target antigen on normal T cells, and a sufficient proportion of the subtypes of the target antigen on normal T cells is present to provide a sufficient number of activated T cells for treating the T cell tumor.

[0042]

[80] A method for treating a T cell tumor in a subject with T cell dysfunction, comprising administering a bispecific antigen-binding molecule to the subject having a T cell tumor, wherein the bispecific antigen-binding molecule comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T cell tumor cells; and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype; provided that the target tumor antigen expressed on T cell tumor cells is not present on normal T cells, or even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells; the normal T cells are activated by the binding of the bispecific antigen-binding molecule to the target antigen on normal T cells; and a sufficient proportion of the subtypes of the target antigen on normal T cells is present to provide a sufficient number of activated T cells for treating the T cell tumor.

[81] The method of treatment according to

[80] , further comprising determining a subtype expressed in the subject's T cell tumor cells, wherein the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to a target antigen on normal T cells of a subtype different from the subtype determined to be expressed in the subject's T cell tumor cells.

[82] Use of a bispecific antigen-binding molecule in the manufacture of a medicament for treating a T cell tumor in a subject with T cell dysfunction, wherein the bispecific antigen-binding molecule comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T cell tumor cells; and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype; provided that the target tumor antigen expressed on T cell tumor cells is not present on normal T cells, or even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells; normal T cells are activated by binding of the bispecific antigen-binding molecule to the target antigen on normal T cells; and a sufficient proportion of subtypes of the target antigen on normal T cells is present to provide a sufficient number of activated T cells for the treatment of the T cell tumor.

[0043] FIG. 1A shows a state in which a bispecific antibody simultaneously binds to CD3 expressed on tumor cells and a target antigen expressed on tumor cells, thereby cross-linking tumor cells. FIG. 1B shows a diagram illustrating the concept of the present invention. The bispecific antigen-binding molecule of the present invention recognizes a target antigen expressed on (T cell) tumor cells and a "target antigen on normal T cells, which has a subtype" (in the figure, the subtype expressed on T lymphocytes is TRBC1), thereby cross-linking tumor cells and T lymphocytes. In the figure, the bispecific antigen-binding molecule simultaneously binds to the TRBC1 antigen of T lymphocytes and the target antigen expressed on tumor cells. The TRBC2 subtype is expressed on tumor cells as well as T lymphocytes. In the figure, the bispecific antigen-binding molecule does not recognize T lymphocytes expressing the TRBC2 subtype. FIG. 2A shows the structure of a bispecific antibody prepared in the Examples. The Fab-scFv type has a structure in which an scFv that binds to one antigen and an Fab portion that binds to the other antigen are linked by a linker. The Fab-scFv-Fc type has two structures in which an scFv that binds to one antigen and an Fab portion that binds to the other antigen are linked by a linker, and also has an Fc. The Fab-Fc-scFv type has a structure in which an scFv that binds to one antigen is linked to the C-terminus of an IgG that binds to the other antigen. A: portion that binds to one antigen, B: portion that binds to the other antigen. Figure 2B shows the VH and VL amino acid sequences of the anti-TRBC1 antibody, humanized anti-TRBC1 antibody, anti-TRBC2 antibody, and anti-CCR4 antibody used to prepare the bispecific antibodies. The underlined parts indicate the respective CDRs. The VH and VL amino acid sequences of the anti-CCR4 antibody are those set forth in SEQ ID NOs: 9 and 14 of Japanese Patent No. 4052515 (Human CDR-grafted antibodies and antibody fragments thereof). The VH and VL amino acid sequences of the humanized anti-TRBC1 antibody are those set forth in SEQ ID NOs: 9 and 19 of Japanese Patent No. 7177794 (corresponding to WO2018 / 224844). The VH and VL amino acid sequences of the anti-TRBC1 antibody are those set forth in SEQ ID NOs: 15 and 16 of US Pat. No. 10,730,942 (Protein-based T-cell receptor knockdown).The VH and VL amino acid sequences of the anti-TRBC2 antibody are the VH domain sequence of hJOV-1 (SEQ ID NO: 1 in WO 2020 / 089644) with three amino acid substitutions T28K, Y32F, and A100N (Table 1 (page 23) in WO 2020 / 089644) and the VL domain sequence of hJOV-1 (SEQ ID NO: 2 in WO 2020 / 089644). The underlined CDRs are defined according to the Kabat numbering scheme. Figures 3A-K show the amino acid sequences constituting the heavy and light chains of the bispecific antibody prepared in Example 1. Figure 4A shows the increase in the rate of TRBC1-positive cells after the addition of an anti-TRBC1 antibody. N=3. FIG. 4B shows live cells stained with Fixable Viability Dye eFluor. ＴＭ Figure 5A shows the increase in cytotoxic T cells by the addition of anti-TRBC1 antibody. Figure 5B shows the increase in cytotoxic T cells by the addition of anti-TRBC1 antibody. Live cells were stained with Fixable Viability Dye eFluor. ＴＭGating was performed using 780 staining, and cells were expanded by FSC and SSC to gate the major cell populations. Then, TRBC1-positive cells were gated and expanded by CD27 and CD45RA, and the proportion of each fraction among TRBC1-positive cells was calculated. *Effector memory (EM): These cells circulate mainly in secondary lymphoid tissues (lymph nodes) and peripheral tissues, and when exposed to the same antigen again, they quickly produce cytokines and mount an immune response. Central memory (CM): These cells reside in secondary lymphoid tissues (lymph nodes) and mount an antigen response. Naive: These are T cells that have never been exposed to an antigen. EM T cells that re-express CD45RA (EMRA): Terminally differentiated effector memory cells. N=3. Figure 6A shows the positivity rate (%) of activation markers in TRBC1-positive cells. (N=3, *Perforin shows Day 0 data only, N=2). Figure 6B shows the results of gating TRBC1-positive cells, then analyzing with CD25 and CD69, and calculating the positivity rate of each marker in TRBC1-positive cells. Figure 6C shows the results of analyzing with TRBC1 and Granzyme B, and calculating the positivity rate of Granzyme B in TRBC1-positive cells. Figure 6D shows the results of analyzing with TRBC1 and Perforin, and calculating the positivity rate of Perforin in TRBC1-positive cells. Data are shown as mean (SD). Figure 7A shows the change in the proportion of PD-1-positive cells with the addition of anti-TRBC1 antibody. N=3. FIG. 7B shows the results of Fixable Viability Dye eFluor on live cells. ＴＭ Figure 7 shows the results of gating by 780 staining, development by FSC and SSC to gate the major cell population, and then development by TRBC1 and PD-1, and calculating the ratio of PD-1 positive cells among TRBC1 positive cells. Figure 8 shows the quantification of cytokine release from T-LAK stimulated with anti-TRBC1 antibody. Figure 9 shows the increase in the number of cells, including TRBC2 positive T cells, after addition of anti-TRBC2 antibody. (a) is a graph showing the increase in cytotoxic T cells by addition of anti-TRBC2 antibody. (b) is a graph showing the increase in cytotoxic T cells by addition of anti-TRBC2 antibody, after addition of live cells with Fixable Viability Dye eFluor. ＴＭCells were gated by 780 staining, expanded by FSC and SSC to determine the major cell population, then gated on CD3-positive, TRBC1-negative cells (gated on TRBC2-positive cells), expanded by CD27 and CD45RA, and the proportion of each fraction in TRBC2-positive cells was calculated (N=3). EM: Effector memory, CM: Central memory, EMRA: EM T cells that re-express CD45RA. (a) shows the positivity rate (%) of activation markers in TRBC2-positive cells (N=3). (b) shows the positivity rate (%) of each marker in TRBC2-positive cells calculated after gating on TRBC2-positive cells and expanding by CD25 and CD69. (c) is a diagram showing the calculation of the Granzyme B positive ratio among TRBC2 positive cells after development with TRBC1 and Granzyme B. (d) is a diagram showing the calculation of the Perforin positive ratio among TRBC2 positive cells after development with TRBC1 and Perforin. Data are shown as mean (SD). (a) shows the change in the ratio of PD-1 positive cells with the addition of anti-TRBC2 antibody (N=3). (b) is a diagram showing the change in the ratio of PD-1 positive cells after the addition of anti-TRBC2 antibody (N=3). ＴＭFigure 13 shows the quantification of cytokine release from T-LAK stimulated with anti-TRBC2 antibodies. The vertical axis of each graph represents concentration. Figures 14A-C show the cytotoxic activity of bispecific antibodies CCR0001 and CCR0004. Figure 15 shows the cytotoxic activity of bispecific antibodies CD1a1001 and CD1a1004. Figure 16 shows the cytotoxic activity of anti-CCR4 / anti-TRBC2 bispecific antibodies against the ATL-derived cell line MT-2. Figure 17 shows the cytotoxic activity of anti-CD1a / anti-TRBC2 bispecific antibodies against the TALL-derived cell line JM (expressing CD1a and TRBC1). Figure 18 shows the cytotoxic activity of anti-CCR9 / anti-TRBC1 bispecific antibodies against the TALL-derived cell line CCRF-CEM9 (expressing CCR9 and TRBC2). Figure 19 shows the cytotoxic activity of anti-CXCR4 / anti-TRBC1 bispecific antibodies against the TALL-derived cell line CCRF-CEM9 (expressing CXCR4 and TRBC2). Figure 20 shows the cytotoxic activity of anti-CCR8 / anti-TRBC1 bispecific antibodies against the ATL-derived cell line MT-1 (expressing CCR8). Figure 21 shows the cytotoxic activity of anti-TIGIT / anti-TRBC1 bispecific antibodies against the ATL-derived cell line ILT-Mat (expressing TIGIT). Figure 22 shows the cytotoxic activity of anti-CD30 / anti-TRBC1 bispecific antibodies against the PTCL-derived cell line DL40 (expressing CD30). Figure 23 shows the cytotoxic activity of anti-CD40L / anti-TRBC1 bispecific antibodies against the TALL-derived cell line MOLT3 (expressing CD40L). Figure 24 shows the results of adding T-LAK to HPB-ALL cells, a CD3-expressing TALL-derived cell line, and HPB-ALL CD3 KO cells in which CD3 has been knocked out, and measuring the cytotoxic activity of the anti-CD1a / anti-CD3 bispecific antibody CD1a1016 (a) and anti-CD1a antibody and the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005 (b).Figure 25 shows the results of measuring the cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 by adding anti-TRBC2 antibody-sensitized T-LAK to CCRF-CEM9 cells, a TALL-derived cell line that expresses TRBC2, and JM cells, a TALL-derived cell line that expresses TRBC1. Figure 26 is a graph of IVIS measurements by group on days 14 and 28 in NOG mice implanted with tumor cells. Figure 27 is an image of tumor distribution in NOG mice implanted with tumor cells. Figure 28 shows the rate of PD-1 and CTLA-4 positive cells in CD3-positive, CD4-positive, CADM1-negative normal T cells. Figure 29 shows the cytotoxic activity of PBMCs derived from a healthy subject (healthy subject 1) and PBMCs derived from ATL patients (ATL patient 1, ATL patient 2) against luciferase-positive MOLT-4-Luc cells in the presence of an anti-CD1a / anti-TRBC1 bispecific antibody (CD1a1005). Figure 30 is a graph showing the analysis of CD8-positive T cell function in PBMCs derived from healthy subjects and ATL patients by single-cell RNA-Seq. Figure 31 is a graph showing the measurement of the cytotoxic activity of PBMCs derived from a healthy subject and PBMCs derived from ATL patients against luciferase-positive MOLT-4-Luc cells in the presence of an anti-CD1a / anti-TRBC2 bispecific antibody.

[0044] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0045] The present invention relates to the treatment of T-cell tumors in subjects with T-cell dysfunction. Hematological malignancies are broadly classified into lymphoid tumors and myeloid tumors based on the origin or maturity of the tumor cells, and lymphoid tumors can be further classified into T-cell tumors and B-cell tumors. Lymphoid tumors originate from lymphoid precursor cells, while myeloid tumors originate from myeloid precursor cells. T-cell tumor cells are formed when T lymphocytes become malignant, and B-cell tumor cells are formed when B lymphocytes become malignant.

[0046] As used herein, "T-cell tumors" include, but are not limited to, T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma and mature T-cell tumors, so long as they are tumors of T cells or their precursor cells.

[0047] Regarding acute lymphoblastic leukemia / lymphoblastic lymphoma (ALL / LBL), when lymphoblasts infiltrate the bone marrow (>25%) and appear in the peripheral blood, it is considered acute lymphoblastic leukemia (ALL), and when they invade the lymph nodes and extranodal organs, it is considered lymphoblastic lymphoma (LBL). Acute lymphoblastic leukemia / lymphoblastic lymphoma includes both B-cell and T-cell types, which are called "B-cell acute lymphoblastic leukemia / lymphoblastic lymphoma" and "T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma," respectively. T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma includes the following (Swerdlow SH, et al (Editors). WHO classification of tumors of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2017): T-lymphoblastic leukemia / lymphoma (Provisional entity: Early T-cell precursor lymphoblastic leukemia, Provisional entity).

[0048] Mature T cell tumor には、The following がまれる (Swerdlow SH, et al (Editors). WHO classification of tumors of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2017). T-cell pro-lymphocytic leukemia, T-cell large granular lymphocytic leukemia, systemic EBV positive T-cell lymphoma of childhood, hydrops vacciniiformis-like lymphoma lymphoproliferative disease), adult T-cell leukemia / lymphoma, extranodal T-cell lymphoma,nasal type), enteropathy-associated T-cell lympoma, monomorphic epithelial tropic intestinal T-cell lympoma, indolent T-cell lympoma (GI tract), hepatosplenic T-cell lympoma, T-cell lympoma), subcutaneous adipose tissue inflammation with T-cell lympoma, mycotic polyposis, sezary syndrome, primary cutaneous CD30 positive T-cell lympoma lesions), lympatoid papulosis, primary cutaneous undifferentiated large cell lympoma, primary cutaneous gamma-delta T-cell lympoma, primary cutaneous CD8 positive rapidly progressive epidermotropic T-cell lympoma aggressive epidemiological cytotoxic T-cell lympoma, primary cutaneous apical CD8-positive T-cell lympoma, primary cutaneous CD4-positive small / medium T-cell lympoma peripheral T-cell lymphoma, nonspecificNOS), Angioimmunoblastic T-cell lymphoma, Follicular T-cell lymphoma, Nodal peripheral T-cell lymphoma with follicular helper T-cell trait (Nodal Peripheral T-cell lymphoma with TFH phenotype), Anaplastic large cell lymphoma, ALK-positive type (ALK) positive), Anaplastic large cell lymphoma (ALK negative type), Anaplastic large cell lymphoma (ALK) negative), breast implant-associated anaplastic large-cell lymphoma.

[0049] As used herein, the term "subject in a state of T cell dysfunction" refers to a subject in which T cell effector function is reduced due to evasion of immune surveillance, the formation of an immunosuppressive environment, or T cell exhaustion in a chronic infection or cancer environment, limiting appropriate immune responses against tumor cells. Subjects in a state of T cell dysfunction include subjects in a state of T cell anergy.

[0050] As used herein, "T cell anergy" refers to a condition in which T cells, part of the immune system, are not activated and do not function properly. Normally, T cells play a role in initiating an immune response against foreign pathogens or substances, but in T cell anergy, this response is suppressed. While T cell anergy is important as a regulatory mechanism for immune responses, excessive T cell anergy can impair immune function and increase the risk of tumor progression, infections, and other immune-related diseases. Therefore, abnormal T cell anergy can contribute to immune dysfunction.

[0051] As used herein, the term "antigen-binding molecule" refers to a molecule that specifically binds to an epitope (antigenic determinant), such as an antibody or antibody fragment. "Bispecific" means that an antigen-binding molecule specifically binds to two different antigenic determinants. A bispecific antigen-binding molecule contains at least two antigen-binding moieties that bind to different antigenic determinants.

[0052] An antibody has a structure in which two heavy chains (H chains) and two light chains (L chains) are bound together. These light and heavy chains are linked by disulfide bonds (SS bonds) to form a heterodimer, and two such heterodimers are further linked to form a Y-shaped heterotetramer. Typically, a heavy chain consists of a heavy chain variable region VH, heavy chain constant regions CH1, CH2, and CH3, and a hinge region located between CH1 and CH2, while a light chain consists of a light chain variable region VL and a light chain constant region CL. The variable regions include complementarity-determining regions (CDRs) and framework regions (FRs). The light chain and heavy chain variable regions each contain three CDRs (heavy chain CDR1-3 and light chain CDR1-3) and four FRs (heavy chain FR1-4 and light chain FR1-4). Methods for identifying CDRs are known, and can be found, for example, in the IMGT / V-QUEST Search page (http: / / www.imgt.org / IMGT_vquest / input), Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008), or in the literature (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). Any other means known in the art can also be used as long as similar results are obtained (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institutes of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342: 877).

[0053] As used herein, an antibody fragment is an antigen-binding antibody fragment. Examples of antibody fragments include Fab and F(ab'). ２Examples of F(ab') include, but are not limited to, Fv, scFv, and the like. Fab refers to an antibody fragment in which the VL-CL and VH-CH1 domains are linked by a disulfide bond. F(ab') ２ refers to an antibody fragment in which two Fab fragments are linked by a disulfide bond at the hinge region. Fv fragments consist of VL and VH. scFv is a fusion protein in which VH and VL are connected by a linker peptide (approximately 10 to 25 amino acids in length).

[0054] A first embodiment of the present invention relates to a therapeutic agent for T cell tumors in a subject with T cell dysfunction, comprising a bispecific antigen-binding molecule. The bispecific antigen-binding molecule constituting the therapeutic agent of the present invention comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T cell tumor cells, and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype; provided that the target tumor antigen expressed on T cell tumor cells is not present on normal T cells, or, even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells; the normal T cells are activated by binding of the bispecific antigen-binding molecule to the target antigen on normal T cells; and a sufficient proportion of subtypes of the target antigen on normal T cells is present to provide a sufficient number of activated T cells for the treatment of the T cell tumor.

[0055] In the present invention, the phrase "a moiety that specifically binds to a target tumor antigen expressed on a T cell tumor cell" (hereinafter referred to as a tumor antigen-binding moiety) refers to a polypeptide molecule that specifically binds to a target tumor antigen expressed on a T cell tumor cell. In one aspect, the tumor antigen-binding moiety can guide normal T cells to T cell tumor cells that express the target tumor antigen. The tumor antigen-binding moiety includes an antibody or a fragment thereof.

[0056] As used herein, "specifically binds" means selectively binding to a specific antigen and is distinguishable 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 binds to a target antigen at a concentration of 1 x 10 －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 x 10 －１０ Dissociation constant (K Ｄ ) can be combined.

[0057] Preferably, the target tumor antigen expressed in T cell tumor cells is not present in normal T cells, or even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in normal T cells. The absence of a 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. Whether an antigen is present or absent in normal T cells, or whether expression of an antigen is positive or negative in normal T cells, can be determined based on a chart obtained by flow cytometry analysis. Details of flow cytometry analysis will be described later. In a preferred embodiment, the target tumor antigen expressed in T cell tumor cells is not present in normal T cells, allowing the bispecific antigen-binding molecule to appropriately crosslink normal T cells and T cell tumor cells (e.g., Figure 1B). Furthermore, the statement "normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells" can be achieved by preparing a tumor antigen-binding moiety based on an antibody that does not have agonistic activity, because activation of normal T cells caused by binding of a bispecific antigen-binding molecule (e.g., an antibody) to an antigen is thought to be largely due to the agonistic activity of the antibody.

[0058] CD3 is expressed on both normal T cells and T cell tumor cells. Bispecific antibodies targeting both CD3 and tumor antigens simultaneously bind to the CD3 antigen expressed on T cell malignancies and the target antigen expressed on T cell malignancies, linking malignant tumor cells together ( Figure 1A ), potentially limiting the therapeutic efficacy of the bispecific antibody. Example 14 below demonstrates that the cytotoxic activity of a bispecific antibody binding to both CD3 and a tumor antigen against CD3-expressing tumor cell lines is reduced compared to that against tumor cell lines that do not express CD3 (CD3 knockout tumor cell lines). This result suggests that CD3 expression on tumor cell lines attenuates cytotoxic activity. Bispecific antigen-binding molecules for use in T cell tumors (which are often CD3-positive) should target molecules other than CD3 for inducing effector T cells.

[0059] The target tumor antigen expressed on T cell tumor cells is a different antigen from the target antigen on normal T cells. Preferably, "(1) at least one portion that specifically binds to the target tumor antigen expressed on T cell tumor cells" and "(2) at least one portion that specifically binds to the target antigen on normal T cells, the antigen having a subtype" of the bispecific antigen-binding molecule are configured to bind to different antigens, and each binds to a different type of antigen expressed on T cell tumor cells or normal T cells.

[0060] In a specific embodiment, it is preferable that the target tumor antigen expressed in T cell tumor cells is expressed at a higher frequency in T cell tumor cells than in normal T cells, even if it is present in normal T cells. For example, for the target tumor antigen, the ratio of the number of molecules expressed per T cell tumor cell to the number of molecules expressed per normal T cell may be 110% or more, and preferably, for example, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more. The number of molecules of the target tumor antigen expressed per cell can be determined by flow cytometry analysis using a fluorescent dye-labeled antibody that specifically binds to the target tumor antigen. The target tumor antigen expressed in T cell tumor cells is expressed at a higher frequency in T cell tumor cells than in normal T cells, so that the bispecific antigen-binding molecule can crosslink a sufficient number of normal T cells and T cell tumor cells for treatment, thereby achieving a therapeutic effect. Specific examples of target tumor antigens are described below.

[0061] In the present invention, the subject to be treated may be a subject suffering from any T-cell tumor, and may be a subject in a state of T-cell dysfunction. In a specific embodiment, the T-cell tumor in a subject in a state of T-cell dysfunction may be adult T-cell leukemia (ATL), peripheral T-cell lymphoma (PTCL), or T-cell acute lymphoblastic leukemia (TALL). Peripheral T-cell lymphoma (PTCL) is a general term for lymphomas originating from T cells that have undergone differentiation and maturation in the thymus and migrated to peripheral organs. PTCL accounts for approximately 10% of all lymphomas in Japan. PTCL is classified 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 histopathologically difficult to differentiate from adult T-cell lymphoma (ATL), and requires screening for anti-HTLV-1 antibodies. In a particular embodiment, the T-cell neoplasm in a subject with T-cell dysfunction is adult T-cell leukemia (ATL).

[0062] A state of T cell dysfunction in a subject can be predicted by detecting elevated expression levels of immunoinhibitory checkpoint molecules (immune exhaustion molecules) in T cells compared to expression levels in T cells in a normal state. Examples of immunoinhibitory checkpoint molecules include, but are not limited to, PD-1, TIM-3, CTLA-4, LAG-3, and TIGIT. It has been reported that high surface expression of PD-1 is associated with T cell exhaustion, and that the expression level of PD-1 correlates with the degree of T cell exhaustion (Wei F, et al. Proc Natl Acad Sci USA. 2013). The expression level of an immunoinhibitory checkpoint molecule in T cells in a normal state can be, for example, the expression level of an immunoinhibitory checkpoint molecule in a normal human, the average expression level of an immunoinhibitory checkpoint molecule in T cells from multiple normal humans, or the expression level of an immunoinhibitory checkpoint molecule in a sample collected before the patient fell into a state of T cell dysfunction. Expression of an immune checkpoint molecule can be determined, for example, by flow cytometry analysis using a fluorescent dye-labeled antibody.

[0063] In Examples 16 and 17 described below, under conditions for adding the bispecific antibody of the present invention, the cytotoxic activity against tumor cells of PBMCs collected from ATL patients, who were suggested to have high PD-1 expression and T cell dysfunction, was equal to or greater than that of PBMCs from healthy individuals. Based on the above, it is believed that some ATL patients are in a state of T cell dysfunction, and that the bispecific antibody of the present invention may induce cytotoxic activity even in patients with T cell dysfunction.

[0064] Furthermore, a subject's T cell dysfunction state can be predicted by comparing the gene expression profile of the subject's T cells with that of T cells from a healthy individual. For example, global gene expression analysis (e.g., RNA-seq) is performed on the subject's T cells, and the resulting expression data is used to classify the expressed genes of all T cells or specific subsets, such as CD8+ T cells, based on their biological function (e.g., activation-related, cell cycle-related, pro-apoptotic, pro-survival, T cell exhaustion-related, and cytotoxicity-related gene groups). By comparing the expression patterns in each of these functional categories with corresponding cells from a healthy individual (T cells or the specific subsets), the functional state of the subject's T cells in terms of immune response can be predicted. Global gene expression analysis can be performed using either bulk RNA sequencing (bulk RNA-seq), which targets RNA obtained from a T cell population, or single-cell RNA sequencing (scRNA-seq), which obtains expression information at the single-cell level.

[0065] In Example 18 described below, single-cell RNA-seq analysis showed that the expression of genes involved in cytotoxicity was reduced in CD8-positive T cells from two ATL patients tested. This is thought to suggest that CD8-positive T cells are unable to exert cytotoxic activity against tumor cells, i.e., that the anti-tumor immune response is limited. In Example 19 described below, it was confirmed that the bispecific antibody of the present invention that recognizes TRBCs can also induce cytotoxic activity in PBMCs derived from patients with T cell dysfunction as described in Example 18.

[0066] <Normal T cell antigen-binding moiety> In the present invention, the phrase "a moiety that specifically binds to a normal T cell target antigen, the antigen having a subtype" (hereinafter referred to as a normal T cell antigen-binding moiety) refers to a polypeptide molecule that specifically binds to a target antigen expressed on normal T cells. The normal T cell antigen-binding moiety comprises an antibody or a fragment thereof. In one aspect, the normal T cell antigen-binding moiety is capable of activating TCR signaling via the normal T cell target antigen (T cell activation). Furthermore, it is preferable that the normal T cell antigen-binding moiety is capable of inducing a cellular response such as cytokine production or cytotoxic activity of T cells.

[0067] "Subtyped antigens" refer to substances (e.g., peptides, proteins, sugars, lipids, or other organic or inorganic substances) that constitute T cells and have variants that have the same or substantially the same function but differ in structure (e.g., the amino acid sequence of a peptide or protein, its three-dimensional structure, the structural units or types of sugars, or the sequence or types of structural elements of lipids), and each variant is called a subtype. In one aspect, it is preferred that only one subtype of a "subtyped antigen" is expressed on each T cell. In one example, a "subtyped antigen" may be, but is not limited to, an antigen with subtypes based on TCR diversity generated by somatic recombination (also known as "V(D)J gene rearrangement"). Most T cell receptors are composed of an α chain and a β chain. The TCR α chain and β chain consist of an N-terminal variable region and a C-terminal constant region. TCR diversity is generated by somatic recombination, which occurs when each TCR chain selects a variable region (V), a diverse region (D), a joining region (J), and a constant region (C). The V, D, and J gene segments are contained in the β chain, and the V and J gene segments are contained in the α chain. For example, one of 30 TRBV polypeptides generated by V(D)J gene rearrangement and allelic exclusion is expressed on each T cell (WO 2022 / 119955). TRBV polypeptides are an example of antigens with 30 subtypes. However, "antigens with subtypes" are antigens expressed anywhere on T cells and are not limited to antigens present on TCRs.

[0068] The number of subtypes possessed by an "antigen having subtypes" is not particularly limited, as long as it is two or more. If the number of subtypes is large, the number of normal T cells to which the normal T cell antigen-binding portion of the bispecific antigen-binding molecule can bind via the target antigen is small, and therefore a sufficient number of activated T cells may not be provided for the treatment of T cell tumors. In one embodiment, the number of subtypes possessed by an "antigen having subtypes" may be, for example, 50 or less, and is 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.

[0069] A normal T cell population contains a mixture of normal T cells expressing any one of the subtypes of a "normal T cell target antigen, which has subtypes." For example, if the "antigen having subtypes" has three subtypes (subtypes A, B, and C), the normal T cell population contains a mixture of cells expressing subtype A, cells expressing subtype B, and cells expressing subtype C. T cell tumor cells also typically express one of the above subtypes (e.g., any of subtypes A to C). The normal T cell 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, if the subtype expressed in the T cell tumor cells is subtype A, the normal T cell antigen-binding portion of the bispecific antigen-binding molecule is configured to specifically bind to subtype B or subtype C. The tumor antigen-binding portion of the bispecific antigen-binding molecule is configured to specifically bind to an antigen that is expressed at a high frequency in T cell tumor cells, other than the "normal T cell target antigen, which has a subtype." This enables the bispecific antigen-binding molecule to appropriately crosslink normal T cells and T cell tumor cells.

[0070] In the present invention, normal T cells are preferably activated by binding of a bispecific antigen-binding molecule to a target antigen on normal T cells. T cell activation can be evaluated, for example, as in the Reference Examples described below, by culturing T cells in the presence of IL-2 and the normal T cell antigen-binding portion of the bispecific antigen-binding molecule, and observing or measuring the cell phenotype, T cell activation markers, cell proliferation, cell viability, and secreted cytokine levels.

[0071] In the present invention, in order to provide a sufficient number of activated T cells for the treatment of T-cell tumors, it is preferable that a sufficient proportion of subtypes of target antigens on normal T cells is present. It is preferable that the subtype of target antigens on normal T cells is expressed in 25% or more of the total number of cells in a normal T cell population, and more preferably in 30% or more, 40% or more, or 50% or more of the cells. When selecting "antigens with subtypes," the proportion of subtypes of target antigens on normal T cells may be determined based on the average value in humans or on analytical values ​​in patients to be treated. In one aspect, in order to provide a sufficient number of activated T cells for the treatment of T-cell tumors, the blood concentration of cells expressing subtypes of target antigens on normal T cells may be 50 cells / μL or more, and preferably 100 cells / μL or more, 200 cells / μL or more, or 300 cells / μL or more. Furthermore, it is preferable that the "antigens with subtypes" are present in αβ T cells, not in γδ T cells. This is because γδT cells account for only a few percent or less of all T cells, and therefore a sufficient number of T cells cannot be mobilized to damage tumor cells and achieve a therapeutic effect.

[0072] In one embodiment, the subtype of the target antigen on normal T cells is any one of the subtypes of antigen subtypes that are not the subtype of the antigen expressed on T cell tumor cells. For example, since a cell population of clonally expanded T cell tumor cells has any one of the subtypes, a subtype different from the subtype expressed on the cell population of T cell tumor cells can be selected as the target antigen on normal T cells of the bispecific antigen-binding molecule.

[0073] In one aspect, a target antigen on a normal T cell is not expressed on a tumor cell when the subtype of the target antigen on a normal T cell is one of the subtypes of an antigen that is not a subtype of an antigen expressed on a T-cell tumor cell.

[0074] In one aspect, the target antigen on normal T cells and the antigen having a subtype is TRBC (T cell receptor beta constant region), the subtype expressed on T cell tumor cells is TRBC1, and the subtype of the target antigen on normal T cells is TRBC2. That is, if the subtype of TRBC expressed on T cell tumor cells in a subject is analyzed and found to be TRBC1, the target antigen on normal T cells of the bispecific antigen-binding molecule can be TRBC2. This allows normal T cells to be mobilized as effector cells by the bispecific antigen-binding molecule.

[0075] In a further aspect, the target antigen on normal T cells and the antigen having a subtype is TRBC, the subtype expressed on T cell tumor cells is TRBC2, and the subtype of the target antigen on normal T cells is TRBC1. That is, if the subtype of TRBC expressed on T cell tumor cells in a subject is analyzed and found to be TRBC2, the target antigen on normal T cells of the bispecific antigen-binding molecule can be TRBC1. This allows normal T cells to be mobilized as effector cells by the bispecific antigen-binding molecule.

[0076] In a further aspect, the target antigen on normal T cells and the antigen having a subtype is TRBC, the T cell tumor cells are TRBC1-negative and TRBC2-negative, and the subtype of the target antigen on normal T cells is TRBC1 or TRBC2. That is, if the subtype of TRBC expressed on T cell tumor cells in a subject is analyzed and they are TRBC1-negative and TRBC2-negative, the target antigen on normal T cells of the bispecific antigen-binding molecule may be either TRBC1 or TRBC2. In this case, since neither TRBC1 nor TRBC2 is expressed on T cell tumor cells but either is expressed on normal T cells, normal T cells can be mobilized as effector cells by the action of the bispecific antigen-binding molecule having a normal T cell antigen-binding portion that binds to either TRBC1 or TRBC2.

[0077] As used herein, TRBC1 and TRBC2 refer to two functionally identical proteins of the TCR β chain constant region. T cell receptors (TCRs) are antigen receptor molecules expressed on T cells and recognize antigens bound to major histocompatibility complex (MHC) molecules. The TCR β chain constant region locus (Chr7:q34) contains two functionally identical genes: TRBC1 (Gene ID: 28639) and TRBC2 (Gene ID: 28636). The mature proteins of TRBC1 and TRBC2 differ by only four amino acid residues. TCRs containing TRBC1 and TRBC2 (TRBC1-TCR and TRBC2-TCR) can be distinguished by antibodies despite having nearly identical amino acid sequences (Maciocia PM, et al., supra). Furthermore, peripheral blood T cells from healthy individuals contain a mixture of approximately 35% TRBC1-positive cells and 65% TRBC2-positive cells, and flow cytometry and immunohistochemistry (IHC) have confirmed the monoclonal nature of TRBCs in many types of T-cell malignancies (Maciocia PM, et al., supra).

[0078] It has been reported that normal T cell populations contain a mixture of cells expressing TRBC1 and cells expressing TRBC2, but that the entire cell population of T cell tumor cells exclusively expresses either TRBC1 or TRBC2 (Maciocia PM, et al., supra). Based on this, it has been proposed to use TRBC1 or TRBC2 as target antigens on tumor cells (Japanese Patent No. 6767872 (Japanese Patent Application No. 2016-554603)). However, it was not known to use a TRBC subtype different from the TRBC subtype expressed on tumor cells as a target antigen on normal T cells or effector cells.

[0079] In a particular embodiment, the therapeutic agent of the present invention for treating T-cell tumors in a subject with T-cell dysfunction consists of a bispecific antigen-binding molecule comprising: at least one moiety that specifically binds to a target tumor antigen expressed on T-cell tumor cells; and at least one moiety that specifically binds to either TRBC1 or 2 expressed on normal T cells.

[0080] In the present invention, the term "moiety that specifically binds to TRBC1 or 2 expressed on normal T cells" (hereinafter referred to as TRBC1 or 2 binding moiety) refers to a polypeptide molecule that specifically binds to TRBC1 or 2 expressed on normal T cells. The TRBC1 or 2 binding moiety includes an antibody or a fragment thereof. In one aspect, the TRBC1 or 2 binding moiety is capable of activating TCR signalling via TRBC1 or 2 (T cell activation). Furthermore, it is preferred that the moiety induces a cellular response such as cytokine production or cytotoxic activity in T cells.

[0081] Preferably, the bispecific antigen-binding molecule simultaneously binds to TRBC1 or 2 expressed on normal T cells and to a target tumor antigen expressed on T-cell tumor cells.

[0082] In one aspect, when T cell tumor cells are TRBC1 positive, the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC2 expressed on normal T cells, and when T cell tumor cells are TRBC2 positive, the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to TRBC1 expressed on normal T cells. By using a TRBC of a different subtype to that expressed on T cell tumor cells as a target for effector cells (T lymphocytes) (when tumor cells express TRBC1, the target for effector cells is TRBC2, and when tumor cells express TRBC2, the target for effector cells is TRBC1), there is no risk of T cell dysfunction and a high therapeutic effect can be achieved.

[0083] In the Examples below, it has been shown that a bispecific antibody whose normal T cell antigen-binding portion binds to TRBC2 has higher cytotoxic activity against TRBC1-positive tumor cell lines compared to a TRBC2-positive tumor cell line (Example 14). It has also been shown that a bispecific antibody whose normal T cell antigen-binding portion binds to TRBC1 has higher cytotoxic activity against TRBC2-positive tumor cell lines (Example 4). These findings confirm that in bispecific antibodies for tumor treatment, high cytotoxic activity can be achieved by having the normal T cell antigen-binding portion recognize a TRBC subtype that is different from the TRBC expressed on T cell tumor cells.

[0084] 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". Also, 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". This is based on the fact that normal T cell populations contain a mixture of cells that express TRBC1 and cells that express TRBC2, but that the entire cell population of T cell tumor cells expresses either TRBC1 or TRBC2 exclusively (Maciocia PM, et al., supra).

[0085] In one embodiment, T cell tumor cells may be TRBC1-negative and TRBC2-negative. When T cell tumor cells are TRBC1-negative and TRBC2-negative, the target antigen on normal T cells of the bispecific antigen-binding molecule may be either TRBC1 or TRBC2, i.e., it may comprise at least one moiety that specifically binds to TRBC1 expressed on normal T cells, or at least one moiety that specifically binds to TRBC2 expressed on normal T cells.

[0086] In one embodiment, the target tumour antigen is neither TRBC1 nor TRBC2, i.e. at least one moiety which specifically binds to a target tumour antigen expressed on said T-cell tumour cells is designed to specifically bind to an antigen selected from among TRBC1 and TRBC2 and therefore does not bind or does not substantially bind to either TRBC1 or TRBC2.

[0087] It has been proposed to treat T-cell lymphoma or leukemia using, for example, a chimeric antigen receptor (CAR) containing an antigen-binding domain that selectively binds to either TRBC1 or TRBC2, with TRBC1 as the target antigen on tumor cells in the case of TRBC1-positive T-cell malignancies, and TRBC2 as the target antigen on tumor cells in the case of TRBC2-positive malignancies (Maciocia PM, et al., supra, 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 be killed, potentially resulting in T-cell immunodeficiency. In the present invention, a bispecific antigen-binding molecule is used that targets tumor cells with an antigen other than TRBC expressed in T-cell malignant tumors, and that targets effector cells (T lymphocytes) with a TRBC subtype different from the TRBC subtype expressed in T-cell malignant tumors, thereby inducing normal T cells to kill tumor cells, thereby reducing the possibility of causing T-cell immunodeficiency.

[0088] It has been reported that T cell tumor cells express either TRBC1 or TRBC2 exclusively in the entire cell population. However, there may be cases where T cell tumor cells express neither TRBC1 nor TRBC2. For example, this occurs when T cells that do not express TCR become malignant. The present invention can treat T cell tumors even when T cell tumor cells do not express TRBC1 or TRBC2. Because normal T cells express either TRBC1 or TRBC2, a bispecific antigen-binding molecule that targets either TRBC1 or TRBC2 as a T cell antigen and an antigen other than TRBC expressed on T cell tumor cells as a tumor cell target can be used to induce and kill approximately half of the normal T cell population.

[0089] In the Examples below, it has been shown that both bispecific antibodies whose normal T cell antigen-binding moiety binds to TRBC1 and bispecific antibodies whose normal T cell antigen-binding moiety binds to TRBC2 have cytotoxic activity against tumour cell lines that express neither TRBC1 nor TRBC2 (Examples 3 and 5).

[0090] Whether a cell is TRBC1 positive or negative can be determined by flow cytometry analysis using a fluorochrome-labeled antibody that specifically binds to TRBC1. Examples of antibodies that specifically bind to TRBC1 include the JOVI-1 clone (US10730942 Protein-based T-cell receptor knockdown SEQ ID No. 15, SEQ ID No. 16). Similarly, whether a T cell is TRBC2 positive or negative can be determined by flow cytometry analysis using a fluorochrome-labeled antibody that specifically binds to TRBC2. Examples of antibodies that specifically bind to TRBC2 include the various antibodies described in WO2020 / 089644.

[0091] Whether a cell antigen is expressed positively or negatively can be determined based on the chart obtained by flow cytometry analysis. The position of the antigen on the chart may vary depending on the instrument's voltage settings, sensitivity settings, antibody clone used, staining conditions, dye used, etc., but those skilled in the art can delineate the chart appropriately so as not to separate cell populations recognized as a single group. Whether a cell antigen is expressed positively or negatively can be determined using an isotype control antibody as a negative control. An isotype control antibody is an antibody that does not react with a specific antigen. Generally, in experiments using antibodies, background signals can arise due to nonspecific binding to proteins other than the target or binding to Fc receptors on the cell surface. By comparing with a system using a negative control antibody, the specificity of the primary antibody's reaction with the antigen of interest can be confirmed. Furthermore, the background effect is eliminated, allowing for accurate interpretation of signal intensity.

[0092] <TRBC1-binding moiety> The bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may comprise a TRBC1-binding moiety and a tumor antigen-binding moiety. The TRBC1-binding moiety may be the anti-TRBC1 antibody described in U.S. Patent No. 10,730,942 (US10,730,942 Protein-based T-cell receptor knockdown SEQ ID No. 15, SEQ ID No. 16) or the humanized anti-TRBC1 antibody described in Japanese Patent No. 7,177,794 (corresponding to WO2018 / 224844) (SEQ ID NOs: 9 and 19 in Japanese Patent No. 7,177,794).

[0093] In one aspect, the bispecific antigen-binding molecule comprises at least one moiety which specifically binds to TRBC1 expressed on normal T cells, said at least one moiety comprising: a VH domain comprising 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, and a VL domain comprising 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.

[0094] In a particular aspect, the at least one moiety which specifically binds to TRBC1 expressed on normal T cells comprises a heavy chain variable region VH which is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region VL which is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5. In a further particular aspect, the at least one moiety which specifically binds to TRBC1 expressed on normal T cells comprises a heavy chain variable region VH which 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 which 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. The amino acid mutations (deletions, insertions and substitutions) to the amino acid sequence of SEQ ID NO: 4 or 5 may be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0095] In a particular aspect, at least one portion which specifically binds to TRBC1 expressed on 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: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149. In a further particular aspect, at least one portion which specifically binds to TRBC1 expressed on 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: 148 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: 149. The amino acid mutations (deletions, insertions, and substitutions) to the amino acid sequence of SEQ ID NO: 148 or 149 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0096] As used herein, "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with those in the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished by a variety of methods within the skill of one in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0097] In certain aspects, amino acid sequence variants of bispecific antigen-binding molecules comprising the TRBC1-binding moiety are generated to improve the binding affinity and / or other biological properties of the TRBC1-binding moiety. Amino acid sequence variants can be generated by introducing appropriate mutations (deletions, insertions, and substitutions) into the nucleotide sequence encoding the anti-TRBC1 antibody or by peptide synthesis. Mutations can be introduced into the complementarity-determining regions (CDRs) or framework regions (FRs) of the antibody, depending on the properties to be improved. Amino acid sequence variants are screened for retention or improvement of binding affinity, retention or improvement of antigen specificity, reduction of immunogenicity, etc.

[0098] <TRBC2 binding moiety> The bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention may comprise a TRBC2 binding moiety and a tumor antigen binding moiety. The TRBC2 binding moiety may be any of the various anti-TRBC2 antibodies or antigen-binding domains that bind to TRBC2 described in WO2020 / 089644. The TRBC2 binding moiety may comprise the VH domain of hJOVI-1 of WO2020 / 089644 (SEQ ID NO: 1 of WO2020 / 089644) with the 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).

[0099] In one aspect, the bispecific antigen-binding molecule comprises at least one moiety which specifically binds to TRBC2 expressed on normal T cells, said at least one moiety comprising: a VH domain comprising 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, and a VL domain comprising 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.

[0100] In a particular aspect, at least one moiety which specifically binds to TRBC2 expressed on 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 particular aspect, at least one moiety which specifically binds to TRBC2 expressed on 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 sequence of SEQ ID NO: 6 or 7 may be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0101] In certain aspects, amino acid sequence variants of bispecific antigen-binding molecules comprising the TRBC2-binding moiety are generated to improve the binding affinity and / or other biological properties of the TRBC2-binding moiety. Amino acid sequence variants can be generated, for example, by introducing appropriate mutations (deletions, insertions, and substitutions) into the nucleotide sequence encoding the anti-TRBC2 antibody or by peptide synthesis. Mutations can be introduced into the complementarity-determining regions (CDRs) or framework regions (FRs) of the antibody, depending on the properties to be improved. Amino acid sequence variants are screened for retained or improved binding affinity, retained or improved antigen specificity, reduced immunogenicity, etc.

[0102] In a particular aspect, at least one moiety that specifically binds to TRBC1 or 2 expressed on normal T cells may be an scFv or Fab fragment.

[0103] The CDR sequences of the TRBC1 or TRBC2 antigen binding site are listed in the table below.

[0104] <Tumor Antigen-Binding Moiety> The bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment of the present invention can comprise a TRBC1 or TRBC2-binding moiety and a tumor antigen-binding moiety. The tumor antigen targeted by the present invention is an antigen expressed on T-cell tumor cells. Many antigens expressed on T-cell tumor cells are known in the art and can be used as target tumor antigens in the present invention. Examples of target tumor antigens are described elsewhere in this application. The tumor antigen-binding moiety can be prepared based on known antibodies. Many antibodies against T-cell tumor antigens are also known in the art and can be used in the present invention. Those skilled in the art can select appropriate light chain variable regions and heavy chain variable regions from the amino acid sequence information of known antibodies and construct bispecific antibodies using genetic engineering techniques. In genetic engineering techniques, a gene sequence encoding the antibody can be inserted into an expression vector, which can be transformed into a host cell, and the host cell can be further cultured to produce the antibody. Examples of Fc domains, peptide linkers, and antibody formats that can be used in the constructed bispecific antibodies are described elsewhere in this application. Non-human antibodies (e.g., mouse antibodies) can be humanized and used to prepare bispecific antibodies. Humanization methods are known, and can be performed, for example, by grafting the CDRs of a non-human antibody onto human FRs. In general, when grafting mouse CDRs onto human FRs, it is considered advantageous to select human FRs that are highly identical to the mouse FRs in order to maintain the function of the CDRs.

[0105] If no monoclonal antibodies that specifically bind to a tumor antigen of interest are known, they can be produced using hybridoma or phage display techniques and used in bispecific antigen-binding molecules. In the hybridoma method, B cells collected from the spleen or lymph nodes of an animal, particularly a rat or mouse, immunized with a peptide of the tumor antigen of interest are fused with myeloma cells to produce hybridomas, and hybridomas that produce antibodies reactive with the antigen are selected. The selected hybridomas can then be used to produce monoclonal antibodies. The phage display method is a technique for selecting antibodies with affinity for a target molecule using a library in which antibody variable regions are functionally displayed on phage. The antibody genes contained in the phage can be sequenced, and monoclonal antibodies can be produced based on the sequence information.

[0106] In one embodiment, the tumor antigen targeted by the present invention is not a pan-T cell antigen such as CD2, CD3, CD5, or CD7, as these antigens may lead to T cell depletion and result in clinically unacceptable levels of immunodeficiency. In one embodiment, when a TRBC1-binding portion is used as the normal T cell antigen-binding portion of a bispecific antibody of the present invention, the tumor antigen targeted by the present invention is not TRBC1, or when a TRBC2-binding portion is used as the normal T cell antigen-binding portion of a bispecific antibody of the present invention, the tumor antigen targeted by the present invention is not TRBC2. These antigens may also lead to normal T cell depletion and result in clinically unacceptable levels of immunodeficiency. For example, WO 2022 / 177889 A1 discloses that a bispecific antibody that binds to both healthy TRBC1-positive T cells and TRBC1-positive cancer cells can kill healthy TRBC1-positive T cells by fratricide.

[0107] (Target Tumor Antigen) 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, CD 49c, CD49d, CD49e, CD49f, CD50, CD52, CD53, CD54, CD55, CD56, CD57, CD58 , CD59, CD60a, CD62L, CD63, CD68, CD69, CD70, CD71, CD73, CD74, CD75S, CD8 0, CD81, CD82, CD84, CD85A, CD85J, CD86, CD87, CD92, CD94, CD95, CD96, CD9 7, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD107a, CD107b, CD108, CD109, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD124, CD126, CD1 27, CD128, CD129, CD130, CD132, CD134, CD137, CD146, CD147, CD148, CD150 , CD152, CD153, CD40L (CD154), CD156b, CD158a, CD158b1, CD158b2, CD158 e1 / e2, CD158f, CD158g, CD158h, CD158i, CD158j, CD158k, CD159a, CD160, C D161, CD162, CD164, CD172g, CD178, CD181, CD182, CD183, CD184, CD185, C D186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198, CDw199, C D205, CD210a, CDw210b, CD212, CD215, CD217, CD218a, CD218b, CD220, CD22 1, CD222, CD223, CD224, CD225, CD226, CD227, CD229, CD230, CD231, CD244,CD245, CD246, CD247, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, C D264, CD267, CD268, CD270, CD272, CD273, CD274, CD275, CD277, CD278, CD279, CD283, CD288, CD289, CD 290, CD294, CD295, CD296, CD298, CD300a, CD300c, CD300e, CD305, CD306, CD307c, CD314, CD316, CD317 , CD319, CD321, CD328, CD351, CD352, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362, CD363. ,

[0108] In one embodiment, the target tumor antigen expressed on the T cell tumor cells may be selected from the group consisting of, but is not limited to, 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).

[0109] 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. CXCL13 (C-X-C chemokine ligand 13) is a chemokine ligand.

[0110] TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an immune checkpoint receptor present on NK cells, cytotoxic T cells, memory T cells, and regulatory T cells (Treg).

[0111] CADM1 (Cell Adhesion Molecule 1) is an intercellular adhesion molecule that belongs to the immunoglobulin superfamily cell adhesion molecule group (IgCAM).

[0112] GPR15 (G protein-coupled receptor 15) is an orphan class A G protein-coupled receptor, and is expressed in epithelial cells, synovial macrophages, endothelial cells, lymphocytes, and particularly T cells.

[0113] SLAM (signaling lymphocytic activation molecule) is a member of the signaling lymphocyte activation molecule (SLAM) family.

[0114] ICOS (inducible T-cell co-stimulator) is a 55-60 kDa disulfide-linked homodimeric T-cell surface glycoprotein, also known as CD278. CD134 is member 4 of the tumor necrosis factor receptor (TNFR) superfamily.

[0115] Anaplastic lymphoma kinase (ALK), also known as CD246, is a 220 kDa transmembrane glycoprotein that is predominantly expressed in the developing nervous system.

[0116] CD30 is a membrane-bound glycoprotein with a molecular weight of 105-120 kD that belongs to the TNF receptor superfamily and is strongly expressed on mononuclear Hodgkin cells and multinuclear Reed-Sternberg cells in Hodgkin lymphoma, as well as on tumor cells of anaplastic large cell lymphoma (ALCL).

[0117] ST2(L) is the product of the ST2 gene and exists in a secreted form (ST2) and a transmembrane receptor form (ST2L). The ST2 gene was cloned as a gene specifically expressed during the initiation of cell proliferation. Notch1 is a member of the Notch family. CD38 is also known as cyclic ADP-ribose hydrolase.

[0118] CD1a is a CD1 isoform. There are four CD1 isoforms in humans (CD1a, CD1b, CD1c, and CD1d). CD1a is a lipid-presenting molecule whose expression is essentially restricted to cortical / thymic T-cell acute lymphoblastic leukemia (cortical T-ALL) and Langerhans cell (LC) histiocytosis, and is virtually absent in human tissues except for developing cortical thymocytes and LC (Blood. 2019;133(21):2291-2304). CD47 is also known as integrin-associated protein (IAP).

[0119] IL-7Rα (CD127) is mainly expressed on thymocytes, dendritic cells, monocytes, mature T cells, etc. IL-7R is a type I cytokine receptor, a heterodimer of an α chain (CD127) and a common γ chain (γc chain). CD40L (CD40 ligand, CD154) is a ligand protein belonging to the TNF superfamily, and is mainly expressed on activated CD4 ＋ It is expressed on T cells. The CD40 / CD40L system has been suggested to be involved in tumorigenesis.

[0120] CCR1, CCR4, CCR7, CCR8, CCR10, CXCR4, CXCR7, TIGIT, CADM1, and GPR15 are expressed on adult T-cell leukemia (ATL) cells and therefore may serve as target antigens for ATL therapy (Leukemia & Lymphoma, 2006; 47(10): 2163-2173).

[0121] CXCR5, CXCL13, SLAM, ICOS, CD134, and CXCR3 are expressed on angioimmunoblastic T-cell lymphoma (AITL) cells and therefore may serve as target antigens for AITL treatment (Blood. 2004;103:236-241).

[0122] Anaplastic lymphoma kinase, CD30, ST2(L), and CCR5 are expressed in anaplastic large cell lymphoma cells and therefore may serve as target antigens for the treatment of peripheral T-cell lymphoma (PTCL), including anaplastic large cell lymphoma (Blood. 2004;103:236-241).

[0123] Notch1, CXCR4, CD38, CD1a, CCR9 (CD199), CD47, IL-7Rα (CD127), and CD40L (CD154) are expressed on T-cell acute lymphoblastic leukemia (TALL) cells and can be target antigens for TALL therapy (for CD1a, see Blood. 2019;133(21):2291-2304; for L-7Rα, see Blood. 2021;138(12):1040-1052; for CCR9 (CD199), see Blood. 2022;140(1):25-37; for CD38 and CD47, see Blood. 2022;140(1):45-57; for Notch1, CXCR4, and CD38, see Blood Cancer Discov 2021;2:19-31.)

[0124] (CCR4-binding moiety) 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 moiety and a TRBC1-binding moiety, or a CCR4-binding moiety and a TRBC2-binding moiety. The CCR4-binding moiety may be the anti-CCR4 antibody mogamulizumab described in Japanese Patent No. 4052515.

[0125] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CCR4, wherein said at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0126] In a particular aspect, at least one portion 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 more particular aspect, at least one portion 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. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 8 or 9 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0127] In certain aspects, at least one portion that specifically binds to CCR4 may be an scFv or Fab fragment.

[0128] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0129] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety that specifically binds to TRBC1 or 2 expressed on normal T cells (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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:149, or (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, and at least one moiety that specifically binds to a target tumor antigen expressed on 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.

[0130] (CD1a-binding moiety) 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 moiety and a TRBC1-binding moiety, or may comprise a CD1a-binding moiety and a TRBC2-binding moiety. The CD1a-binding moiety may be the anti-CD1a antibody SC02-113 described in WO2005 / 063819.

[0131] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD1a, wherein said at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0132] In a particular aspect, at least one portion 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 more particular aspect, at least one portion 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. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 40 or 41 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0133] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39.

[0134] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0135] (CCR9-binding moiety) 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 a CCR9-binding moiety and a TRBC2-binding moiety. The CCR9-binding moiety may be the anti-CCR9 antibody 9G7 described in WO2023 / 037125.

[0136] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CCR9, wherein said at least one portion comprises a VH domain comprising 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, and a VL domain comprising 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.

[0137] In a particular aspect, at least one portion 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 more particular aspect, at least one portion 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. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 131 or 132 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0138] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises: a VH domain comprising 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; and a VL domain comprising 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.

[0139] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0140] (CXCR4 binding moiety) 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 moiety and a TRBC1-binding moiety, or may comprise a CXCR4-binding moiety and a TRBC2-binding moiety. The CXCR4-binding moiety may be the anti-CXCR4 antibody (hz515H7 VH1 D76N-VL2) described in WO2010 / 125162.

[0141] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CXCR4, wherein said at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0142] In certain aspects, at least one portion 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 more particular aspects, at least one portion 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. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 133 or 134 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0143] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109; and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0144] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0145] (TIGIT-binding moiety) 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 moiety and a TRBC1-binding moiety, or a TIGIT-binding moiety and a TRBC2-binding moiety. The TIGIT-binding moiety may be the anti-TIGIT antibody Vibostolimab described in WO2016 / 028656.

[0146] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to TIGIT, wherein said at least one portion comprises: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:94.

[0147] In a specific aspect, at least one portion 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 a more specific aspect, at least one portion 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) relative to the amino acid sequence of SEQ ID NO: 135 or 136 can be present in the complementarity-determining regions (CDRs) or framework regions (FRs).

[0148] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91; and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:94.

[0149] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0150] (CCR8-binding moiety) 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 moiety and a TRBC1-binding moiety, or a CCR8-binding moiety and a TRBC2-binding moiety. The CCR8-binding moiety may be the anti-CCR8 antibody ABBV-514 described in WO2023 / 010054.

[0151] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CCR8, wherein said at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:97, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:100.

[0152] In a particular aspect, at least one portion 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 a more particular aspect, at least one portion 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. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 137 or 138 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0153] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:97; and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:100.

[0154] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0155] (CD30-binding moiety) 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 moiety and a TRBC1-binding moiety, or a CD30-binding moiety and a TRBC2-binding moiety. The CD30-binding moiety may be the anti-CD30 antibody AC10 described in Japanese Patent No. 4303964.

[0156] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD30, wherein said at least one portion comprises a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115, and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0157] In a particular aspect, at least one portion 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 a more particular aspect, at least one portion 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. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 139 or 140 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0158] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises: a VH domain comprising 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 a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115; and a VL domain comprising 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 a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0159] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0160] (CD40L-binding moiety) The target tumor antigen of the bispecific antigen-binding molecule constituting 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 moiety and a TRBC1-binding moiety, or may comprise a CD40L-binding moiety and a TRBC2-binding moiety. The CD40L-binding moiety may be the anti-CD40L (CD154) antibody ABI793 described in Japanese Patent Application Laid-Open No. 2003-526371 (WO 01 / 068860).

[0161] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD40L (CD154), wherein said at least one portion comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119, 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, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, 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.

[0162] In a particular aspect, at least one portion that specifically binds 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 more particular aspect, at least one portion that specifically binds 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. The amino acid mutations (deletions, insertions, and substitutions) to the amino acid sequence of SEQ ID NO: 141 or 142 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0163] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one portion which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) a VH domain comprising 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, and a VL domain comprising 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; or (b) a VH domain comprising 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, and a VL domain comprising 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; and At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119, 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; and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, 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.

[0164] In a particular aspect, in the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention, at least one moiety which specifically binds to TRBC1 or 2 expressed on normal T cells comprises: (a) at least one moiety which specifically binds to TRBC1 expressed on normal T cells, and 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 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 148 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149, or (b) at least one moiety which specifically binds to TRBC2 expressed on 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 At least one portion that specifically binds to a target tumor antigen expressed on a T-cell tumor cell 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.

[0165] In a more specific embodiment, amino acid sequence variants of a bispecific antigen-binding molecule comprising a tumor antigen-binding portion are generated to improve the binding affinity and / or other biological properties of the tumor antigen-binding portion. Amino acid sequence variants can be generated, for example, by introducing appropriate mutations (deletions, insertions, and substitutions) into the nucleotide sequence encoding the antibody or by peptide synthesis. Depending on the properties to be improved, mutations can be introduced into the complementarity-determining regions (CDRs) or framework regions (FRs) of the antibody. Amino acid sequence variants are screened for retention or improvement of binding affinity, retention or improvement of antigen specificity, reduction of immunogenicity, etc.

[0166] The CDR sequences of each tumor antigen-binding moiety are listed in the table below.

[0167] (Format) In a specific embodiment, in a bispecific antigen-binding molecule constituting a therapeutic agent of the present invention, at least one portion that specifically binds to an antigen having a subtype expressed on normal T cells is an scFv, and at least one portion that specifically binds to a target tumor antigen expressed on 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 embodiment, the bispecific antigen-binding molecule has at least one portion that specifically binds to an antigen having a subtype expressed on normal T cells is an scFv, and at least one portion that specifically binds to a target tumor antigen expressed on T cell tumor cells is an scFv, and the two scFvs are linked via a peptide linker.

[0168] A "peptide linker" is a peptide containing about 2 to 20 amino acids, e.g., (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 the group consisting of GGGGS (SEQ ID NO:51), GGGGSGGGGS (SEQ ID NO:52), GGGGSGGGGSGGGGGS (SEQ ID NO:64), SGGGGGSGGGG (SEQ ID NO:53) and GGGGSGGGGSGGGG (SEQ ID NO:54), although the sequences GSPGSSSSGSGS (SEQ ID NO:55), (G ４ S) ４ , GSGSGSG (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) may also be used.

[0169] In a specific embodiment, the bispecific antigen-binding molecule constituting the therapeutic agent of the present invention further comprises an Fc domain. In a more specific embodiment, the Fc domain is an Fc domain of IgG1, IgG2, IgG3, or IgG4, and preferably an IgG1 Fc domain. In a more specific embodiment, the polypeptide having the IgG1 Fc domain can have one or more amino acid mutations, particularly amino acid mutations that alter the binding ability to Fc receptors, and more preferably amino acid mutations that reduce the binding ability to Fc receptors.

[0170] In certain embodiments, the Fc domain is of the human IgG1 isotype and can have a mutation of leucine at positions 234 and 235 to alanine, L234AL235A (LALA mutation), or a corresponding mutation. These mutations can occur at equivalent positions in other isotypes and subtypes. The LALA mutation abolishes binding to complement component (C1q) and Fc gamma receptor (FcgR), thereby preventing in vitro FcgR-mediated coactivation of innate immune effector cells, including natural killer (NK) cells, monocytes / macrophages, and neutrophils, without altering functional binding to FcRn (fetal Fc receptor) (Clin Cancer Res (2016) 22 (13): 3286-3297.). Thus, bispecific antigen-binding molecules having an Fc domain with the LALA mutation lack complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) activity, and the only immune effector cells involved may be T cells.

[0171] In certain embodiments, the format of the bispecific antigen-binding molecule may be any one of the structures / formats 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. 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.

[0172] In a particular embodiment, the bispecific antigen-binding molecule is monovalent for both a target antigen on normal T cells and a target tumor antigen expressed on T-cell tumor cells.

[0173] In a particular embodiment, the bispecific antigen-binding molecule is bivalent for both a target antigen on normal T cells and a target tumor antigen expressed on T-cell tumor cells.

[0174] In a particular embodiment, the bispecific antigen-binding molecule is trivalent to both a target antigen on normal T cells and a target tumor antigen expressed on T-cell tumor cells.

[0175] In a particular embodiment, the bispecific antigen-binding molecule is monovalent for a target antigen on normal T cells and bivalent for a target tumor antigen expressed on T cell tumor cells.

[0176] In a particular embodiment, the bispecific antigen-binding molecule is bivalent for a target antigen on normal T cells and monovalent for both target tumor antigens expressed on T cell tumor cells.

[0177] In a specific aspect, the bispecific antigen-binding molecule has two structures (i.e., bivalent + bivalent) in which an scFv that specifically binds to a target antigen on normal T cells (e.g., TRBC1 or 2) and a Fab fragment that specifically binds to a target tumor antigen expressed on T-cell tumor cells are linked by a peptide linker, and further has an Fc domain (Fab-scFv-Fc).

[0178] In certain embodiments, the therapeutic agents of the present invention can be administered in combination with chemotherapy agents, radiation and / or other agents used in cancer immunotherapy.

[0179] The dosage and interval of the therapeutic agent of the present invention may be adjusted individually to provide a plasma level of the bispecific antigen-binding molecule of the present invention sufficient to maintain the therapeutic effect. The dosage varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is approximately 0.1 to 50 mg / kg / day. A therapeutically effective plasma level can be achieved by administering multiple doses daily. The plasma level can be measured, for example, by HPLC.

[0180] In combination therapy, the therapeutic agent of the invention and one or more other agents or therapies can be administered simultaneously or sequentially. When administered simultaneously or sequentially, all agents or therapies can be administered by the same route or by different routes.

[0181] As used herein, "co-administration" refers to the administration of two or more drugs or therapies separated by a time interval of a few minutes to a few seconds or less. For example, two drugs or therapies are administered about 15 minutes to 1 minute or less apart. As used herein, "sequential administration" refers to the administration of two drugs or therapies separated by a time interval of a few minutes, a few hours, a few days, or a few weeks. For example, two drugs or therapies are administered at intervals of 15 minutes or more, 30 minutes or more, 60 minutes or more, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 2 weeks, 3 weeks, or 4 weeks.

[0182] The therapeutic agent of the present invention may be a pharmaceutical composition. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Formulation of pharmaceutical compositions with pharmaceutically acceptable carriers is known in the art (e.g., Remington: The Science and Practice of Pharmacy (23rd edition (2020))). Pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citrate, 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0183] The pharmaceutical composition of the present invention is preferably produced under conditions that comply with the manufacturing control and quality control regulations for drugs and quasi-drugs (good manufacturing practice, GMP).

[0184] In one embodiment, the pharmaceutical composition is a liquid such as a solution, suspension, emulsion, microemulsion, or gel. The pharmaceutical composition may be an aqueous formulation and may contain at least 50% (w / w) water. In one embodiment, the pharmaceutical composition is in a dosage form suitable for injection. The injection may be, for example, subcutaneous, intramuscular, intraperitoneal, intravitreal, or intravenous.

[0185] In one aspect, the pharmaceutical composition may be in a solid dosage form, e.g., a freeze-dried or spray-dried form, which may be administered, e.g., by adding solvents and / or diluents prior to administration.

[0186] The pharmaceutical composition may be provided in packaging such as a syringe, vial, or infusion bag. The syringe may contain the pharmaceutical composition in lyophilized form (which must be solubilized, for example, with water for injection, before administration) or in aqueous form. Other solid dosage forms may be powders, granules, tablets, or capsules.

[0187] In a specific embodiment, the therapeutic agent of the present invention can be used in a method for treating a T-cell tumor in a subject with T-cell dysfunction, the method comprising determining the subtype expressed in the subject's T-cell tumor cells and administering a therapeutic agent to the subject, the therapeutic agent comprising a bispecific antigen-binding molecule comprising at least one portion that specifically binds to a target antigen on normal T cells of a subtype different from the subtype determined to be expressed in the subject's T-cell tumor cells. The subtype of the antigen expressed in the subject's T-cell tumor cells 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, and when the subtype is TRBC2, various antibodies described in WO2020 / 089644 can be used.

[0188] A second embodiment of the present invention is a method for treating a T-cell malignancy in a subject with T-cell dysfunction, comprising administering a bispecific antigen-binding molecule to the subject having a T-cell malignancy.

[0189] In a particular aspect, the method of treatment further comprises determining the subtype of the target antigen on normal T cells expressed on the subject's T cell tumor cells, wherein the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to the target antigen on normal T cells of a subtype different from the subtype determined to be expressed on the subject's T cell tumor cells.

[0190] In certain embodiments, the treatment method can further include determining that the subject is in a state of T cell dysfunction. A subject's state of T cell dysfunction can be detected, for example, by comparing the expression level of an immunoinhibitory checkpoint molecule (e.g., PD-1, TIM-3, CTLA-4, LAG-3, or TIGIT) in the subject's T cells with the expression level in T cells in a normal state. An increase in the expression level of an immunoinhibitory checkpoint molecule in T cells compared to the expression level in T cells in a normal state suggests that the subject is in a state of T cell dysfunction. The expression level of an immunoinhibitory checkpoint molecule in T cells in a normal state can be, for example, the expression level of an immunoinhibitory checkpoint molecule in a normal human, the average expression value of an immunoinhibitory checkpoint molecule in T cells of multiple normal humans, or the expression level of an immunoinhibitory checkpoint molecule in a sample collected before the patient fell into a state of T cell dysfunction. The expression of an immune checkpoint molecule can be determined, for example, by flow cytometry analysis using a fluorescent dye-labeled antibody.

[0191] A state of T cell dysfunction in a subject can also be detected by comparing the gene expression profile of T cells derived from the subject with that of T cells derived from a healthy individual. Gene expression profiling can be performed by bulk RNA-seq analysis or scRNA-seq analysis, the details of which are as described above.

[0192] A third embodiment of the present invention is the use of a bispecific antigen-binding molecule in the manufacture of a medicament for treating a T-cell tumor in a subject with T-cell dysfunction. The bispecific antigen-binding molecule in the second and third embodiments of the present invention is the same as the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment described hereinabove. As used herein, the term "subject" refers to a mammal, such as a mouse, rat, or primate, and particularly a human.

[0193] <Bispecific antigen-binding molecules> A fourth embodiment of the present invention relates to bispecific antigen-binding molecules for use in a method for treating T-cell mediated malignancies in subjects with T-cell dysfunction.

[0194] The bispecific antigen-binding molecule of the present invention for use in the method for treating a T-cell tumor in a subject with T-cell dysfunction comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T-cell tumor cells, and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype; wherein the at least one moiety that specifically binds to a target antigen on normal T cells having a subtype is at least one moiety that specifically binds to TRBC1 or 2 (T-cell receptor beta constant region 1 or 2) of normal T cells (TRBC1-binding moiety or TRBC2-binding moiety). The amino acid sequences of the TRBC1-binding moiety or TRBC2-binding moiety and the target tumor antigen-binding moiety 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. The format that the bispecific antigen-binding molecule of the present invention can take is the same as that described above for the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment.

[0195] Furthermore, the present invention provides nucleic acid molecules encoding the heavy and light chains of the bispecific antigen-binding molecule of the fourth embodiment. The nucleic acid molecules of the present invention may be RNA, DNA, or cDNA.

[0196] The nucleic acid molecules of the present invention may be in the form of a vector, may be present in a vector, and / or may be part of a vector such as a plasmid, cosmid, or YAC. The vector may be, in particular, an expression vector, which provides expression of the bispecific antigen-binding molecules of the present invention in a host cell, host organism, and / or expression system. Expression vectors typically contain at least one nucleic acid of the present invention operably linked to one or more appropriate expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of elements and their sequences for expression in a particular host is within the knowledge of those skilled in the art. Specific examples of regulatory elements and other elements useful or essential for expression of the heavy and light chains of the bispecific antigen-binding molecules of the present invention include promoters, enhancers, terminators, integration elements, selection markers, leader sequences, reporter genes, etc.

[0197] The nucleic acid molecules 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 regarding the amino acid sequences of the heavy and light chains of the bispecific antigen-binding molecules of the present invention disclosed herein, and / or can be isolated from suitable natural sources.

[0198] Furthermore, the present invention provides a host cell that expresses or is capable of expressing one or more of the heavy chain and light chain of the bispecific antigen-binding molecule of the fourth aspect. 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.

[0199] Suitable bacterial cells include cells of gram-negative bacterial strains (eg, E. coli, Proteus, and Pseudomonas) and gram-positive bacterial strains (eg, Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0200] Suitable fungal and yeast cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or Saccharomyces, e.g., Saccharomyces cerevisiae, Schizosaccharomyces, e.g., Schizosaccharomyces pombe, Pichia, e.g., Pichia pastoris, and Pichia methanolica. Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0201] However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins can also be used in the present invention.

[0202] The heavy and light chains of the bispecific antigen-binding molecules of the present invention can be produced intracellularly as described above and then isolated from the host cells and optionally further purified, or can be produced extracellularly (e.g., in the medium in which the host cells are cultured) and then isolated from the medium and optionally further purified.

[0203] Methods and reagents for recombinant production of polypeptides are known in the art, such as particular appropriate expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques suitable for methods of making the bispecific antigen-binding molecules of the present invention are well known to those skilled in the art.

[0204] However, the bispecific antigen-binding molecules 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.

[0205] (Cytotoxic Activity) The bispecific antigen-binding molecules of the present invention having a TRBC1-binding portion or a TRBC2-binding portion can recruit TRBC1-positive T cells or TRBC2-positive T cells and induce tumor cell cytotoxicity and / or cell death. The cytotoxic activity of the bispecific antigen-binding molecule can be determined in vitro by culturing target tumor cells with the bispecific antigen-binding molecule in the presence of effector cells and measuring the viability of the target tumor cells. For example, activated T cells (e.g., T-LAK) can be used as effector cells. The mixing ratio of effector cells to target tumor cells may be 10:1 to 1:10. In one aspect, the bispecific antigen-binding molecule of the present invention is capable of inducing tumor cell cytotoxicity in vitro with an EC50 of less than about 500 pM, and in a preferred aspect, is capable of inducing tumor cell cytotoxicity 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.

[0206] The anti-TRBC1 antibody used in the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention can increase the rate of TRBC1-positive cells in human peripheral blood mononuclear cells (PBMCs). Specifically, as shown in Reference Example 1 below, when 10 nM anti-TRBC1 antibody was added to human PBMCs and cultured in the presence of IL-2, the rate of TRBC1-positive cells increased from 30% on Day 0 to just over 80% on Day 15. This suggests that the addition of the anti-TRBC1 antibody selectively proliferated TRBC1-positive cells. It is believed that the addition of a bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody also selectively proliferated TRBC1-positive cells.

[0207] The anti-TRBC1 antibody used in the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention is capable of increasing cytotoxic T cells in the presence of IL-2. Specifically, as shown in Reference Example 1 below, on Day 15, the proportion of effector memory T cells significantly increased from approximately 20% to approximately 70% in the presence of an anti-TRBC1 antibody and IL-2. This result suggests that stimulation with an anti-TRBC1 antibody can increase T cells that cause cytotoxic effects. Stimulation with a bispecific antibody that has the same antigen-binding portion as the anti-TRBC1 antibody is also thought to similarly increase T cells that cause cytotoxic effects.

[0208] The anti-TRBC1 antibody used in the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention is capable of activating T cells. Specifically, as shown in Reference Example 1 below, stimulation with the anti-TRBC1 antibody increased the ratio of CD69, Granzyme B, and Perforin-positive cells, suggesting that TRBC1-positive cells are activated by the anti-TRBC1 antibody. Bispecific antibodies having the same antigen-binding portion as the anti-TRBC1 antibody used in this example are also expected to have a similar T cell activation effect.

[0209] As shown in Reference Example 1 below, stimulation with the anti-TRBC1 antibody used in the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention did not significantly change the ratio of PD-1 positive cells. This suggests that T cells are less likely to become exhausted even when activated with an anti-TRBC1 antibody. It is also thought that T cells are less likely to become exhausted when a bispecific antibody having the same antigen-binding portion as the anti-TRBC1 antibody used in this example is used.

[0210] As shown in Reference Example 1 below, the anti-TRBC1 antibody used in the TRBC1-binding portion of the bispecific antigen-binding molecule of the present invention is able to induce T cell activation and release cytokines, but the amount of IL-6, a pro-inflammatory cytokine, released was very small. This indicates that anti-TRBC1 antibodies may have a low risk of cytokine release syndrome. This suggests that bispecific antibodies having the same antigen-binding portion as anti-TRBC1 antibodies also have a similarly low risk of cytokine release syndrome.

[0211] The anti-TRBC2 antibody used in the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention can increase the rate of TRBC2-positive cells in PBMCs. Specifically, as shown in Reference Example 2 below, cells that bind to TRBC1 antibodies (TRBC1-positive T cells) were removed from human PBMCs using beads, and then 10 nM of anti-TRBC2 antibody was added and cultured in the presence of IL-2. On Day 0, approximately 5 x 10 ６ ~7 x 10 ６ The number of cells was about 18 x 10 cells by Day 15. ６ ~42 x 10 ６ The number of cells increased to 100. This suggests that the addition of the anti-TRBC2 antibody selectively proliferated TRBC2-positive cells. It is thought that the addition of a bispecific antibody that has the same antigen-binding moiety as the anti-TRBC2 antibody also selectively proliferated TRBC2-positive cells.

[0212] The anti-TRBC2 antibody used in the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention is capable of increasing cytotoxic T cells in the presence of IL-2. Specifically, as shown in Reference Example 2 below, on Day 15, the proportion of effector memory T cells significantly increased from approximately 27% to approximately 81% in the presence of an anti-TRBC2 antibody and IL-2. This result suggests that stimulation with an anti-TRBC2 antibody can increase T cells that cause cytotoxic effects. Stimulation with a bispecific antibody that has the same antigen-binding portion as the anti-TRBC2 antibody is also thought to increase T cells that cause cytotoxic effects.

[0213] The anti-TRBC2 antibody used in the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention is capable of activating T cells. Specifically, as shown in Reference Example 2 below, stimulation with the anti-TRBC2 antibody increased the ratio of CD69, Granzyme B, and Perforin-positive cells, suggesting that TRBC2-positive cells are activated by the anti-TRBC2 antibody. Bispecific antibodies that have the same antigen-binding portion as the anti-TRBC2 antibody are also expected to have a similar T cell activation effect.

[0214] As shown in the Reference Examples below, stimulation with the anti-TRBC2 antibody used in the TRBC2-binding portion of the bispecific antigen-binding molecule of the present invention did not significantly change the ratio of PD-1 positive cells. This suggests that T cells are less likely to become exhausted even when activated with an anti-TRBC2 antibody. It is also thought that bispecific antibodies having the same antigen-binding portion as the anti-TRBC2 antibody are less likely to exhaust T cells.

[0215] As shown in Reference Example 2 below, the anti-TRBC2 antibody used as the TRBC2-binding moiety of the bispecific antigen-binding molecule of the present invention is able to induce T cell activation and release cytokines, but the amount of IL-6, a pro-inflammatory cytokine, released was very small. This indicates that anti-TRBC2 antibodies may have a low risk of cytokine release syndrome. This suggests that bispecific antibodies having the same antigen-binding moiety as anti-TRBC2 antibodies also have a similar low risk of cytokine release syndrome.

[0216] The bispecific antigen binding molecules having a TRBC1-binding moiety or a TRBC2-binding moiety of the present invention may be used in a method for (i) inducing cytotoxicity of tumour cells, (ii) increasing the rate of TRBC1-positive cells or increasing the rate of TRBC2-positive cells, (iii) increasing cytotoxic T cells, (iv) activating T cells, (v) stimulating or enhancing a cellular response without T cell exhaustion, (vi) stimulating or enhancing a cellular response without increasing the risk of cytokine release syndrome, (vii) treating T cell tumours in subjects with T cell dysfunction, (viii) delaying the progression of T cell tumours, or (ix) prolonging the survival of a subject suffering from a T cell tumour.

[0217] The bispecific antigen-binding molecules of the present invention can have antitumor activity. The antitumor activity can be measured in vivo, for example. For example, 1 x 10 human PBMCs were transfected into immunodeficient NSG mice (Jackson Laboratory Japan). ７ 1x10 cells / mouse were intravenously transplanted, and 7-10 days later, luciferase-stably expressing T cell tumor line was transplanted into the cells. ７ The mice are intravenously transplanted with 150 mg / kg of luciferin per mouse. Subsequently, the bispecific antigen-binding molecule of the present invention is administered at a dose of 0.5 mg / kg twice a week. Two to three weeks after the start of administration, the mice are intraperitoneally administered with 150 mg / kg of luciferin, and the T cell tumor burden is measured by quantifying in vivo luciferase activity using an IVIS imaging system (Revvity) or similar. The antitumor effect can be evaluated by comparing the T cell tumor burden between the control group and the bispecific antigen-binding molecule-administered group of the present invention.

[0218] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0219] Example 1: Construction of bispecific antibodies 1.1 Bispecific antibodies In this example, bispecific antibodies that bind to TRBC1 or 2 and a target tumor antigen are constructed. TRBC1 or 2 binding moiety The TRBC1 binding moiety was constructed based on the sequence information of the anti-TRBC1 antibody JOVI-1 clone described in US 10730942 (US 10730942 Protein-based T-cell receptor knockdown SEQ ID No. 15, SEQ ID No. 16). The TRBC2-binding moiety was generated based on the VH domain of hJOVI-1 (SEQ ID NO: 1 of WO2020 / 089644) with 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 VH and VL amino acid sequences are set forth in SEQ ID NOs: 6 and 7 of the present application).

[0220] The portion that binds to the target tumor antigen expressed on tumor cells was prepared based on the sequence information disclosed in the literature listed in the table below.

[0221] The heavy and light chain sequences of the bispecific antibody prepared in this example are shown below. FIG. 2A shows schematic diagrams of the Fab-scFv type, the Fab-scFv-Fc type, and the Fab-Fc-scFv type.

[0222] CCR0010 has the T255K, Y259F, and A327N mutations introduced into the heavy chain of CCR0001, and has a Fab-scFv structure. Previously developed anti-TRBC-2 antibodies were obtained by introducing mutations into the anti-TRBC-1 antibody (JOVI-1) (e.g., WO2015 / 132598A1, WO2020 / 089644A1), and the amino acid sequences of the CDRs of both antibodies share high homology.

[0223] 1.2 Production and Purification of Recombinant Antibodies The bispecific antibodies listed in Table 4 were constructed by adding signal sequences to the heavy and light chains listed in Table 4 and expressing them in expression vectors. Details of the heavy and light chain configurations of each bispecific antibody are as shown in Figures 3A-K. Specifically, bispecific antibody (heavy and light chain) expression vectors were constructed using DNA encoding the bispecific antibody and an expression vector (pcDNA3.4, Thermo Fisher Scientific). The combinations of heavy and light chains of the bispecific antibodies are as shown in Table 4. The above expression vectors were mixed at a 1:1 ratio and transfected into CHO cells at 0.8 μg of DNA per mL of culture medium using ExpiFectamine CHO Reagent (Thermo Fisher Scientific). The day after transfection and 5 days later, ExpiCHO Feed and ExpiFectamine CHO Enhancer were added, and the cells were cultured at 32°C for 10 to 12 days. The cells were removed from the culture medium by centrifugation and filtration, and the culture supernatant was collected. The antibody was purified by a combination of affinity chromatography using nickel-conjugated agarose and gel filtration chromatography, or a combination of Protein A affinity chromatography and gel filtration chromatography.

[0224] Example 2: Cell Preparation This example describes the preparation of cells used in the experiment. The following reagents were used: RPMI 1640 medium (Nacalai Tesque, 30264-56) FBS (fetal bovine serum) (Nichirei Biosciences, 175012): Protein was inactivated by incubation in a 56°C water bath for 30 minutes. Penicillin-streptomycin mixed solution (Nacalai Tesque, 2625384) MEM non-essential amino acid solution (100X) (Gibco, 11140-050) Sodium pyruvate solution 100mM (Gibco, 11360-070) 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 NOs: 144 and 145.) Imunase Injection 35 (IL-2, interleukin 2) (Shionogi Kyowa Pharmaceutical Industries): Dissolved in PBS to 250 IU / μL and stored at -25°C. PBS (phosphate buffered saline) (Gibco, 10010-023) Human PBMC (peripheral blood mononuclear cells) (CTL, CTL-UP1)

[0225] Various cell types were prepared as follows. T-LAK is a T cell population that expresses lymphokine-activated killer (LAK) function. T-LAK was prepared by optimizing the experimental system 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.). Specifically, human PBMCs were thawed in a 37°C water bath and washed once with culture medium. After culturing for 2 days in RPMI 1640 medium containing 10% FBS and supplemented with 10 nM anti-TRBC1 antibody and 100 IU / mL IL-2, the cells were further cultured in RPMI 1640 medium containing IL-2 and 10% FBS (for up to 17 days) to induce T-LAK development. The cells were passaged every 2 to 3 days, and 100 IU / mL of IL-2 was added when the culture medium was changed. The cell numbers on the initiation day (Day 0) and 15 days (Day 15) of culture were measured using an automatic cell counter (BIO RAD, TC20).

[0226] 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 Institutes of Therapeutic Research and Development, Health and Nutrition) (cell number JCRB1210). MT-2 cells are CCR4-positive (Haematologica 2018 Volume 103(1):126-135). They were cultured in RPMI medium containing 10% FBS, non-essential amino acids, and sodium pyruvate at 5% CO. ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0227] The MOLT-4 cells used in Example 3 were purchased from the National Institute of Therapeutic Research and Development, Japan. ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0228] CCRF-CEM9 used in the following examples expresses CD1a and TRBC2. CCRF-CEM9 was obtained by single-cell cloning of CCRF-CEM, a TALL-derived cell line, using the limiting dilution method. Specifically, a cell suspension of CCRF-CEM was cultured at 1 x 10 ５ The cells were diluted repeatedly from 0.1 cells / mL until the concentration reached 2.5 cells / mL. 100 μL of the 2.5 cells / mL diluted solution was added to two 96-well plates and cultured at 37°C. Each well (clone) in which cells had proliferated was expanded and analyzed for expression of TRBC1 and CD1a. As a result, CCRF-CEM9 was obtained, in which expression of both antigens was confirmed. Cultures were grown in RPMI medium containing 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0229] Reference Example 1 Verification of effector activity of anti-TRBC1 antibody and quantification of cytokine release amount Reagents used: Flow Cytometry Staining Buffer (eBioscience, 00-4222-26), Clear Back (MBL, MT-001), Fixable Viability Dye eFluor780 (invitrogen, 65-0865-14), Transcription Factor Buffer Set (BD, 562574), TF Fix / Perm Buffer (4x) (51-9008100): Used after diluting 4-fold with TF Diluent Buffer. TF Diluent Buffer (51-9008101) TF Perm / Wash Buffer (5x) (51-9008102): Used after diluting 5 times with distilled water. MILLIPLEX Human Cytokine / Chemokine / Growth Factor Panel A (Millipore, HCYTA-60K)

[0230] The staining antibodies are shown in the table below. *BV421, BV711: abbreviations for Brilliant Violet 421 (registered trademark) and Brilliant Violet 711 (registered trademark)

[0231] The anti-TRBC1 antibody was produced and purified by the method described in Example 1, and then dye-labeled using HiLyte Fluor 647 Labeling Kit-NH2 (Dojindo, LK15). The heavy and light chain sequences of the anti-TRBC1 antibody are SEQ ID NOs: 144 and 145.

[0232] As an isotype control, human IgG (ChromPure Human IgG, whole molecule, Jackson Immuno Research, 009-000-003) was similarly dye-labeled. Method The experiment was performed with reference to WO2021 / 173896 A1.

[0233] Cell surface staining method: Human PBMC or T-LAK were centrifuged (300g x 5 minutes), the supernatant was removed, and ClearBack was added. After 5 minutes of incubation at room temperature, various antibodies were added in Flow Cytometry Staining Buffer and allowed to react for 30 minutes at 4°C. After the reaction, the cells were washed twice by adding Flow Cytometry Staining Buffer, centrifuging (500g x 2 minutes), and discarding the supernatant. Signals were quantified by flow cytometry analysis using a BD LSRFortessa X-20 (BD Biosciences). Flow Jo_v10.6.1 was used as the analysis software.

[0234] Intracellular staining method: Human PBMC or T-LAK were centrifuged (300g x 5 minutes), the supernatant was removed, and ClearBack was added. After 5 minutes of incubation at room temperature, anti-CD3 antibody and anti-TRBC1 antibody were added in Flow Cytometry Staining Buffer and allowed to react for 30 minutes at 4°C. After the reaction, the cells were washed twice by adding Flow Cytometry Staining Buffer, centrifuging (500g x 2 minutes), and discarding the supernatant. TF Fix / Perm Buffer in the Transcription Factor Buffer Set was added, and the cells were fixed and permeabilized by incubating on ice for 30 minutes. After centrifugation (700g x 2 minutes), the cells were washed twice by adding TF Perm / Wash Buffer, centrifuging (700g x 2 minutes), and discarding the supernatant. Anti-Granzyme B antibody or anti-Perforin antibody was added to the TF Perm / Wash Buffer and allowed to react on ice for 30 minutes. After the reaction, the cells were washed twice by adding TF Perm / Wash Buffer, centrifuging (700g x 2 minutes), and discarding the supernatant. Signals were quantified by flow cytometry analysis using a BD LSRFortessa X-20 (BD Biosciences). Flow Jo_v10.6.1 was used as the analysis software.

[0235] Quantification of cytokine release: After culturing T-LAK with anti-TRBC1 antibody for 24 hours, the amount of released cytokines was quantified using MILLIPLEX Human Cytokine / Chemokine / Growth Factor Panel A. T-LAK was washed once with RPMI1640 medium (culture medium) containing 10% FBS, and the appropriate number of cells was counted and added to a 384-well plate. Anti-TRBC1 antibody was adjusted to final concentrations of 0, 0.1, 1.0, and 10 nM in the culture medium and added to the wells containing T-LAK. The incubation was carried out at 5% CO ２The plates were incubated at 37°C for 24 hours. The 384-well plate was centrifuged (300g, 5 minutes), and the supernatant was collected. IFN-γ, IL-2, IL-6, IL-10, and TNF-α were quantified using a MILLIPLEX Human Cytokine / Chemokine / Growth Factor Panel A. Quantitative values ​​were calculated automatically using a Luminex® 200x PONET® 3.1 system.

[0236] Results TRBC1-positive cell rate Figures 4A and 4B show the increase in the TRBC1-positive cell rate after the addition of anti-TRBC1 antibody. When 10 nM anti-TRBC1 antibody was added to human PBMCs and cultured in the presence of IL-2, the rate of TRBC1-positive cells increased from 30% on Day 0 to just over 80% by Day 15. This suggests that the addition of anti-TRBC1 antibody resulted in the selective proliferation of TRBC1-positive cells. It is thought that the addition of a bispecific antibody that has the same antigen-binding moiety as the anti-TRBC1 antibody used in this experiment would also result in the selective proliferation of TRBC1-positive cells.

[0237] Increase in Cytotoxic T Cells As shown in Figures 5A and B, T-LAK cells on Day 0 and Day 15 were stained with CD27 and CD45RA, and the cell ratios of effector memory, central memory, naive, and EMRA fractions were calculated. As a result, on Day 15, the effector memory ratio significantly increased from approximately 20% to approximately 70%, while the ratios of other fractions decreased. These results suggest that stimulation with anti-TRBC1 antibodies can increase T cells that cause cytotoxic effects. Stimulation with a bispecific antibody that has the same antigen-binding moiety as the anti-TRBC1 antibody used in this experiment is also likely to increase T cells that cause cytotoxic effects.

[0238] Evaluation of T Cell Activation Markers CD69 is a cell surface glycoprotein that is expressed very early after T cell or B cell activation, but is not expressed on resting lymphocytes. Cytotoxic T cells possess intracellular granules containing cytotoxic proteins (Granzyme B, Perforin). Granzyme B is a serine protease that, once delivered into target cells, cleaves various intracellular substrates, including caspases, to induce apoptosis in the target cells. Perforin disrupts the target cell membrane, facilitating the transport of Granzyme B into the target cells.

[0239] FIG. 6 shows the results of analyzing the percentage of positive cells for CD25, CD69, Granzyme B, and Perforin using T-LAK on Day 0 and Day 15.

[0240] In this experiment, although the ratio of CD25-positive cells decreased, the ratios of CD69, Granzyme B, and Perforin-positive cells increased, suggesting that TRBC1-positive cells were activated by the anti-TRBC1 antibody. Similar effects are expected for bispecific antibodies that have the same antigen-binding moiety as the anti-TRBC1 antibody used in this experiment. In this experiment, it was speculated that the addition of IL-2 during culture caused internalization and downregulation of CD25, resulting in a decrease in CD25 expression.

[0241] Changes in the Proportion of PD-1-Positive Cells Figure 7 shows the results of an analysis of the proportion of PD-1-positive cells, one of the immunosuppressive checkpoint molecules of T cells, using T-LAK on Day 0 and Day 15. Effector memory (EM) cells, which are functional cytotoxic T cells, show almost no expression of immunosuppressive checkpoint molecules such as PD-1 or TIGIT, whereas exhausted T cells, which are not functional, show high expression of the above immunosuppressive checkpoint molecules. The results in Figure 7 showed no significant change in the proportion of PD-1-positive cells. This suggests that T cells are less likely to become exhausted even when activated with an anti-TRBC1 antibody. It is also believed that bispecific antibodies with the same antigen-binding moiety as the anti-TRBC1 antibody used in this experiment also show less likelihood of T cell exhaustion.

[0242] Quantification of Cytokine Release Figure 8 shows the results of quantifying the release of IFN-γ, IL-2, IL-6, IL-10, and TNFα using Day 15 T-LAK cells after 24 hours of stimulation with 0.1, 1.0, or 10 nM anti-TRBC1 antibody. The results confirmed an antibody concentration-dependent increase in the release of each cytokine, suggesting that the anti-TRBC1 antibody induced T cell activation and cytokine release. In this experimental system, the release of the inflammatory cytokine IL-6 was minimal. These data suggest that anti-TRBC1 antibodies may pose a low risk of cytokine release syndrome. This suggests that bispecific antibodies with the same antigen-binding moiety as the anti-TRBC1 antibody used in this example also pose a low risk of cytokine release syndrome.

[0243] Reference Example 2: Evaluation of anti-TRBC2 antibody agonist activity Anti-TRBC2 antibodies were produced and purified by the method described in Example 1. The heavy and light chain sequences of the anti-TRBC2 antibodies are SEQ ID NOs: 146 and 147. The reagents and staining antibodies used in this Reference Example were the same as those described in Reference Example 1.

[0244] Results: Number of TRBC2-positive cells Human PBMCs were lysed and washed in a 37°C water bath. The washed human PBMCs were reacted with JOVI-1 mIgG (Santa Cruz) for 30 minutes at 4°C. After washing, the PBMCs reacted with the antibody were suspended in medium and reacted with Dynabeads M-280 Anti-Mouse IgG (Invitrogen) for 30 minutes at 4°C. Bead-unwanted cell complexes were collected on the wall of the tube using DynaMag (Invitrogen), and the supernatant containing TRBC2-positive T cells was collected. The collected cells were cultured for 2 days in RPMI 1640 medium containing 10% FBS and supplemented with 10 nM anti-TRBC2 antibody and 100 IU / mL IL-2, and then further cultured (for up to 17 days) in RPMI 1640 medium containing IL-2 and 10% FBS to induce T-LAK. Cells were passaged every 2-3 days, and 100 IU / mL IL-2 was added freshly when the culture medium was changed.

[0245] The number of cells on the initiation day of culture (Day 0) and on the 15th day (Day 15) was measured using an automatic cell counter (BIO RAD, TC20). On Day 0, approximately 5 × 10 ６ ~7 x 10 ６ The number of cells was about 18 x 10 cells by Day 15. ６ ~42 x 10 ６ The number of cells increased to 100 (Figure 9). This suggests that the addition of the anti-TRBC2 antibody resulted in the selective proliferation of TRBC2-positive cells. It is thought that the addition of a bispecific antibody with the same antigen-binding moiety as the anti-TRBC2 antibody used in this experiment would also result in the selective proliferation of TRBC2-positive cells.

[0246] Increase in Cytotoxic T Cells As shown in Figure 10, anti-TRBC2 antibody-sensitized T-LAK cells on Day 0 and Day 15 were stained with CD27 and CD45RA, and the cell ratios of effector memory (EM), central memory (CM), naive, and EMRA fractions were calculated. As a result, on Day 15, the effector memory ratio significantly increased from approximately 27% to approximately 81%, while the ratios of other fractions decreased. These results suggest that stimulation with anti-TRBC2 antibodies can increase the number of T cells that cause cytotoxicity. Stimulation with a bispecific antibody that has the same antigen-binding moiety as the anti-TRBC2 antibody used in this experiment is also likely to increase the number of T cells that cause cytotoxicity. Note that the CD3-positive, TRBC1-negative cell population was defined as TRBC2-positive cells.

[0247] Evaluation of T cell activation markers Figure 11 shows the results of analyzing the percentage of CD25-, CD69-, Granzyme B-, and Perforin-positive cells using anti-TRBC2 antibody-sensitized T-LAK on Day 0 and Day 15. In this experiment, the percentage of CD25-positive cells decreased, while the percentages of CD69-, Granzyme B-, and Perforin-positive cells increased, suggesting that TRBC2-positive cells were activated by the anti-TRBC2 antibody. Similar effects are expected for bispecific antibodies that share the same antigen-binding moiety as the anti-TRBC2 antibody used in this experiment. In this experiment, the addition of IL-2 during culture presumably led to internalization and downregulation of CD25, resulting in reduced CD25 expression. Note that the CD3-positive, TRBC1-negative cell population was defined as TRBC2-positive cells.

[0248] Changes in the Proportion of PD-1-Positive Cells Figure 12 shows the results of an analysis of the proportion of PD-1-positive cells, one of the immunosuppressive checkpoint molecules of T cells, using anti-TRBC2 antibody-sensitized T-LAK on Day 0 and Day 15. Effector memory (EM) cells, which are functional cytotoxic T cells, show almost no expression of immunosuppressive checkpoint molecules such as PD-1 or TIGIT, whereas exhausted T cells, which are functionally inactive, show high expression of the above immunosuppressive checkpoint molecules. The results in Figure 12 showed no significant change in the proportion of PD-1-positive cells. This suggests that T cells are less likely to become exhausted when activated with an anti-TRBC2 antibody, similar to an anti-TRBC1 antibody. It is believed that bispecific antibodies with the same antigen-binding moiety as the anti-TRBC2 antibody used in this experiment also show less likelihood of T cell exhaustion.

[0249] Quantification of cytokine release. Figure 13 shows the results of quantifying the release of IFN-γ, IL-2, IL-6, IL-10, and TNFα using Day 15 T-LAK cells after 24 hours of stimulation with 0.1, 1.0, or 10 nM anti-TRBC2 antibody. The results confirmed an antibody concentration-dependent increase in the release of each cytokine, suggesting that the anti-TRBC2 antibody induces T cell activation and cytokine release. In this experimental system, the release of the inflammatory cytokine IL-6 was minimal. These data suggest that anti-TRBC2 antibodies may pose a low risk of cytokine release syndrome. This suggests that bispecific antibodies with the same antigen-binding moiety as the anti-TRBC2 antibody used in this experiment also pose a low risk of cytokine release syndrome.

[0250] Example 3 Cytotoxic Activity of Anti-CCR4 / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the bispecific antibody prepared in Example 1, target cells were cultured with T-LAK (effector cells) in the presence of the specific antibody, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. The experiment was performed with reference to Neri S et al. (Clin Diagn Lab Immunol. 2001 Nov;8(6):1131-5).

[0251] The following reagents were used: HBSS (Hank's Balanced Salt Solution), no phenol red (Gibco, 14025092), RPMI1640 Medium, no phenol red (Gibco, 11835-030), Probenecid Water Soluble (Invitrogen, P36400), Calcein-AM (Invitrogen, C1430), Lysis Solution 10X (Promega, G1821).

[0252] 3.1 Cytotoxic Activity of Bispecific Antibodies against MT-2 Cells. Bispecific antibodies were adjusted to a final concentration of 100 nM to 100 fM using RPMI non-phenolic medium (assay medium) containing 5 mM probenecid and 0.5% FBS and added to a 96-well round-bottom culture plate. Control wells were set up with assay medium alone. For the cytotoxicity assay using bispecific antibodies bearing 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 by incubation at 37°C for 30 minutes with 10 μM calcein-AM supplemented with 5 mM probenecid. After incubation, the cells were washed three times with HBSS and, after the final wash, suspended in assay medium. The number of labeled target cells was counted and then added to the prepared 96-well round-bottom culture plate. After recovering the required amount of effector cells from the culture medium, they were washed once with assay medium, counted, and then added to a 96-well round-bottom culture plate containing the bispecific antibody. The effector cells and labeled target cells were added at a ratio of 10:1. The plate containing the bispecific antibody, labeled target cells, and effector cells was incubated in a 5% CO atmosphere. ２ The cells were incubated at 37°C for 2 hours. The 96-well round-bottom culture plate was centrifuged (300 g, 5 minutes), and the supernatant was collected. The fluorescence of calcein eluted from the labeled target cells contained in the supernatant was measured using a plate reader (BioTek, SYNERGY H1). The calcein fluorescence value was used as an index to calculate the cytotoxicity rate of the target cells, and bispecific antibody-dependent cytotoxicity was evaluated.

[0253] The cytotoxicity rate was calculated as follows: The minimum eluted fluorescence value of labeled target cells (lysis rate = 0%) was obtained from wells in which labeled target cells and effector cells were incubated without any bispecific antibody. The maximum eluted fluorescence value (lysis rate = 100%) was obtained by adding 10x Lysis Solution to wells under the same conditions as the minimum eluted fluorescence value. The cytotoxicity rate was calculated using the following formula: Cytotoxicity rate = [(eluted fluorescence value of target cells in the presence of bispecific antibody) - (minimum eluted fluorescence value)] / [(maximum eluted fluorescence value) - (minimum eluted fluorescence value)].

[0254] Sigmoidal dose-response curves were calculated using Prism Software (GraphPad Software Inc.), and EC50 values ​​(50% effective concentration) and Emax values ​​(maximum activity) were calculated.

[0255] Results FIG. 14A and the table below show the results of adding T-LAK to MT-2 cells, an ATL-derived cell line that expresses CCR4, and measuring the cytotoxic activity of the bispecific antibodies CCR0001 and CCR0004.

[0256] The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-CCR4 and anti-TRBC1 bispecific antibodies CCR0001 and CCR0004 were considered to exhibit very potent cytotoxic activity against CCR4-expressing cells.

[0257] 3.2 Cytotoxic activity of negative control antibodies against MT-2 cells Cytotoxic activity was measured using an anti-CCR4 antibody, an anti-TRBC1 antibody, and a combination thereof (anti-CCR4 antibody + anti-TRBC1 antibody) as negative control antibodies using the method described above. The anti-CCR4 antibody used in this example was mogamulizumab. The anti-TRBC1 antibody was produced and purified by the method described in Example 1. The heavy and light chain sequences of the anti-TRBC1 antibody are SEQ ID NOs: 144 and 145.

[0258] Figure 14B and the table below show the results of adding T-LAK to MT-2 cells, an ATL-derived cell line that expresses CCR4, and measuring the cytotoxic activity of the negative control antibody. The EC50 and Emax values ​​(N=3) are shown in the table below. ＊ For anti-CCR4 antibody, calculation was not possible for one of the three measurements, so the average value of two measurements was shown. NC: Not calculated

[0259] T-LAK was added to MT-2 cells, an ATL-derived cell line that expresses CCR4, and then anti-CCR4 antibody alone, anti-TRBC1 antibody alone, or both anti-CCR4 antibody and anti-TRBC1 antibody were added, and cytotoxic activity was measured. The EC50 values ​​and Emax were significantly lower than when the anti-CCR4 / anti-TRBC1 bispecific antibodies CCR0001 and CCR0004 were added. The anti-CCR4 antibody did not exert sufficient cytotoxic activity in this experimental system. The anti-TRBC1 antibody did not show sufficient cytotoxic activity.

[0260] 3.3 Cytotoxic activity of bispecific antibodies against negative control cells MOLT-4 cells, a T cell tumor-derived cell line that does not express CCR4, were used as a negative control cell instead of MT-2 cells to measure the cytotoxic activity of bispecific antibodies CCR0001 and CCR0004. MOLT-4 cells were labeled with calcein-AM in the same manner as for MT-2 cells above and suspended in assay medium. Figure 14C and the table below show the results of measuring the cytotoxic activity of CCR0001 and CCR0004 after adding T-LAK to the negative control MOLT-4 cells.

[0261] The EC50 and Emax values ​​(N=3) are shown in the table below. ＊ Since calculation was not possible for one of the three tests, the average value of two tests is shown, and SD was not calculated. NC: Not calculated

[0262] Both the EC50 value and Emax of the cytotoxic activity against MOLT-4 cells, a T cell tumor cell line that does not express CCR4, were significantly lower than those against CCR4-expressing cells. These results demonstrate that the bispecific antibodies CCR0001 and CCR0004 act specifically on CCR4-expressing cells.

[0263] Example 4 Cytotoxic Activity of Anti-CD1a / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-CD1a / anti-TRBC1 bispecific antibody prepared in Example 1, CCRF-CEM9 target cells were cultured with anti-TRBC1 antibody-sensitized T-LAK (effector cells) in the presence of the specific antibody, and the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity.

[0264] The target cells CCRF-CEM9 express CD1a and TRBC2 and were obtained by single-cell cloning of CCRF-CEM, a TALL-derived cell line, as described in Example 2. Cytotoxic activity was measured in the same manner as described in Example 3.

[0265] 15 and the table below show the results of adding T-LAK to CCRF-CEM9 cells, a TALL-derived cell line that expresses CD1a, and measuring the cytotoxic activity of the bispecific antibodies CD1a1001 and CD1a1004. The EC50 and Emax values ​​(N=3) are shown in the table below.

[0266] T-LAK was added to CCRF-CEM9 cells and the cytotoxic activity of CD1a1001 and CD1a1004 was measured. The EC50 values ​​were both 2.9 pM and the Emax were 94.2% and 77.0%, respectively. These results suggest that the anti-CD1a and anti-TRBC1 bispecific antibody exhibits very potent cytotoxic activity against CD1a and TRBC2 expressing cells.

[0267] Example 5 Cytotoxic activity of anti-CCR4 / anti-TRBC2 bispecific antibody In this example, to evaluate the in vitro efficacy of the anti-CCR4 / anti-TRBC2 bispecific antibody prepared in Example 1, target cells MT-2 were cultured with anti-TRBC2 antibody-sensitized T-LAK cells (effector cells) in the presence of the bispecific antibody, and the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity.

[0268] Results Figure 16 shows the results of measuring the cytotoxic activity of CCR0010 when anti-TRBC2 antibody-sensitized T-LAK was added to MT-2 cells, an ATL-derived cell line that expresses CCR4. The EC50 and Emax values ​​(N=3) are shown in the table below.

[0269] Anti-TRBC2 antibody-sensitized T-LAK cells were added to MT-2 cells and the cytotoxic activity of CCR0010 was measured. As a result, the bispecific antibody CCR0010, which recognises both CCR4 and TRBC2, showed activity against CCR4-expressing cells with an EC50 of 1677.0 pM and an Emax of 81.4%. Therefore, it is believed that the bispecific antibody recognizing CCR4 and TRBC2, like the bispecific antibody recognizing CCR4 and TRBC1, exhibits cytotoxic activity against CCR4-expressing ATL cell lines.

[0270] Example 6: Cytotoxic Activity of Anti-CD1a / Anti-TRBC2 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-CD1a / anti-TRBC2 bispecific antibody prepared in Example 1, target JM cells 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 the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. Cytotoxic activity was measured using the same method as described in Example 3. Target JM cells are a TALL-derived cell line expressing CD1a and TRBC1. JM cells were purchased from the RIKEN Bioresearch Research Center (RCB0537). They were cultured in RPMI medium containing 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0271] Results Figure 17 shows the results of measuring the cytotoxic activity of bispecific antibodies CD1a1003 and CD1a1006 when anti-TRBC2 antibody-sensitized T-LAK was added to JM cells, a TALL-derived cell line that expresses CD1a. The EC50 and Emax values ​​(N=3) are shown in the table below.

[0272] T-LAK was added to JM cells and the cytotoxic activity of CD1a1003 and CD1a1006 was measured. The EC50 values ​​were 22.5 pM and 12.9 pM, respectively, and the Emax was 90.1% and 70.1%, respectively. These results suggest that the anti-CD1a and anti-TRBC2 bispecific antibody exhibits very potent cytotoxic activity against CD1a and TRBC1 expressing cells.

[0273] Example 7 Cytotoxic Activity of Anti-CCR9 / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-CCR9 / anti-TRBC1 bispecific antibody prepared in Example 1, CCRF-CEM9 target cells were cultured with T-LAK (effector cells) in the presence of the specific antibody, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. CCRF-CEM9 target cells express CCR9 and TRBC2 and were obtained by single-cell cloning of CCRF-CEM, a TALL-derived cell line, as described in Example 2. Cytotoxic activity was measured using a method similar to that described in Example 3.

[0274] Figure 18 shows the results of adding T-LAK to CCRF-CEM9 cells, a TALL-derived cell line that expresses CCR9, and measuring the cytotoxic activity of the bispecific antibodies CCR9-1002 and CCR9-1004. The EC50 and Emax values ​​(N=3) are shown in the table below. T-LAK was added to CCRF-CEM9 cells and the cytotoxic activity of CCR9-1002 and CCR9-1004 was measured. The EC50 values ​​were 29.1 pM and 6.6 pM, respectively, and the Emax was 85.9% and 94.5%, respectively. These results suggest that the anti-CCR9 and anti-TRBC1 bispecific antibody exhibits very potent cytotoxic activity against cells expressing both CCR9 and TRBC2.

[0275] Example 8 Cytotoxic Activity of Anti-CXCR4 / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-CXCR4 / anti-TRBC1 bispecific antibody prepared in Example 1, CCRF-CEM9 target cells were cultured with T-LAK (effector cells) in the presence of the specific antibody, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. CCRF-CEM9 target cells express CXCR4 and TRBC2 and were obtained by single-cell cloning of CCRF-CEM, a TALL-derived cell line, as described in Example 2. Cytotoxic activity was measured using a method similar to that described in Example 3.

[0276] Figure 19 shows the results of adding T-LAK to CCRF-CEM9 cells, a TALL-derived cell line that expresses CXCR4, and measuring the cytotoxic activity of the bispecific antibodies CXCR4-1002 and CXCR4-1004. The EC50 and Emax values ​​(N=3) are shown in the table below. T-LAK was added to CCRF-CEM9 cells and the cytotoxic activity of CXCR4-1002 and CXCR4-1004 was measured. The EC50 values ​​were 0.31 pM and 0.73 pM, respectively, and the Emax was 88.3% and 95.6%, respectively. These results suggest that the anti-CXCR4 / anti-TRBC1 bispecific antibody exhibits very potent cytotoxic activity against CXCR4 and TRBC2 expressing cells.

[0277] Example 9 Cytotoxic Activity of Anti-CCR8 / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-CCR8 / anti-TRBC1 bispecific antibody prepared in Example 1, target cells were cultured with T-LAK cells (effector cells) 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 effector cell-dependent cytotoxic activity. The target cells, MT-1, are an ATL-derived cell line expressing CCR8. Cytotoxic activity was measured using the same method as described in Example 3. MT-1 cells were purchased from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition) (JCRB1209). The cells were cultured in RPMI medium containing 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0278] Results Figure 20 shows the results of adding T-LAK to MT-1 cells, an ATL-derived cell line that expresses CCR8, and measuring the cytotoxic activity of CCR81001 and CCR81002. The EC50 and Emax values ​​(N=3) are shown in the table below. T-LAK was added to MT-1 cells and the cytotoxic activity of CCR81001 and CCR81002 was measured. The EC50 values ​​were 23.5 pM and 31.4 pM, respectively, and the Emax was 89.9% and 97.1%, respectively. These results suggest that the bispecific antibodies CCR81001 and CCR81002, which recognize both CCR8 and TRBC1, exhibit very potent cytotoxic activity against CCR8-expressing cells.

[0279] Example 10: Cytotoxic activity of anti-TIGIT / anti-TRBC1 bispecific antibody In this example, to evaluate the in vitro efficacy of the anti-TIGIT / anti-TRBC1 bispecific antibody prepared in Example 1, target cells were cultured with T-LAK cells (effector cells) 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 effector cell-dependent cytotoxic activity. The target cells, ILT-Mat, are an ATL-derived cell line expressing TIGIT. Cytotoxic activity was measured using a method similar to that described in Example 3. ILT-Mat cells were purchased from the RIKEN Bioresearch Research Center (RCB0475). The cells were cultured in RPMI medium containing 320 IU / mL IL-2 and 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0280] Figure 21 shows the results of adding T-LAK to ILT-Mat cells, an ATL-derived cell line that expresses TIGIT, and measuring the cytotoxic activity of TIGIT2001 and TIGIT2002. The EC50 and Emax values ​​(N=3) are shown in the table below. T-LAK was added to ILT-Mat cells, and the cytotoxic activity of TIGIT2001 and TIGIT2002 was measured. The EC50 values ​​were 3.4 pM and 5.6 pM, respectively, and the Emax was 105.1% and 91.9%, respectively. These results suggest that the bispecific antibodies TIGIT2001 and TIGIT2002, which recognize both TIGIT and TRBC1, exhibit very potent cytotoxic activity against TIGIT-expressing cells.

[0281] Example 11: Cytotoxic activity of anti-CD30 / anti-TRBC1 bispecific antibody In this example, to evaluate the in vitro efficacy of the anti-CD30 / anti-TRBC1 bispecific antibody prepared in Example 1, target cells were cultured with T-LAK cells (effector cells) in the presence of the specific antibody, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. The target cell, DL40, is a PTCL-derived cell line expressing CD30. Cytotoxic activity was measured using a method similar to that described in Example 3. DL40 cells were purchased from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition) (JCRB1337). The cells were cultured in RPMI medium containing 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0282] Figure 22 shows the results of adding T-LAK to DL40 cells, a PTCL-derived cell line that expresses CD30, and measuring the cytotoxic activity of CD301001 and CD301003. The EC50 and Emax values ​​(N=3) are shown in the table below. T-LAK was added to DL40 cells, and the cytotoxic activity of CD301001 and CD301003 was measured. The EC50 values ​​were 151.1 pM and 14.5 pM, respectively, and the Emax was 89.1% and 95.7%, respectively. These results suggest that the anti-CD30 antibody and anti-TRBC1 bispecific antibodies CD301001 and CD301003 exhibit very potent cytotoxic activity against CD30-expressing cells.

[0283] Example 12: Cytotoxic activity of anti-CD40L / anti-TRBC1 bispecific antibody In this example, to evaluate the in vitro efficacy of the anti-CD40L / anti-TRBC1 bispecific antibody prepared in Example 1, target cells were cultured with T-LAK cells (effector cells) in the presence of the specific antibody, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. The target cell MOLT3 is a TALL-derived cell line that expresses CD40L (CD154). Cytotoxic activity was measured using a method similar to that described in Example 3. MOLT3 cells were purchased from the RIKEN Bioresearch Research Center (RCB9048). They were cultured in RPMI medium containing 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0284] Figure 23 shows the results of adding T-LAK to MOLT3 cells, a TALL-derived cell line that expresses CD40L (CD154), and measuring the cytotoxic activity of CD40L1007 and CD40L1008. The EC50 and Emax values ​​(N=3) are shown in the table below. T-LAK was added to MOLT3 cells, and the cytotoxic activity of CD40L1007 and CD40L1008 was measured. The EC50 values ​​were 125.0 pM and 49.1 pM, respectively, and the Emax was 67.7% and 72.7%, respectively. These results suggest that the anti-CD40L and anti-TRBC1 bispecific antibodies CD40L1007 and CD40L1008 exhibit very potent cytotoxic activity against CD40L (CD154)-expressing cells.

[0285] Example 13: Evaluation of the cytotoxic activity of anti-CD3 bispecific antibodies against T cell tumors. CD3 is expressed similarly on both normal and tumor T cells. Bispecific antibodies targeting both CD3 and tumor antigens simultaneously bind to the CD3 antigen expressed on T cell malignancies and the target antigen expressed on T cell malignancies, linking malignant tumor cells together (Figure 1A), making it highly likely that the therapeutic effect of the bispecific antibody will be insufficient. In this example, the cytotoxic activity of anti-CD3 bispecific antibodies against CD3-expressing tumor cell lines and CD3-knockout tumor cell lines was evaluated. Similar experiments were also performed using anti-TRBC1 bispecific antibodies.

[0286] The anti-CD1a / anti-CD3 bispecific antibody CD1a1016 and the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005 were produced using the method described in Example 1. 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. HPB-ALL target cells and HPB-ALL CD3 KO cells were cultured with T-LAK (effector cells) in the presence of each bispecific antibody, and the cell viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. HPB-ALL target cells express CD1a, CD3, and TRBC2. HPB-ALL CD3 KO target cells are a cell line in which the CD3 gene was knocked out of HPB-ALL, a TALL-derived cell line, using CRSPR-Cas9. Cytotoxic activity was measured using a method similar to that described in Example 3. HPB-ALL target cells were purchased from the RIKEN Bioresearch Research Center (RCB1935). They were cultured in RPMI 1640 medium containing 10% FBS at 5% CO ２ The cells were cultured in an incubator at 37°C and subcultured 2 to 3 times a week.

[0287] Results Figure 24 shows the results of adding T-LAK to HPB-ALL cells, a TALL-derived cell line that expresses CD3, and HPB-ALL CD3 KO cells in which CD3 has been knocked out, and measuring the cytotoxic activity of the anti-CD1a / anti-CD3 bispecific antibody CD1a1016 and the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005. The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-CD1a / anti-CD3 bispecific antibody CD1a1016 had an EC50 approximately 10-fold higher and a lower Emax when CD3 was expressed on target cells compared to when CD3 was not expressed. These results suggest that the cytotoxic activity of bispecific antibodies employing anti-CD3 antibodies on the effector cell side is attenuated when CD3 is expressed on target cells. The EC50 and Emax of the anti-CD1a / anti-TRBC1 bispecific antibody CD1a1005 did not differ significantly depending on the target cell type.

[0288] Example 14: Cytotoxic activity of anti-TRBC2 bispecific antibodies against TRBC1-expressing tumor cells and TRBC2-expressing tumor cells. In this example, the cytotoxic activity 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 was compared. CCRF-CEM9 and JM target cells were cultured with anti-TRBC2 antibody-sensitized T-LAK (effector cells) in the presence of the bispecific antibody, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxic activity. CCRF-CEM9 target cells are a TALL-derived cell line that expresses CD1a and TRBC2. JM target cells are a TALL-derived cell line that expresses CD1a and TRBC1. Cytotoxic activity was measured in the same manner as described in Example 3. 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. The cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 against the TRBC1-positive tumor cell line JM was also evaluated in Example 6.

[0289] Results Figure 25 shows the results of measuring the cytotoxic activity of the anti-CD1a / anti-TRBC2 bispecific antibody CD1a1006 when anti-TRBC2 antibody-sensitized T-LAK cells were added to CCRF-CEM9 cells, a TALL-derived cell line that expresses TRBC2, and JM cells, a TALL-derived cell line that expresses TRBC1. The EC50 and Emax values ​​(N=3) are shown in the table below. The EC50 values ​​for the target cells CCRF-CEM9 and JM were 28.4 pM and 18.0 pM, respectively, and the Emax was 33.0% and 72.8%, respectively. These results demonstrate that the anti-CD1a / anti-TRBC2 bispecific antibody exhibits high cytotoxic activity against cells expressing CD1a and TRBC1, but low cytotoxic activity against cells expressing CD1a and TRBC2. Furthermore, as shown in Example 4, when the effector antibody was TRBC1, it exhibited very potent cytotoxic activity against the target cells CCRF-CEM9. These results demonstrate that it is important that the effector antibody recognizes a subtype not expressed on tumor cells.

[0290] Example 15: Evaluation of in vivo antitumor activity In this example, the in vivo antitumor activity of CD1a1006, an anti-CD1a / anti-TRBC2 bispecific antibody, was examined. Cell preparation JM cells are a TALL-derived cell line that expresses CD1a and TRBC1. They were purchased from the Cell Materials Research Laboratory of RIKEN (cell number RCB1164, product number RCB0537). JM-Luc cells, in which the luciferase gene was stably expressed, were used. JM-Luc cells were grown in RPMI1640 medium containing 10% FBS under 5% CO ２ The cells were cultured in an incubator at 37°C and used for testing the in vivo antitumor effect.

[0291] Test animals Animal species: Mouse Strain: NOD / Shi-scid, IL-2RγKO JiC (NOG) Supplier: CLEA Japan, Inc. Age: 6 weeks (at time of receipt)

[0292] Preparation of PBMCs PBMCs were purchased from Hemacare (product number: PB009C-3, Lot No.: 22074722). Frozen PBMCs (1 x 10 ８ The cells (25 mL / vial) were thawed in a constant temperature bath at approximately 37°C, and 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 the cells were slowly added dropwise to the centrifuge tube and mixed. The mixture was centrifuged (room temperature, 400 x g, 5 minutes), and the supernatant was aspirated. The cells were then resuspended in washing media (25 mL of D-PBS containing 0.005% FBS + 100 μL of 0.5 M EDTA). The solution was diluted 2-fold with trypan blue, and the viable cell count was calculated. The mixture was centrifuged again (room temperature, 400×g, 5 minutes), the supernatant was removed by suction, and the mixture was resuspended in RPMI 1640 medium at 1×10 ８ A cell suspension of 1000 cells / mL was prepared.

[0293] The prepared PBMC suspension was transported to the breeding room on ice, and within 2 hours after preparation, 0.1 mL (1 × 10) of PBMC was injected into the NOG mice using a 1 mL disposable syringe and a 27 G needle (Terumo Corporation). ７ Each mouse was transplanted into the tail vein with 1000 cells / mouse, and this was designated as "Day 10."

[0294] Preparation of JM-Luc Cells JM-Luc cells in culture were collected on Day 0. They were centrifuged (room temperature, 400 × g, 5 minutes), the supernatant was removed by suction, washed with PBS, and then centrifuged again (room temperature, 400 × g, 5 minutes). The supernatant was removed by suction, diluted appropriately with PBS, stained with trypan blue, and the cells were counted to obtain a cell count of 1 × 10 ７ The prepared cell suspension was stored on ice until transplantation.

[0295] Transplantation of JM-Luc cells The prepared JM-Luc cell suspension was transported to the breeding room on ice, and within 2 hours after preparation, the tails of NOG mice were disinfected with alcohol swabs, and 0.1 mL (1 × 10) of JM-Luc cells was injected into the mice using a 1 mL disposable syringe and a 27 G needle (Terumo Corporation). ６ The cells were transplanted into the tail vein of each mouse (1000 cells / mouse), and this was designated as "Day 0."

[0296] Preparation of antibodies CD1a1006 was prepared using PBS so that the doses of each antibody were 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).

[0297] Antibody Administration On days 1, 7, 10, 14, 17, 21 and 25, the tail was disinfected with alcohol swabs, and 0.1 mL of the prepared antibody solution was implanted into the tail vein using a 1 mL disposable syringe and a 27G needle (Terumo Corporation).

[0298] Analysis method of antitumor effect On days 14 and 28, the luminescence intensity of JM-Luc cells was measured for all surviving individuals using an IVIS imaging system (Revvity). On each IVIS measurement day, luciferin 150 mg / kg (10 mL / kg) was administered intraperitoneally, and the animals were placed in a supine position in an IVIS chamber under isoflurane inhalation anesthesia. Starting approximately 5 minutes after luciferin administration, the whole body was designated as the region of interest (ROI) and the luminescence intensity (total flux) was measured approximately every minute for 20 minutes. For each measurement day and for each individual, the maximum luminescence intensity approximately every minute was taken as the measured value for that individual.

[0299] Results The IVIS measurement values ​​for each group on Days 14 and 28 are graphed in Figure 26, and an image of tumor distribution is shown in Figure 27. The IVIS measurement value for Group 1 on Day 14 was 56.34 ± 68.05 × 10 ５ p / s, whereas the IVIS measurements for groups 2 to 6 administered CD1a1006 were 11.74 ± 2.59 × 10 ５ p / s, 7.17±0.14×10５ 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. The IVIS measurement value for Group 1 on Day 28 was 3951.23±5229.34×10 ５ p / s, whereas the IVIS measurements for groups 2 to 6 administered CD1a1006 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 These results demonstrated that CD1a1006 has the ability to suppress the engraftment and subsequent proliferation of JM-Luc cells.

[0300] Example 16 Identification of ATL Patients with T Cell Dysfunction Reagents Used Buffer: Prepared by mixing 500 mL of PBS (Nacalai Tesque, 14249-94) and 10 mL of HI-FBS (SIGMA) Clear Back (MBL, MT-001) Fixable Viability Dye eFluor780 (Invitrogen, 65-0865-14) Transcription Factor Buffer Set (BD, 562574) TF Fix / Perm Buffer (4x) (51-9008100): Used after diluting 4-fold with TF Diluent Buffer. TF Diluent Buffer (51-9008101) TF Perm / Wash Buffer (5x) (51-9008102): Used after diluting 5 times with distilled water. The staining antibodies are shown in the table below. *BV421, BV711: abbreviations for Brilliant Violet 421 (registered trademark) and Brilliant Violet 711 (registered trademark)

[0301] Cell surface and intracellular staining method: PBMCs from healthy donors or ATL patients were centrifuged (500g x 2 minutes), the supernatant removed, and ClearBack was added. After 5 minutes of incubation at room temperature, various cell surface antibodies were added in buffer and allowed to react for 30 minutes at 4°C. After the reaction, buffer was added, the cells were centrifuged (500g x 2 minutes), and the supernatant was discarded, thereby washing the cells twice. TF Fix / Perm Buffer in the Transcription Factor Buffer Set was added, and the cells were fixed and permeabilized by incubating on ice for 45 minutes. After centrifugation (700g x 2 minutes), TF Perm / Wash Buffer was added, the cells were centrifuged (700g x 2 minutes), and the supernatant was discarded, thereby washing the cells three times. Anti-CTLA-4 antibody or isotype control antibody was added in TF Perm / Wash Buffer and incubated at room temperature for 45 minutes. After incubation, the cells were washed twice by adding TF Perm / Wash Buffer, centrifuging (700g x 2 minutes), and discarding the supernatant. Signals were quantified by flow cytometry analysis using a BD FACSymphony A1 (BD Biosciences). Flow Jo_v10.8.1 was used as the analysis software.

[0302] result Figure 28 and the table above show the PD-1 and CTLA-4 positive cell rates in CD3-positive, CD4-positive, CADM1-negative normal T cells. The PD-1 positive cell rate in normal T cells derived from PBMCs of healthy individuals was 1.5-6.0%, whereas the PD-1 positive cell rate in ATL patient 1 was 16.0%, demonstrating a significantly higher rate. Furthermore, there was almost no difference in the CTLA-4 positive cell rate between healthy individuals and ATL patients. These findings suggest that some ATL patients exhibit T cell dysfunction due to increased PD-1 expression.

[0303] Example 17 Analysis of bispecific antibody-dependent cytotoxicity in ATL patients The following reagents were used: FBS (NICHIREI, 175012, Lot. 19J00C), Steady-Glo (Promega, E2520), RPMI1640 (Nacalai Tesque, 30264-85), 1 M Hepes (Nacalai, 17557-94), 100x sodium pyruvate (Gibco, 11360-070), 100x 2-ME (Wako, 198-15781).

[0304] In this example, the anti-CD1a / anti-TRBC1 bispecific antibody prepared in Example 1 was used to verify the cytotoxic activity of effector cells derived from ATL patients (ATL patient 1, ATL patient 2) and healthy subject (healthy subject 1) used in Example 16 against target cells.

[0305] MOLT-4-Luc target cells were cultured with PBMCs derived from healthy individuals or PBMCs derived from ATL patients (effector cells) in the presence of the bispecific antibody, and the cell viability of the target cells was measured using luciferase activity as an indicator to evaluate effector cell-dependent cytotoxicity. MOLT-4-Luc target cells are a cell line in which the luciferase gene has been introduced into MOLT-4 cells, a TALL-derived cell line, using a plasmid vector.

[0306] Methods: Bispecific antibodies were adjusted to a final concentration of 1 nM to 10 fM in RPMI 1640 medium containing 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 0.1 mM 2-ME (TDCC medium) and added to 384-well flat-bottom culture plates. For the cytotoxicity assay using bispecific antibodies with antigen-binding sites for CD1a and TRBC1, the required amount of MOLT-4-Luc cells (target cells) was collected from the culture medium, suspended in TDCC medium, and counted. The cells were then added to the 384-well flat-bottom culture plates containing the bispecific antibodies. The required amount of effector cells was collected, suspended in TDCC medium, and counted. The cells were then added to the 384-well flat-bottom culture plates containing the bispecific antibodies. The effector cells were added at a ratio of 10:1 to labeled target cells. Plates containing bispecific antibodies, labeled target cells, and effector cells were incubated at 37°C in 5% CO2. After 72 hours, 25 μL of Steady-Glo was added to each well, and 5 minutes later, luciferase luminescence in surviving target cells was measured using a plate reader (BioTek, Cytation 5). The cytotoxicity rate of target cells was calculated using the luciferase luminescence value as an index, and bispecific antibody-dependent cytotoxicity was evaluated.

[0307] The cytotoxicity rate was determined as follows: The maximum luminescence value of labeled target cells (cytotoxicity rate = 0%) was obtained from wells in which labeled target cells and effector cells were incubated without any bispecific antibody. The cytotoxicity rate was calculated using the following formula: Cytotoxicity rate = 100 - [100 × (luminescence value of target cells in the presence of bispecific antibody) / (maximum luminescence value)]. A sigmoidal 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.

[0308] Results Figure 29 shows the results of measuring the cytotoxic activity of PBMCs derived from a healthy subject (healthy subject 1) and PBMCs derived from ATL patients (ATL patient 1, ATL patient 2) against luciferase-positive MOLT-4-Luc cells in the presence of an anti-CD1a / anti-TRBC1 bispecific antibody (CD1a1005). The EC50 and Emax values ​​are shown in the table below.

[0309] The Emax in healthy subject 1 was 79.3%, while the Emax in ATL patient 1 was 83.7%, showing activity comparable to that in healthy subjects. The Emax in ATL patient 2 was 45.3%, showing cytotoxic activity against target cells.

[0310] The results of Example 16 suggest that ATL patient 1 has higher PD-1 expression than healthy subjects, possibly resulting in T cell dysfunction. Furthermore, the results of Example 17 indicate that ATL patient 1 exhibited bispecific antibody-dependent cytotoxicity against target cells equivalent to or greater than that of healthy subjects. These findings suggest that some ATL patients are in a state of T cell dysfunction, and that bispecific antibodies that recognize TRBCs may induce cytotoxic activity even in patients with T cell dysfunction.

[0311] Example 18: Single-cell analysis of T cells from ATL patients PBMCs were obtained from peripheral blood of healthy individuals or ATL patients by standard methods, and dead cells were removed using a Miltenyi Biotec Dead Cell Removal Kit. T cells were then isolated and collected using a Miltenyi Biotec Pan T Cell Isolation Kit. Single-cell droplets were prepared using Chromium Next GEM Single Cell 5' Reagent Kits v2, and cDNA was produced. The obtained cDNA was analyzed using a next-generation sequencer, HiSeq. （Ｒ） Sequence analysis was performed using a system (Illumina).

[0312] The obtained sequencing data were preprocessed and analyzed using R software (version 4.1.3). After analysis using Cell Ranger, secondary analysis was performed using Seurat (version 4.3.0.1). Cell quality control was performed by removing extracellular mRNA contamination using SoupX and predicting and removing doublets using DoubletFinder. Data were filtered based on RNA counts and the proportion of mitochondrial genes. GEX data were normalized using SCTransform, and principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) were performed for dimensionality reduction. Harmony was applied to integrate the data and correct for batch effects. Then, the expression levels of the following genes were analyzed for the CD8-positive T cell data, and the biological functions of the CD8-positive T cells were scored using Seurat's AddModuleScore.

[0313] The results are shown in Figure 30 and Table 27. The activation, survival-promoting, and cytotoxicity items of PBMCs from ATL patients 3 and 4 were significantly lower than those of healthy controls, indicating that CD8-positive T cells from ATL patients 3 and 4 were in a dysfunctional state.

[0314] Example 19 Analysis of bispecific antibody-dependent cytotoxic activity in ATL patients The cytotoxic activity of PBMCs against target cells was measured in the same manner as in Example 17, except that an anti-CD1a / anti-TRBC2 bispecific antibody (CD1a1003) was used instead of the anti-CD1a / anti-TRBC1 bispecific antibody, and the PBMCs obtained in Example 18 were used as effector cells.

[0315] The results are shown in Figure 31. Despite the CD8-positive T cells in PBMCs from ATL patient 3 and ATL patient 4 being in a dysfunctional state, the anti-CD1a / anti-TRBC2 bispecific antibody induced concentration-dependent cytotoxic activity, similar to when PBMCs from healthy individuals were used. Therefore, it was confirmed that bispecific antibodies that recognize TRBCs can induce cytotoxic activity even in patients with T cell dysfunction.

Claims

1. A therapeutic agent for T-cell tumors in a subject with T-cell dysfunction, comprising a bispecific antigen-binding molecule, wherein the bispecific antigen-binding molecule comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on T-cell tumor cells; and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype; provided that the target tumor antigen expressed on T-cell tumor cells is not present on normal T cells, or, even if present, the normal T cells are not substantially activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present on normal T cells; the normal T cells are activated by the binding of the bispecific antigen-binding molecule to the target antigen on normal T cells; and a sufficient proportion of the subtypes of the target antigen on normal T cells is present to provide a sufficient number of activated T cells for the treatment of the T-cell tumor.

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

3. The therapeutic agent according to claim 1 or 2, wherein the target antigen on normal T cells and having a subtype is TRBC (T cell receptor beta 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 target antigen on normal T cells and having a subtype 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 target antigen on normal T cells and having a subtype is TRBC, the T cell tumor cells are TRBC1-negative and TRBC2-negative, 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, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to TRBC1 on normal T cells, said at least one portion comprising a VH domain comprising 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, and a VL domain comprising 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 of claim 6, wherein at least one portion which specifically binds to TRBC1 on 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, or a heavy chain variable region (VH) that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:148 and a light chain variable region (VL) that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

149.

8. The therapeutic agent according to claim 1 or 2, wherein the subtype of the target antigen on normal T cells is TRBC2, and the bispecific antigen-binding molecule comprises at least one portion that specifically binds to TRBC2 on normal T cells, said at least one portion comprising a VH domain comprising 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, and a VL domain comprising 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 which specifically binds to TRBC2 on 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 according to claim 1 or 2 for use in a method for treating a T-cell tumor in a subject with T-cell dysfunction, the method comprising determining the subtype expressed in the subject's T-cell tumor cells and administering the therapeutic agent to the subject, wherein the therapeutic agent comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to a target antigen on normal T cells of a subtype different from the subtype determined to be expressed in the subject's T-cell tumor cells.

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