Bispecific antibody effective for patients with malignant tumors other than t-cell tumors

Abstract

The present invention addresses the problem of providing a new tumor treatment method that is capable of inducing cytotoxicity to tumor cells and is unlikely to cause immunodeficiency and cytokine release syndrome. The present invention provides a therapeutic agent for tumors (tumors excluding T-cell tumors), the therapeutic agent comprising a bispecific antigen-binding molecule. The bispecific antigen-binding molecule includes (1) at least one portion that specifically binds to a target tumor antigen expressed by tumor cells, and (2) at least one portion that specifically binds to a normal T-cell-side target antigen having subtypes. The target tumor antigen expressed by the tumor cells is not present in normal T-cells. Even if the target tumor antigen is present in normal T-cells, the normal T-cells are substantially not activated when the bispecific antigen-binding molecule binds to the same antigen as the target tumor antigen present in the normal T-cells. The normal T-cells are activated by having the bispecific antigen-binding molecule bind to the normal T-cell-side target antigen. In order to provide a sufficient number of activated T-cells for the treatment of a tumor, the subtypes of the normal T-cell-side target antigen are present at sufficient proportions.

Description

Bispecific antibodies effective in patients with malignancies other than T-cell neoplasms

[0001] This application claims priority from Japanese Patent Application No. 2024-066506, filed April 17, 2024, the entire disclosure of which is expressly incorporated herein by reference.

[0002] The present invention relates to a tumor therapeutic agent 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 tumor cells, and at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype (with the proviso that the tumor is not a T-cell tumor).

[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 the structure of a single-chain antibody in which two single-chain variable region fragments (scFv) are linked by a short linker.

[0004] Lymphoid malignancies are broadly divided into B-cell malignancies and T-cell malignancies. Most bispecific antibodies have been developed for the treatment of B-cell malignancies. In immunotherapy 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 precursors, resulting in B-cell immunodeficiency and the inability to produce antibodies. However, B-cell immunodeficiency can be controlled by regular administration of immunoglobulin preparations (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] In addition to blinatumomab, many bispecific antibodies targeting both the CD3 antigen and tumor cell antigens have been developed. For example, an anti-DLL3 / CD3 bispecific antibody has been developed for the immunotherapy of solid tumors and is currently undergoing clinical trials (Martin Wermke et al., First-in-human dose-escalation trial of BI 764532, a delta-like ligand 3 (DLL3) / CD3 IgG-like T-cell engager in patients (pts) with DLL3-positive (DLL3+) small-cell lung cancer (SCLC) and neuroendocrine carcinoma (NEC). JCO 41, 8502-8502(2023).DOI:10.1200 / JCO.2023.41.16_suppl.8502). 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.

[0006] Immunotherapy is widely used to treat solid tumors and hematological malignancies, but one of the most concerning complications is cytokine release syndrome (CRS), a paraphysiological response resulting in excessive cytokine release and widespread systemic symptoms (Ojemolon PE, et al. (January 25, 2022) Cytokine Release Syndrome Following Blinatumomab Therapy. Cureus 14(1): e21583. DOI 10.7759 / cureus.21583). For example, it has been reported that 11-15% of patients receiving blinatumomab infusion develop CRS, and 2-5% develop severe (grade 3 or higher) CRS (Ojemolon PE, et al., supra). Among the cytokines involved in CRS, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) are products of activated T cells that induce the secretion of interleukin (IL)-1 and IL-6 from macrophages and monocytes, and are thought to play a crucial role in the downstream processes that lead to the clinical symptoms of CRS (Ojemolon PE, et al., supra).

[0007] There is a need for new tumor treatment methods that are less likely to cause serious immunodeficiency and cytokine release syndrome problems and that can provide high therapeutic efficacy.

[0008] Therefore, an object of the present invention is to provide a new method for treating tumors that can induce cytotoxicity in tumor cells and is less likely to cause immunodeficiency and cytokine release syndrome.

[0009] The present inventors conducted extensive research to solve the above-mentioned problems and found that a bispecific antigen-binding molecule that specifically binds to a target tumor antigen expressed on tumor cells and to an antigen with a subtype on normal T cells recruits effector cells (normal T cells) and induces cytotoxicity of tumor cells. Furthermore, the inventors found that the antigen-binding portion of the bispecific antigen-binding molecule on the normal T cell side binds to a specific subtype and selectively activates a subset of T cells, thereby reducing the likelihood of immunodeficiency and cytokine release syndrome. The present invention was completed based on these findings.

[0010] The present invention provides the following: [1] A therapeutic agent for tumors (excluding T-cell tumors) 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 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 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 tumor treatment.

[0011] [2] The therapeutic agent according to [1], wherein the target antigen on normal T cells and having a subtype is TRBC (T cell receptor beta constant region), and the subtype of the target antigen on normal T cells is TRBC1 or TRBC2. [3] The therapeutic agent according to [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 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 of SEQ ID NO: 5, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0012] [4] The therapeutic agent according to [3], 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: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91. [5] The therapeutic agent according to [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 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: 11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0013] [6] The therapeutic agent according to [5], 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: 3, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4. [7] The therapeutic agent according to any one of [1] to [6], wherein at least one moiety that specifically binds to TRBC1 or 2 expressed on normal T cells is an scFv or Fab fragment. [8] The therapeutic agent according to any one of [1] to [7], wherein the tumor is a hematological malignancy excluding T-cell tumors. [9] The therapeutic agent according to [8], wherein the hematological malignancy is a myeloid tumor or a B-cell tumor.

[10] The therapeutic agent according to any one of [1] to [7], wherein the tumor is a solid cancer.

[0014]

[11] The therapeutic agent according to any one of [1] to [7], wherein the target tumor antigen is CD19, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD19, 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: 31, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[12] The therapeutic agent according to

[11] , wherein the at least one moiety that specifically binds to CD19 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38.

[13] The therapeutic agent according to any one of [1] to [7], wherein the target tumor antigen is BCMA, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to BCMA, 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: 39, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44.

[14] The therapeutic agent according to

[13] , wherein the at least one moiety that specifically binds to BCMA comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 45, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.

[0015]

[15] The therapeutic agent according to any one of [1] to [7], wherein the target tumor antigen is CD33, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD33, 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: 47, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 48, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 50, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 51, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 52.

[16] The therapeutic agent according to

[15] , wherein the at least one moiety that specifically binds to CD33 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 53, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 54.

[17] The therapeutic agent according to any one of [1] to [7], wherein the target tumor antigen is CD123, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to CD123, 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: 55, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60.

[18] The therapeutic agent according to

[17] , wherein the at least one moiety that specifically binds to CD123 comprises: a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 61, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 62.

[19] The therapeutic agent according to any one of [1] to [7], wherein the target tumor antigen is HER2, and the bispecific antigen-binding molecule comprises at least one moiety that specifically binds to HER2, 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: 63, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68.

[20] The therapeutic agent according to

[19] , wherein the at least one moiety that specifically binds to HER2 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 69, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[21] A bispecific antigen-binding molecule comprising: (1) at least one moiety that specifically binds to a target tumor antigen expressed on tumor cells (excluding 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 and 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.

[22] The bispecific antigen-binding molecule of

[21] , 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: 5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[23] The bispecific antigen-binding molecule of

[22] , wherein at least one portion that specifically binds to TRBC1 expressed in normal T cells comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91.

[0016]

[24] The bispecific antigen-binding molecule of

[21] , 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: 11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[25] The bispecific antigen-binding molecule of

[24] , 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: 3, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4.

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

[21] to

[25] , wherein the target tumor antigen expressed on tumor cells is any one selected from the group consisting of CD19, BCMA, CD33, CD123, and HER2.

[27] The bispecific antigen-binding molecule of

[26] , wherein at least one portion that specifically binds to CD19 comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[28] The bispecific antigen-binding molecule of

[27] , wherein at least one portion that specifically binds to CD19 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38.

[29] The bispecific antigen-binding molecule of

[26] , wherein at least one portion that specifically binds to BCMA comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44.

[30] The bispecific antigen-binding molecule of

[29] , wherein at least one portion that specifically binds to BCMA comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 45, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.

[31] The bispecific antigen-binding molecule of

[26] , wherein at least one portion that specifically binds to CD33 comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 48, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 49, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 50, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 51, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 52.

[32] The bispecific antigen-binding molecule of

[31] , wherein at least one portion that specifically binds to CD33 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 53, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 54.

[33] The bispecific antigen-binding molecule of

[26] , wherein at least one portion that specifically binds to CD123 comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60.

[34] The bispecific antigen-binding molecule of

[33] , wherein at least one portion that specifically binds to CD123 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 61, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 62.

[35] The bispecific antigen-binding molecule of

[26] , wherein at least one portion that specifically binds to HER2 comprises a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68.

[36] The bispecific antigen-binding molecule of

[35] , wherein at least one portion that specifically binds to HER2 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 69, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0017]

[37] A method for treating a tumor in a subject (excluding T-cell tumors), comprising administering a bispecific antigen-binding molecule to a tumor-bearing subject, wherein the bispecific antigen-binding molecule comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on 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 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 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 tumor treatment.

[38] Use of a bispecific antigen-binding molecule in the manufacture of a medicament for treating a tumor (provided that the tumor does not include 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 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 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 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 tumor treatment.

[0018] Figure 1 shows the structures of the bispecific antibodies prepared in the Examples. The Fab-scFv type has a structure in which an scFv that binds to one antigen and a 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 a 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 2 shows the VH and VL amino acid sequences of the anti-TRBC1 antibody, humanized anti-TRBC1 antibody, and anti-TRBC2 antibody used to prepare the bispecific antibodies. The underlined parts indicate the respective CDRs. 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 humanized anti-TRBC1 antibody are those set forth in SEQ ID NOs: 9 and 19 of Japanese Patent No. 7,177,794 (corresponding to WO 2018 / 224844). 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-G 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 the results of Fixable Viability Dye eFluor on live cells. ＴＭFigure 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. ＴＭ Gating 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. ＴＭFigure 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. ＴＭ Cells 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. Figure 14 shows the cytotoxic activity of bispecific antibodies CCR190001 and blinatumomab. Figure 15 shows the cytotoxic activity of bispecific antibodies CCR190001 and blinatumomab. Figure 16A shows the changes in T cell activation markers induced by bispecific antibodies CCR190001 and blinatumomab. Figure 16B is a continuation of Figure 16A. Figure 17A shows a comparison of cytokine release from bispecific antibodies CCR190001 and blinatumomab. Figure 17B is a continuation of Figure 17A. Figure 18 shows the cytotoxic activity of an anti-CD19 / anti-TRBC1 bispecific antibody against Raji cells (expressing CD19), a Burkitt's lymphoma-derived cell line. Figure 19 shows the cytotoxic activity of an anti-BCMA / anti-TRBC1 bispecific antibody against U266 cells (expressing BCMA), a multiple myeloma-derived cell line. Figure 20 shows the cytotoxic activity of an anti-CD33 / anti-TRBC1 bispecific antibody against MOLM13 cells (expressing CD33), an acute myeloid leukemia-derived cell line. Figure 21 shows the cytotoxic activity of an anti-CD123 / anti-TRBC1 bispecific antibody against MOLM13 cells (expressing CD123), an acute myeloid leukemia-derived cell line. Figure 22 shows the cytotoxic activity of anti-HER2 / anti-TRBC1 bispecific antibodies against MKN7 cells (expressing HER2), a tubular adenocarcinoma-derived cell line.

[0019] 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.

[0020] The present invention relates to the treatment of tumors. The bispecific antibodies of the present invention can be used to treat, for example, any hematological malignancies and solid cancers. However, to avoid duplication with prior inventions, T-cell tumors may be excluded from the tumors. Note that when the phrase "excluding ..." is used in this specification, it is also intended to avoid duplication with prior inventions.

[0021] 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.

[0022] 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).

[0023] 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).

[0024] A first embodiment of the present invention relates to a tumor therapeutic agent comprising a bispecific antigen-binding molecule. The tumor does not include T-cell tumors. 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 tumor cells; and (2) at least one moiety that specifically binds to a target antigen on normal T cells, the antigen having a subtype. The target tumor antigen expressed on 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. 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 tumor treatment.

[0025] In the present invention, the phrase "a moiety that specifically binds to a target tumor antigen expressed on tumor cells" (hereinafter referred to as a tumor antigen-binding moiety) refers to a polypeptide molecule that specifically binds to a target tumor antigen expressed on tumor cells. In one aspect, the tumor antigen-binding moiety can guide normal T cells to tumor cells expressing the target tumor antigen. The tumor antigen-binding moiety includes an antibody or a fragment thereof.

[0026] 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.

[0027] Preferably, the target tumor antigen expressed on 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 absence of a target tumor antigen expressed on tumor cells on normal T cells can be determined by flow cytometry analysis using a fluorescent dye-labeled antibody that specifically binds to the target tumor antigen. The presence or absence of a certain antigen on normal T cells, or whether expression of a certain antigen is positive or negative on 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 absence of a target tumor antigen expressed on tumor cells on normal T cells allows the bispecific antigen-binding molecule to appropriately crosslink normal T cells and tumor cells. 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.

[0028] The target tumor antigen expressed on 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 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 tumor cells or normal T cells.

[0029] In certain embodiments, even if the target tumor antigen is present in normal T cells, it is preferably expressed at a higher frequency in tumor cells than in normal T cells. For example, the ratio of the number of molecules of the target tumor antigen expressed per tumor cell to the number of molecules expressed per normal T cell may be 110% or more, and is 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. When the target tumor antigen is expressed at a higher frequency in tumor cells than in normal T cells, the bispecific antigen-binding molecule can crosslink a sufficient number of normal T cells and tumor cells for treatment, thereby achieving a therapeutic effect. Specific examples of target tumor antigens are described below.

[0030] <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.

[0031] "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.

[0032] 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 a sufficient number of activated T cells for tumor treatment may not be provided. 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.

[0033] 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. Tumor cells also typically express one of the above subtypes (e.g., any of subtypes A to C). When an antigen with subtypes is expressed in tumor cells, 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 tumor cells. For example, if the subtype expressed in 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 on tumor cells, other than the "target antigen on normal T cells, which antigen has a subtype," thereby enabling the bispecific antigen-binding molecule to appropriately crosslink normal T cells and tumor cells.

[0034] 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.

[0035] In the present invention, in order to provide a sufficient number of activated T cells for tumor treatment, 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 tumor treatment, 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.

[0036] In one aspect, the target antigen on normal T cells and the antigen having a subtype is TRBC (T cell receptor beta constant region), and the subtype of the target antigen on normal T cells is TRBC1 or TRBC2. Within the scope of the present application, tumor cells are not T cell tumor cells and are TRBC1-negative and TRBC2-negative, and the target antigen on normal T cells of the bispecific antigen-binding molecule may be either TRBC1 or TRBC2. Since normal T cells express either TRBC1 or TRBC2, normal T cells can be mobilized as effector cells by the action of a bispecific antigen-binding molecule having a normal T cell antigen-binding portion that binds to either TRBC1 or TRBC2.

[0037] 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).

[0038] It has been reported 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 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 that TRBC1 or TRBC2 could be used as target antigens on normal T cells or effector cells.

[0039] In a specific embodiment, the therapeutic agent for treating tumors of the present invention (with the proviso that the tumors exclude T-cell tumors) comprises a bispecific antigen-binding molecule comprising: at least one moiety that specifically binds to a target tumor antigen expressed on tumor cells; and at least one moiety that specifically binds to either TRBC1 or 2 expressed on normal T cells.

[0040] 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.

[0041] 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 tumor cells.

[0042] 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.

[0043] 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.

[0044] Currently, anti-CD3 antibodies are the main bispecific antibodies used as T cell engagers on the market. The Examples below demonstrate that bispecific antibodies prepared using anti-TRBC1 or anti-TRBC2 antibodies can induce high cytotoxic activity similar to that of blinatumomab prepared using an anti-CD3 antibody. Furthermore, bispecific antibodies using anti-TRBC1 antibodies have been shown to produce a lower percentage of T cells expressing activation markers compared to bispecific antibodies using anti-CD3 antibodies. This suggests that bispecific antibodies using anti-TRBC1 or anti-TRBC2 antibodies can selectively activate some T cells, thereby reducing the risk of cytokine release syndrome and other conditions.

[0045] The therapeutic agents of the present invention comprise bispecific antibodies prepared using an anti-TRBC1 antibody or an anti-TRBC2 antibody, and when administered to a subject, they activate approximately half the number of T cells compared to bispecific antibodies using an anti-CD3 antibody, thereby reducing the risk of cytokine release syndrome. The number of activated T cells can be determined by measuring cells expressing T cell activation markers CD4, CD8, CD25, and / or CD69. Furthermore, despite activating approximately half the number of T cells, the therapeutic agents of the present invention are able to induce sufficiently high cytotoxic activity.

[0046] <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).

[0047] 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10.

[0048] 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 that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2. 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 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: 1 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: 2. The amino acid mutations (deletions, insertions and substitutions) to the amino acid sequence of SEQ ID NO: 1 or 2 may be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0049] In a particular aspect, at least one moiety 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91. In a further particular aspect, at least one moiety 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: 90 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: 91. The amino acid mutations (deletions, insertions, and substitutions) to the amino acid sequence of SEQ ID NO: 90 or 91 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0050] 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.

[0051] 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.

[0052] <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).

[0053] 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: 11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0054] 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: 3, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4. 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: 3, 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: 4. Amino acid mutations (deletions, insertions and substitutions) to the amino acid sequence of SEQ ID NO: 3 or 4 may be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0055] 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.

[0056] 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.

[0057] The CDR sequences of the TRBC1 or TRBC2 antigen binding portions are listed in the table below.

[0058] <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 in tumor cells, excluding T-cell tumor cells. Numerous antigens expressed in 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. Tumor antigen-binding moieties can be constructed based on known antibodies. Those skilled in the art can construct bispecific antibodies by genetic engineering techniques by selecting appropriate light chain variable regions and heavy chain variable regions from the amino acid sequence information of known antibodies. Genetic engineering techniques involve incorporating a gene sequence encoding the antibody into an expression vector, transforming it into a host cell, and culturing the host cell 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., murine antibodies) can be humanized and used to generate bispecific antibodies. Humanization methods are known, and can be performed, for example, by grafting 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.

[0059] 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.

[0060] (Hematologic Malignancies) The therapeutic agent of the present invention can be used to treat hematologic malignancies, excluding T-cell tumors.

[0061] 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.

[0062] Hematological malignancies (excluding T-cell tumors) that can be treated with the therapeutic agent of the present invention include, but are not limited to, any selected from the group consisting of acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, myeloproliferative neoplasm, acute lymphoblastic leukemia / lymphoblastic lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, mature B-cell tumors, Hodgkin's lymphoma, and multiple myeloma.

[0063] Myeloid tumors are classified as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, and myeloproliferative neoplasms. B-cell tumors include chronic lymphocytic leukemia / small lymphocytic lymphoma, mature B-cell neoplasms, Hodgkin's lymphoma, and multiple myeloma. T-cell tumors include mature T-cell tumors and NK-cell tumors, although these are excluded from the scope of this application. Acute lymphoblastic leukemia / lymphoblastic lymphoma includes both B-cell and T-cell tumors (which are excluded from the scope of this application), and these can be described as "B-cell acute lymphoblastic leukemia / lymphoblastic lymphoma" and "T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma," respectively.

[0064] Acute myeloid leukemia (AML) includes the following (Swerdlow SH, et al (Editors). WHO classification of tumors of hematopoietic and lymphoid tissues. Lyon: IARC Press; 2017): AML with recurrent genetic abnormalities (AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1, AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11, APL with PML-RARA, AML with t (9; 11) (p21.3; q23.3); MLLT3-KMT2A, AML with inv (6; 9) (p23; q34.1); DEK-NUP214, AML with inv (3) (q21.3q26.2) or t(3;3) (q21.3;q26.2); GATA2, MECOM, AML (megakaryoblastic) with t(1;22) (p13.3; q13.3);RBM15-MKL1, Provisional entity, AML with BCR-ABL1, AML with mutated NPM1, AML with biallelic mutations of CEBPA, Provisional entity, AML with mutated RUNX1), AML with myelodysplasia-related changes Therapy-related myeloid neoplasms, AML-NOS (AML with minimal differentiation, AML without maturation, AML with maturation, Acute myelomonocytic leukemia, Acute monoblastic / monocytic leukemia, Pure erythroid leukemia, Acute megakaryoblastic leukemia, Acute basophilic leukemia, Acute panmyelosis with myelofibrosis), Myeloid sarcoma, Myeloid proliferations related to Down syndrome (Transient abnormal myelopoiesis (TAM), Myeloid leukemia associated with Down syndrome). ;

[0065] Myelodysplastic syndromes (MDS) are diseases in which abnormalities occur in hematopoietic stem cells (the cells that become blood cells), resulting in a decrease in normal blood cells (red blood cells, white blood cells, and platelets).

[0066] Regarding chronic myeloid leukemia / myeloproliferative neoplasms (CML / MPN), myeloproliferative neoplasms (MPN) include the following (Swerdlow SH, et al (Editors). WHO classification of tumors of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2017): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).

[0067] Acute lymphoblastic leukemia / lymphoblastic lymphoma (ALL / LBL) is classified as acute lymphoblastic leukemia (ALL) when lymphoblasts infiltrate the bone marrow, and as lymphoblastic lymphoma (LBL) when there is no bone marrow involvement. 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).B-lymphoblastic leukemia / lymphoma, NOS, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, B-lymphoblastic leukemia / lymphoma with t(9;22)(q34.1;q11.2);BCR-ABL1, B-lymphoblastic leukemia / lymphoma with t(v;11q23.3);KMT2A rearranged, B-lymphoblastic leukemia / lymphoma with t(12;21) (p13.2;q22.1); ETV6-RUNX1, B-lymphoblastic leukemia / lymphoma with hyperdiploidy, B-lymphoblastic leukemia / lymphoma with hyperdiploidy, B-lymphoblastic leukemia / lymphoma with t(5;14)(q31.1;q32.3);IL3-IGH, B-lymphoblastic leukemia / lymphoma with t(1;19)(q23;p13.3);TCF3-PBX1, Provisional entity: B-lymphoblastic leukemia / lymphoma, BCR-ABL1-like, Provisional entity: B-lymphoblastic leukemia / lymphoma with iAMP21).

[0068] Chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL) is a disease in which mature lymphocytes proliferate monoclonally in the bone marrow and lymphoid tissues.

[0069] Mature B-cell neoplasms include the following (Swerdlow SH, et al (Editors). WHO classification of tumors of haematopoietic and lymphoid tissues. Lyon: IARC Press; 2017): Monoclonal B-cell lymphocytosis, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma Splenic B-cell lymphoma / leukemia, hairy cell leukemia, splenic B-cell lymphoma / leukemia, Splenic diffuse red pulp small B-cell lymphoma lymphoma), hairy cell leukemia-variant, lymphoplasmacytic lymphoma, monoclonal gammopathy of undetermined significance (MGUS) IgM type (monoclonal gammopathy of undetermined significance,IgM), μ heavy chain disease, λ heavy chain disease, α heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraskeletal plasmacytoma, monoclonal immunoglobulin deposition disease disease, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, follicular lymphoma, pediatric type follicular lymphoma, large B-cell lymphoma with IRF4 rearrangement primary cutaneous follicle center lymphoma, mantle cell lymphoma lymphoma), Diffuse large B-cell lymphoma, NOS, T-cell / histiocyte-rich large B-cell lymphoma lymphoma), primary central nervous system diffuse large B-cell lymphoma (DLBCL of the central nervous system) system), primary cutaneous diffuse large B-cell lymphoma, leg type, EBV positive diffuse large B-cell lymphoma, unspecified type (EBV positive DLBCL,NOS), EBV positive mucocutaneous ulcer (EBV positive mucocutaneous ulcer), chronic inflammation associated diffuse large B cell lymphoma (DLBCL associated with chronic lymphomatoid granulomatosis, primary mediastinal [thymic] large-B cell lymphoma lymphoma), intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma (ALK Plasmablastic lymphoma, primary effusion lymphoma, HHV8-positive diffuse large B-cell lymphoma, not otherwise specified (HHV8-positive DLBCL, NOS), Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, and high-grade B-cell lymphoma with MYC and / or BCL2 and / or BCL6 rearrangements. lymphoma, with MYC and BCL2 and / or BCL6 rearrangement), high-grade B-cell lymphoma, not otherwise specified (High-grade B-cell lymphoma, NOS), B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma).

[0070] Hodgkin lymphoma (HL) includes the following (Swerdlow SH, et al (Editors). WHO classification of tumors of hematopoietic and lymphoid tissues. Lyon: IARC Press; 2017): nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (CHL).

[0071] Multiple myeloma (MM) includes the following (Swerdlow SH, et al (Editors). WHO classification of tumors of hematopoietic and lymphoid tissues. Lyon: IARC Press; 2017). Non-IgM monoclonal gammopathy of undetermined significance (Non-IgM MGUS), IgM monoclonal gammopathy of undetermined significance (IgM MGUS), light-chain monoclonal gammopathy of undetermined significance (Light-chain MGUS), solitary plasmacytoma of bone / soft tissue, solitary plasmacytoma with minimal marrow involvement of bone / soft tissue tissue), smoldering (asymptomatic) multiple myeloma, (symptomatic) multiple myeloma (secretory / non-secretory), multiple solitary plasmacytoma, plasma cell leukemia, POEMS syndrome, systemic AL amyloidosis. The bispecific antibodies constituting the therapeutic agents of the present invention can target antigens expressed in the above-mentioned hematological malignancies.

[0072] For purposes of this application, the subject of treatment does not include "T-cell neoplasms," which include, but are not limited to, T-cell acute lymphoblastic leukemia / lymphoblastic lymphoma and mature T-cell neoplasms, as long as they are tumors of T cells or their precursor cells.

[0073] (Solid Cancer) The therapeutic agent of the present invention can be used to treat solid cancers. Solid cancers that can be treated with the therapeutic agent of the present invention include breast cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer (NSCLC)), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastroesophageal junction cancer, esophageal cancer, gastric cancer (including papillary adenocarcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet ring cell carcinoma, and mucinous carcinoma), gastrointestinal cancer, pancreatic cancer, brain tumor, glioblastoma, cervical cancer, endometrial cancer or uterine cancer, ovarian cancer, liver cancer, hepatoma (hepatoma), biliary tract cancer (gallbladder cancer, bile duct cancer), bladder cancer, colon cancer, rectal cancer, large intestine cancer, small intestine cancer, duodenal cancer, salivary gland cancer, kidney cancer, and prostate cancer. The cancer or tumor may be any cancer or tumor selected from the group consisting of prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, testicular germ cell tumor, urethral cancer, head and neck cancer, melanoma, orbital cancer, paranasal cancer, nasopharyngeal cancer, laryngeal cancer, parotid gland cancer, submandibular gland cancer, tongue cancer, glioma, schwannoma, thyroid cancer, parathyroid cancer, soft tissue sarcoma, skin cancer, cutaneous squamous cell carcinoma, papillary thyroid carcinoma, papillary renal cell carcinoma, hemangioma, pediatric solid tumors, lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, and Kaposi's sarcoma. These cancers or tumors may be primary or metastatic. The bispecific antibody constituting the therapeutic agent of the present invention can target an antigen expressed in the above solid tumors.

[0074] (Target Tumor Antigen) In the present invention, examples of target tumor antigens expressed on tumor cells include, but are not limited to, the following B cell antigens: CD1a, CD1b, CD1c, CD1d, CD2, CD5, CD6, CD9, CD11a, CD11b, CD11c, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD29, CD30, CD31, CD32a, CD32b, CD35, CD37, CD38, CD39, CD40, CD45, CD45RA, CD45RB, and CD4 5RC, CD45RO, CD46, CD47, CD48, CD49b, CD49c, CD49d, CD50, CD52, CD53, CD 54, CD55, CD58, CD60a, CD62L, CD63, CD68, CD69, CD70, CD72, CD73, CD74, CD 75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85E, CD85I , CD85J, CD86, CD92, CD95, CD97, CD98, CD99, CD100, CD102, CD108, CD119, C D120a, CD120b, CD12lb, CD122, CD124, CD125, CD126, CD130, CD132, CD137 , CD138, CD139, CD147, CD148, CD150, CD152, CD162, CD164, CD166, CD167a, CD170, CD171, CD175, CD175s, CD180, CD184, CD185, CD192, CD196, CD197, C D200, CD205, CD20la, CDw210b, CD212, CD213al, CD213a2, CD215, CD217, CD 218a, CD218b, CD220, CD221, CD222, CD224, CD225, CD226, CD227, CD229, C D230, CD232, CD252, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD2 61, CD262, CD263, CD264, CD267, CD268, CD279 (BCMA), CD270, CD272, CD274 , CD275, CD277, CD279, CD283, CD289, CD290, CD295, CD298, CD300, CD300c,CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD314, CD315, CD316, CD317, CD319, CD321, CD327, CD328, CD329, CD338, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD360, CD361, CD362, or CD363.

[0075] In the present invention, examples of target tumor antigens expressed on tumor cells include, but are not limited to, the following macrophage or monocyte antigens: CD1a, CD1b, CD1c, CD4, CD9, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD16, CD17, CD18, CD23, CD25, CD26, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD33, CD35, CD36, CD37, CD38, CD39, CD40, CD44, CD45, CD D45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, 15CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD58, CD59, CD60a , CD61, CD63, CD64a, CD65, CD66, CD68, CD69, CD72, CD74, CD75, CD75S, CD80 , CD81, CD82, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85I, CD85J , CD85K, CD86, CD87, CD88, CD89, CD91, CD92, CD93, CD95, CD97, CD98, CD99, CD99R, CDl00, CD101, CD102, CD105, 20CDl11, CDl12, CDl14, CDl15, CDl16, CDl19, CD120a, CD120b, CD121b, CD122, CD124, CD127, CD130, CD131, CD132 , CD136, CD137, CD139, CD141, CD142, CD143, CD147, CD148, CD153, CD155, CD 156a, CD156b, CD156c, CD157, CD162, CD163, CD164, CD165, CD166, CD168, C D169, CDl70, CDl71, CD172a, CD172b, CD180, CD181, CD182, CD184, CD185, CD 191, CD192, CD194, CD195, CDw198, CD24, CD205, CD206, CD209, CD210a, CDw 210b, CD213al, CD213a2, CD217, CD220, CD221, CD222, CD224, CD226, CD227,CD230, CD232, CD244, CD252, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD267, CD268, CD270, CD272, CD273, CD274, CD275, CD276, CD277, CD280, CD281, CD282, CD284, CD286, CD288, CD289, C CD295, CD297, CD298, CD300a, CD300c, CD300e, CD301, CD302, CD305, CD306, CD312, CD214, CD315, CD317, CD319, CD321, CD328, CD329, CD338, CD351, CD352, CD354, CD357, CD358, CD360, CD361, or CD362.

[0076] In the present invention, examples of target tumor antigens expressed on tumor cells include, but are not limited to, the following granulocyte antigens: CD4, CD9, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD23, CD24, CD29, CD31, CD32a, CD32b, CD32c, CD33, CD35, CD37, CD43, CD44, CD45, CD45RB, and CD45. RO, CD46, CD47, CD50, CD53, CD55, CD58, CD59, CD60a, CD62L, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, 15CD68, CD 69, CD75S, CD82, CD85A, CD85D, CD85K, CD87, CD88, CD89, CD92, CD93, CD95, CD97, CD98, CDl00, CD101, CD107a, CD107b, CD114, C Dl16, CD119, CD120a, CD120b, CD123, CD125, CD130, CD131, CD132, CD139, CD141, CD147, CD148, CD153, CD156a, CD156b, CD157, CD162, CD170, CD171, CD172a, CD177, CD178, CD181, CD182, CD183, CD192, CD193, 20CD195, CD203c, CD217, CD218a, CD218b, CD2 20, CD221, CD222, CD230, CD232, CD244, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD268, CD270, CD274, CD275, CD281, CD282, CD289, CD290, CD294, CD295, CD298, CD302, CD305, CD312, CD314, CD321, CD328, CD329, CD352, CD354, CD360, or CD362.

[0077] In one embodiment, the target tumor antigen expressed on tumor cells is, for example, DLL3, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine receptor type 5), CD19, HER (human epidermal growth factor receptor)-2, HER-3, HER-4, EGFR (epidermal growth factor receptor), FLT3 (Fms-like tyrosine kinase 3), PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, hCG, Lewis-Y, ganglioside GD3, 9-O-acetyl-GD3, GM2, globo H, fucosyl GM1, poly SA, GD2, carbonic anhydrase IX (MN / CAIX), CD44v6, Shh (serotonin receptor 1), or the like. These antigens include, but are not limited to, IgE, IgE-1, IgE (membrane-bound IgE), MCSP (melanoma chondroitin sulfate proteoglycan), CCR8, TNF-α (tumor necrosis factor alpha) precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6, desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker, MIS (Mullerian inhibitory substance) receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, and CD63. The therapeutic agents of the present invention can be used to treat cancers that overexpress any of these antigens (e.g., HER2-overexpressing cancers).

[0078] (CD19-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 CD19. Thus, the bispecific antigen-binding molecule may comprise a CD19-binding moiety and a TRBC1-binding moiety, or a CD19-binding moiety and a TRBC2-binding moiety. The CD19-binding moiety may be the CD19-binding moiety of the bispecific antibody (blinatumomab) described in U.S. Patent No. 7,635,472 B2. Therapeutic agents comprising bispecific antigen-binding molecules comprising a CD19-binding portion and a TRBC1-binding portion or a TRBC2-binding portion can be used to treat B-cell tumors, including, but not limited to, mature B-cell tumors (e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), MALT lymphoma, and Burkitt's lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), and acute lymphoblastic leukemia / lymphoblastic lymphoma (ALL / LBL).

[0079] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD19, 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: 31, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0080] In a particular aspect, at least one portion that specifically binds to CD19 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38. In a more particular aspect, at least one portion that specifically binds to CD19 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: 37 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: 38. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 37 or 38 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0081] In certain aspects, at least one portion that specifically binds to CD19 may be an scFv or Fab fragment.

[0082] 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; or (b) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16; and At least one portion that specifically binds to a target tumor antigen expressed on a tumor cell comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0083] 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: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91, 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: 3 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, and at least one moiety that specifically binds to a target tumor antigen expressed on 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: 37 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 38.

[0084] (BCMA-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 BCMA (B-cell maturation antigen). Thus, the bispecific antigen-binding molecule may comprise a BCMA-binding moiety and a TRBC1-binding moiety, or may comprise a BCMA-binding moiety and a TRBC2-binding moiety. The BCMA-binding moiety may be the BCMA-binding moiety of the bispecific antibody Teclistamab described in U.S. Patent No. US10072088 B2. A therapeutic agent comprising a bispecific antigen-binding molecule comprising a BCMA-binding moiety and a TRBC1-binding moiety or a TRBC2-binding moiety can be used to treat tumors expressing BCMA, for example, but not limited to, multiple myeloma (MM).

[0085] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to BCMA, 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: 39, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44.

[0086] In certain aspects, at least one portion that specifically binds to BCMA comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 46. In even more specific aspects, at least one portion that specifically binds to BCMA 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: 45 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: 46. Amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 45 or 46 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0087] In certain aspects, at least one portion that specifically binds to BCMA may be an scFv or Fab fragment.

[0088] 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; or (b) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16; and At least one portion that specifically binds to a target tumor antigen expressed on a tumor cell comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 39, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41; and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44.

[0089] 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: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91, 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: 3 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, and at least one moiety that specifically binds to a target tumor antigen expressed on 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: 45 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 46.

[0090] (CD33-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 CD33. Thus, the bispecific antigen-binding molecule may comprise a CD33-binding moiety and a TRBC1-binding moiety, or may comprise a CD33-binding moiety and a TRBC2-binding moiety. The CD33-binding moiety may be an anti-CD33 antibody described in U.S. Patent No. 5,585,089. A therapeutic agent comprising a bispecific antigen-binding molecule comprising a CD33-binding moiety and a TRBC1-binding moiety or a TRBC2-binding moiety can be used for the treatment of, but is not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia / myeloproliferative neoplasm (CML / MPN), and myelodysplastic syndrome (MDS).

[0091] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD33, 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:47, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:48, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:49, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:50, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:51, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:52.

[0092] In a particular aspect, at least one portion that specifically binds to CD33 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 54. In a more particular aspect, at least one portion that specifically binds to CD33 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: 53 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: 54. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 53 or 54 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0093] In certain aspects, at least one portion that specifically binds to CD33 may be an scFv or Fab fragment.

[0094] 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; or (b) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16; and At least one portion that specifically binds to a target tumor antigen expressed on a tumor cell comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:47, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:48, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:49; and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:50, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:51, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:52.

[0095] 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: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91, 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: 3 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, and at least one moiety that specifically binds to a target tumor antigen expressed on 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: 53 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 54. (CD123-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 CD123. Thus, the bispecific antigen-binding molecule may comprise a CD123-binding moiety and a TRBC1-binding moiety, or may comprise a CD33-binding moiety and a TRBC2-binding moiety. The CD123-binding moiety may be the CD123-binding moiety of the bispecific antibody Vibecotamab described in U.S. Patent No. US9850320 B2. The therapeutic agent comprising a bispecific antigen-binding molecule comprising a CD123-binding moiety and a TRBC1-binding moiety or a TRBC2-binding moiety can be used for the treatment of, but is not limited to, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia / lymphoblastic lymphoma (ALL / LBL), and chronic myeloid leukemia / myeloproliferative neoplasm (CML / MPN).

[0096] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to CD123, 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:55, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:56, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:57, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:58, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:59, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:60.

[0097] In a particular aspect, at least one portion that specifically binds to CD123 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 61 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 62. In a more particular aspect, at least one portion that specifically binds to CD123 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: 61 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: 62. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 61 or 62 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0098] In certain aspects, at least one portion that specifically binds to CD123 may be an scFv or Fab fragment.

[0099] 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; or (b) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16; and At least one portion that specifically binds to a target tumor antigen expressed on a tumor cell comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:55, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:56, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:57; and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:58, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:59, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:60.

[0100] 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: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91, 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: 3 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, and at least one moiety that specifically binds to a target tumor antigen expressed on 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: 61 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 62.

[0101] (HER2 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 HER2. Thus, the bispecific antigen-binding molecule may comprise a HER2-binding moiety and a TRBC1-binding moiety, or may comprise a HER2-binding moiety and a TRBC2-binding moiety. The HER2-binding moiety may be the anti-HER2 antibody trastuzumab described in U.S. Patent No. 5,821,337 B2. A therapeutic agent comprising a bispecific antigen-binding molecule comprising a HER2-binding moiety and a TRBC1-binding moiety or a TRBC2-binding moiety can be used to treat HER2-positive tumors, including, but not limited to, breast cancer, lung cancer, ovarian cancer, gastric cancer, urothelial cancer, gastroesophageal junction cancer, esophageal cancer, cervical cancer, endometrial cancer, biliary tract cancer, pancreatic cancer, and bladder cancer.

[0102] In one aspect, the bispecific antigen-binding molecule comprises a bispecific antigen-binding molecule comprising at least one portion that specifically binds to HER2, 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:63, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:64, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:65, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:66, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:67, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:68.

[0103] In certain aspects, at least one portion that specifically binds to HER2 comprises a heavy chain variable region VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 69 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 70. In even more certain aspects, at least one portion that specifically binds to HER2 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: 69 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: 70. The amino acid mutations (deletions, insertions, and substitutions) relative to the amino acid sequence of SEQ ID NO: 69 or 70 can be present in the complementarity determining regions (CDRs) or framework regions (FRs).

[0104] In certain aspects, at least one portion that specifically binds to HER2 may be an scFv or Fab fragment.

[0105] 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; or (b) a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:16; and At least one portion that specifically binds to a target tumor antigen expressed on a tumor cell comprises: a VH domain comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65; and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68.

[0106] 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: 1 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, 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: 90 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 91, 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: 3 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, and at least one moiety that specifically binds to a target tumor antigen expressed on 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: 69 and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0107] 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.

[0108] The CDR sequences of each tumor antigen-binding moiety are listed in the table below.

[0109] (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 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 tumor cells is an scFv, and the two scFvs are linked via a peptide linker.

[0110] A "peptide linker" is a peptide containing about 2 to 20 amino acids, such as (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: 17), GGGGSGGGGS (SEQ ID NO: 18), GGGGSGGGGSGGGGGS (SEQ ID NO: 30), SGGGGGSGGGG (SEQ ID NO: 19) and GGGGSGGGGSGGGG (SEQ ID NO: 20), although the sequences GSPGSSSSGSGS (SEQ ID NO: 21), (G ４ S) ４ , GSGSGSG (SEQ ID NO: 22), GSGSGNGS (SEQ ID NO: 23), GGSGSGSG (SEQ ID NO: 24), GGSGSG (SEQ ID NO: 25), GGSG (SEQ ID NO: 26), GGSGNGSG (SEQ ID NO: 27), GGNGSGSG (SEQ ID NO: 28) and GGNGSG (SEQ ID NO: 29) may also be used.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] In certain embodiments, the bispecific antigen-binding molecule is monovalent for both the target antigen on normal T cells and the target tumor antigen expressed on tumor cells.

[0115] In certain embodiments, the bispecific antigen-binding molecule is bivalent for both a target antigen on normal T cells and a target tumor antigen expressed on tumor cells.

[0116] In certain embodiments, the bispecific antigen-binding molecule is trivalent to both a target antigen on normal T cells and a target tumor antigen expressed on tumor cells.

[0117] In a particular embodiment, the bispecific antigen-binding molecule is monovalent for a target antigen on normal T cells and bivalent for both target tumor antigens expressed on tumor cells.

[0118] In a particular embodiment, the bispecific antigen-binding molecule is bivalent for the target antigen on normal T cells and monovalent for both target tumor antigens expressed on tumor cells.

[0119] 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 tumor cells are linked by a peptide linker, and further has an Fc domain (Fab-scFv-Fc).

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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).

[0125] The pharmaceutical composition of the present invention is preferably produced under conditions that comply with regulations for manufacturing and quality control of drugs and quasi-drugs (good manufacturing practice, GMP).

[0126] 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.

[0127] 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.

[0128] 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.

[0129] In a specific embodiment, the therapeutic agent of the present invention can be used in a method for treating a tumor in a subject, the method comprising administering the therapeutic agent to the subject. The tumor excludes T cell tumors. The therapeutic method can also determine the proportion of T cells expressing each subtype in the subject's T cell population. The subtype of the antigen expressed in the subject's T 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.

[0130] A second embodiment of the present invention is a method of treating a tumor in a subject, comprising administering a bispecific antigen-binding molecule to a tumor-bearing subject, with the proviso that the tumor is not a T-cell tumor.

[0131] A third embodiment of the present invention is a bispecific antigen-binding molecule for use in a method for treating a tumor, with the proviso that the tumor is not a T-cell tumor. A fourth embodiment of the present invention is use of a bispecific antigen-binding molecule in the manufacture of a medicament for treating a tumor, with the proviso that the tumor is not a T-cell tumor. The bispecific antigen-binding molecule in the second, third, and fourth embodiments of the present invention is the same as the bispecific antigen-binding molecule constituting the therapeutic agent of the first embodiment described herein above.

[0132] As used herein, the term "subject" means a mammal, such as a mouse, rat, primate, or especially a human.

[0133] <Bispecific antigen-binding molecule> A fifth embodiment of the present invention relates to a bispecific antigen-binding molecule. According to the present invention, there is provided a bispecific antigen-binding molecule that constitutes the therapeutic agent of the first embodiment.

[0134] The bispecific antigen-binding molecule of the present invention comprises: (1) at least one moiety that specifically binds to a target tumor antigen expressed on tumor cells (excluding T-cell tumor cells); and (2) at least one moiety that specifically binds to a normal T-cell target antigen, the antigen having a subtype, wherein the at least one moiety that specifically binds to a normal T-cell target antigen 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.

[0135] Furthermore, the present invention provides nucleic acid molecules encoding the heavy and light chains of the bispecific antigen-binding molecule of the fifth aspect. The nucleic acid molecules of the present invention may be RNA, DNA, or cDNA.

[0136] 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.

[0137] 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.

[0138] 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 fifth 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.

[0139] 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).

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] (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 (lymphokine-activated killer cells)) 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] The bispecific antigen binding molecules having a TRBC1-binding moiety or a TRBC2-binding moiety of the present invention may be used in methods 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 tumours, (viii) delaying tumour progression, or (ix) prolonging the survival of a subject afflicted with a tumour.

[0157] 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 tumor cells were transplanted into the mice. ７ The mice are intravenously transplanted with the bispecific antigen-binding molecule of the present invention 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 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 tumor burden between the control group and the group administered with the bispecific antigen-binding molecule of the present invention.

[0158] 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 portion was constructed 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: 3 and 4 of the present application).

[0159] 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.

[0160] The heavy and light chain sequences of the bispecific antibody prepared in this example are shown below. FIG. 1 shows schematic diagrams of the Fab-scFv type, the Fab-scFv-Fc type, and the Fab-Fc-scFv type.

[0161] 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-G. 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.

[0162] 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: 72 and 73.) Immunase 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) Various cells were prepared as follows. T-LAK is a T cell population that expresses lymphokine-activated killer (LAK) function. T-LAK was prepared using an optimized 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, they 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 freshly when the culture medium was changed.

[0163] 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)

[0164] The staining antibodies are shown in the table below. *BV421, BV711: abbreviations for Brilliant Violet 421 (registered trademark) and Brilliant Violet 711 (registered trademark)

[0165] The anti-TRBC1 antibody was produced and purified using 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: 72 and 73. As an isotype control, human IgG (ChromPure Human IgG, whole molecule, Jackson Immuno Research, 009-000-003) was similarly dye-labeled. Method: Experiments were performed with reference to WO2021 / 173896 A1. 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, Flow Cytometry Staining Buffer was added, followed by centrifugation (500g x 2 minutes). The supernatant was discarded, washing the cells twice, and the signal was quantified by flow cytometry analysis using a BD LSRFortessa X-20 (BD Biosciences). Flow Jo_v10.6.1 was used as the analysis software.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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. Functional cytotoxic T cell effector memory (EM) cells show almost no expression of immunosuppressive checkpoint molecules such as PD-1 or TIGIT, whereas functionally inactive exhausted T cells 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 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.

[0174] 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.

[0175] 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: 74 and 75. The reagents and staining antibodies used in this Reference Example were the same as those described in Reference Example 1.

[0176] 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.

[0177] 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.

[0178] 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 T-LAK cell population was defined as TRBC2-positive cells.

[0179] 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.

[0180] 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. Functional cytotoxic T cell effector memory (EM) cells show almost no expression of immunosuppressive checkpoint molecules such as PD-1 and TIGIT, whereas functionally inactive exhausted T cells 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 the anti-TRBC1 antibody. It is also 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.

[0181] 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.

[0182] Example 3: Comparison of the Activity of Existing Bispecific Antibodies and Bispecific Antibodies Using TRBC1 In this Example, to evaluate the in vitro efficacy of the bispecific antibodies prepared in Example 1, target cells were cultured with T-LAK (effector cells) in the presence of the specific antibodies, and the viability of the target cells was measured using a fluorescent dye (calcein activity) as an indicator to evaluate effector cell-dependent cytotoxicity. The experiment was performed with reference to Neri S et al. (Clin Diagn Lab Immunol. 2001 Nov;8(6):1131-5). Currently, anti-CD3 antibodies are the primary bispecific antibodies used as T cell engagers on the market. In this Example, the cytotoxic activity of an anti-CD19 / anti-TRBC1 bispecific antibody (CD190001) and blinatumomab, an anti-CD19 / CD3 bispecific antibody marketed as a treatment for B cell tumors, was examined. These results suggest that the creation of bispecific antibodies using agonistic anti-TRBC1 or anti-TRBC2 antibodies may be able to suppress cytokine release while retaining sufficient tumor cytotoxicity.

[0183] 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).

[0184] Cytotoxicity of bispecific antibodies against Raji cells: Bispecific antibodies were adjusted to a final concentration of 1 fM to 100 nM (concentration varied depending on the antigen) in 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 CD19 binding moieties, the required amount of Raji cells (target cells) was recovered from the culture medium and washed twice with HBSS. The target cells were labeled with calcein by incubation with 10 μM calcein-AM containing 5 mM probenecid at 37°C for 30 minutes. 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.

[0185] 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)]. Sigmoidal dose-response curves were calculated using Prism Software (GraphPad Software Inc.), and the EC50 value (50% effective concentration) and Emax value (maximum activity value) were calculated.

[0186] Results FIG. 14 and the table below show the results of adding T-LAK to Raji cells, a Burkitt's lymphoma-derived cell line that expresses CD19, and measuring the cytotoxic activity of the bispecific antibodies CD190001 and Blinatumomab. When sufficient T cells expressing CD3 and TRBC1 were present, both CD190001 and Blinatumomab showed similar activity, with Emax of approximately 99%.

[0187] Example 4 Comparison of activity between existing bispecific antibodies and bispecific antibodies using TRBC1 The following reagents were used. PBS (Nacalai Tesque, 14249-24), Ficoll-Paque™ PLUS (GE Healthcare, 17-1440-02), Leucosep (Grener bio-one, 227290), FBS (NICHIREI, 175012, Lot. 19J00C), 2% FBS-PBS, Red Blood Cell Lysis Buffer (Roche, 11814389001), CELLBUNKER2 (Takara, 11914), RPMI1640 (Nacalai Tesque, 30264-85), 1M Hepes (Nacalai, 17557-94) 100x Sodium pyruvate (Gibco, 11360-070) 100x MEM NEAA (Gibco, 11140-050) 100x 2-ME (Wako, 198-15781) PKH26 Red Fluorescent Cell Linker Kit (Sigma; PKH26GL) In this example, the cytotoxic activity of the anti-CD19 / anti-TRBC1 bispecific antibody of the present invention and blinatumomab, a marketed therapeutic agent for B-cell malignancies, was examined. Blinatumomab is a bispecific antibody targeting CD19 and CD3 for use in the treatment of B-cell malignancies. It is believed that the use of human T cells in this example will enable evaluation of cytotoxic activity under conditions closer to those in vivo. An anti-CD19 / anti-CD3 bispecific antibody was produced by the method described in Example 1.

[0188] Methods: For the cytotoxicity assay using bispecific antibodies bearing antigen-binding moieties for CD19 and TRBC1 or CD3, CD190001 and Blinatumomab prepared in Example 3 were used. The bispecific antibodies were adjusted to a final concentration of 1 fM to 1 nM in RPMI 1640 medium containing 5% FBS, 10 mM HEPES, 1 mM sodium pyruvate, MEM NEAA, and 0.1 mM 2-ME (TDCC medium) and added to a 96-well U-bottom culture plate. Control wells contained TDCC medium only. The required amount of Raji cells (target cells) was recovered from the culture medium, labeled with PKH26 according to the PKH26 Red Fluorescent Cell Linker Kit protocol, suspended in TDCC medium, and counted. The bispecific antibody was then added to the prepared 96-well U-bottom culture plate. After collecting the required amount of PBMCs (effector cells) from healthy donors, they were suspended in TDCC medium and counted. The bispecific antibody was then added to the prepared 96-well flat-bottom culture plate. The effector cells were added at a ratio of 20:1 to labeled target cells. The plate containing the bispecific antibody, labeled target cells, and effector cells was incubated at 37°C with 5% CO2. After 72 hours, the cells from each well were transferred to a 96-well V-bottom plate, washed once with 2% FBS / PBS, and diluted antibodies for dead cell staining and T cell activation markers were added. After incubation at 4°C for 30 minutes, the cells were washed twice with 2% FBS / PBS, and CountBright Absolute Counting Beads (Invitrogen, C36950) were added to the samples and measured using a flow cytometer (BD Symphony A1). PKH26-positive and Fixable Viability Dye eFluor 780-negative fractions were gated as viable target cells, and cell number was corrected using CountBright Absolute Counting Beads to evaluate cytotoxicity. Flow Jo_v10.9.0 was used as the analysis software.

[0189] The cytotoxicity rate was calculated as follows: The number of viable target cells was determined by correcting the number of cells in the PKH26-positive and Fixable Viability Dye eFluor 780-negative fraction to the number of cells per 3,000 CountBright Absolute Counting Beads. The maximum viable number 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 × (viable number of target cells in the presence of bispecific antibody) / (maximum viable number)]. The 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. Preparation of PBMCs from Healthy Volunteers PBMCs were prepared from blood collected from healthy volunteers as follows. Heparinized blood was diluted 3-fold with PBS, added to a Leucosep tube filled with Ficoll, and made up to 50 mL with PBS. The mixture was then centrifuged (1000 × g, 10 min, 20°C), the upper layer was removed, and the PBMC layer was transferred to a separate tube. After washing twice with PBS, the mixture was suspended in 3 mL of 2% FBS-PBS, 6 mL of RBC buffer was added, and the mixture was allowed to stand for 10 minutes. After washing with 10 mL of 2% FBS / PBS, the cells were suspended in CELLBUNKER 2 and stored in liquid nitrogen until use.

[0190] Figure 15 shows the results of measuring the cytotoxic activity of anti-CD19 / anti-TRBC1 bispecific antibody (CD190001) and anti-CD19 / anti-CD3 bispecific antibody (Blinatumomab) when PBMCs derived from healthy individuals were added to Raji cells, a Burkitt's lymphoma-derived cell line. The EC50 and Emax values ​​(N=4) are shown in the table below. Both the anti-CD19 / anti-TRBC1 bispecific antibody and the anti-CD19 / anti-CD3 bispecific antibody showed sufficient cytotoxic activity. Although the TRBC1-positive cell rate among T cells was approximately 50%, the activity of the bispecific antibody using TRBC1 is thought to be similar to that of the bispecific antibody using CD3.

[0191] Example 5: Changes in T cell activation markers using existing bispecific antibodies and bispecific antibodies using TRBC1 This Example showed that a lower percentage of T cells expressing activation markers was observed when using a bispecific antibody using TRBC1 compared to a bispecific antibody using CD3. This suggests that selective activation of a portion of T cells using a bispecific antibody using TRBC1 or TRBC2 may reduce the risk of cytokine release syndrome, etc.

[0192] Methods: The antibodies used in Example 4 were used to evaluate T cell activation by bispecific antibodies bearing antigen-binding moieties for CD19 and TRBC1 or CD3. The bispecific antibodies were adjusted to a final concentration of 1 fM to 1 nM in RPMI 1640 medium containing 5% FBS, 10 mM HEPES, 1 mM sodium pyruvate, MEM NEAA, and 0.1 mM 2-ME (TDCC medium) and added to 96-well U-bottom culture plates. Control wells contained TDCC medium only. The required amount of Raji cells (target cells) was recovered from the culture medium, labeled with PKH26 according to the PKH26 Red Fluorescent Cell Linker Kit protocol, suspended in TDCC medium, and counted. The bispecific antibodies were then added to the prepared 96-well U-bottom culture plates. After collecting the required amount of PBMCs (effector cells) from healthy individuals, they were suspended in TDCC medium and counted. Then, bispecific antibodies were added to prepared 96-well flat-bottom culture plates. Effector cells were added at a 20:1 ratio to labeled target cells. The plates containing bispecific antibodies, labeled target cells, and effector cells were incubated at 37°C with 5% CO2. After 72 hours, cells from each well were transferred to a 96-well V-bottom plate, washed once with 2% FBS / PBS, and diluted antibodies for dead cell staining and T cell activation markers were added. After 30 minutes of incubation at 4°C, the plates were washed twice with 2% FBS / PBS. CountBright Absolute Counting Beads (Invitrogen, C36950) were added to the samples, and the results were analyzed using a flow cytometer (BD Symphony A1). The PKH26-negative and Fixable Viability Dye eFluor780-negative fraction was gated as viable effector cells, and the positivity rates of CD25 and CD69, which are activation markers for CD4- or CD8-positive T cells, were evaluated. *BV421, BV650: abbreviations for Brilliant Violet 421 (registered trademark) and Brilliant Violet 650 (registered trademark)

[0193] Results The results are shown in Figure 16. When the expression of CD25 and CD69, which are activation markers for CD4 or CD8 positive T cells, was measured for the target cells and effector cells after reaction with the bispecific antibody used in this example, expression of both activation markers was induced in an antibody concentration-dependent manner. Comparing CD190001 and Blinatumomab, the proportion of activated T cells was 50% or less in the presence of the same antibody concentration. This result is thought to suggest that when using a bispecific antibody with TRBC1, only T cells expressing TRBC1 crosslink with target cells and function as effector cells.

[0194] Example 6 Comparison of cytokine release between an existing bispecific antibody and a bispecific antibody using TRBC1 The following reagents were used. PBS (Nacalai Tesque, 14249-24), Ficoll-Paque™ PLUS (GE Healthcare, 17-1440-02), Leucosep (Grener bio-one, 227290), FBS (NICHIREI, 175012, Lot. 19J00C), 2% FBS-PBS, Red Blood Cell Lysis Buffer (Roche, 11814389001), CELLBUNKER2 (Takara, 11914), RPMI1640 (Nacalai Tesque, 30264-85), 1M Hepes (Nacalai, 17557-94) 100x Sodium pyruvate (gibco, 11360-070) 100x MEM NEAA (gibco, 11140-050) 100x 2-ME (Wako, 198-15781) PKH26 Red Fluorescent Cell Linker Kit (Sigma; PKH26GL) MILLIPLEX (registered trademark) Human Cytokine / Chemokine / Growth Factor Panel A (Millipore, HCYTA-60K)

[0195] Methods: The antibodies used in Example 4 were used to evaluate T cell activation by bispecific antibodies containing antigen-binding moieties for CD19 and TRBC1 or CD3. Bispecific antibodies were adjusted to a final concentration of 1 fM to 1 nM in RPMI 1640 medium (TDCC medium) containing 5% FBS, 10 mM HEPES, 1 mM sodium pyruvate, MEM NEAA, and 0.1 mM 2-ME, and added to a 96-well U-bottom culture plate. Control wells were set up with TDCC medium alone. The required amount of Raji cells (target cells) was collected from the culture medium, suspended in TDCC medium, and counted. The bispecific antibodies were then added to the 96-well U-bottom culture plate. The required amount of PBMCs (effector cells) from healthy individuals was collected, suspended in TDCC medium, and counted. The bispecific antibody was then added to the prepared 96-well flat-bottom culture plate. Effector cells were added at a 20:1 ratio of labeled target cells. The plate containing the bispecific antibody, labeled target cells, and effector cells was incubated at 37°C with 5% CO2. After 24 hours, the collected culture supernatant was frozen and stored in a -80°C freezer until measurement. Samples were prepared according to the MILLIPLEX® Human Cytokine / Chemokine / Growth Factor Panel A protocol, and IFN-γ, IL-2, and TNF-α were quantified. Quantitative values ​​were calculated automatically using the Luminex® 200x PONET® 3.1 system.

[0196] Results The results are shown in Figure 17. When comparing the major cytokines IFN-γ, TNFα, and IL-2, both Blinatumomab and CD190001 increased the amount of cytokine released in an antibody concentration-dependent manner. When comparing the maximum release amounts within the measurement range, the cytokine amount of CD190001 was less than half that of Blinatumomab for all cytokines. Because the rate of TRBC1-positive cells among T cells was approximately 50%, the number of cells that were activated as effector cells and released cytokines was also low, which is thought to enable the final total amount of cytokine release to be suppressed.

[0197] Example 7 Cytotoxic Activity of Anti-CD19 / 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). 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)

[0198] Cytotoxicity of bispecific antibodies against Raji cells: Bispecific antibodies were adjusted to a final concentration of 1 fM to 100 nM (concentration varied depending on the antigen) in 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 CD19 binding moieties, the required amount of Raji cells (target cells) was recovered from the culture medium and washed twice with HBSS. The target cells were labeled with calcein by incubation with 10 μM calcein-AM containing 5 mM probenecid at 37°C for 30 minutes. 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.

[0199] The cytotoxicity rate was calculated as follows: The minimum elution 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 elution fluorescence value (lysis rate = 100%) was obtained by adding 10x Lysis Solution to wells under the same conditions as the minimum elution fluorescence value. The cytotoxicity rate was calculated using the following formula: Cytotoxicity rate = [(elution fluorescence value of target cells in the presence of bispecific antibody) - (minimum elution fluorescence value)] / [(maximum elution fluorescence value) - (minimum elution fluorescence value)]. Sigmoidal dose-response curves were calculated using Prism Software (GraphPad Software Inc.), and the EC50 value (50% effective concentration) and Emax value (maximum activity value) were calculated.

[0200] 18 and the table below show the results of adding T-LAK to Raji cells, a Burkitt's lymphoma-derived cell line that expresses CD19, and measuring the cytotoxic activity of the bispecific antibody CD190001. The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-CD19 and anti-TRBC1 bispecific antibody CD190001 was thought to exhibit very potent cytotoxic activity against CD19-expressing cells.

[0201] Example 8 Cytotoxic Activity of Anti-BCMA / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-BCMA / anti-TRBC1 bispecific antibody prepared in Example 1, target cells U266 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. The target cells U266 are a cell line that expresses BCMA. Cytotoxic activity was measured using a method similar to that described in Example 3.

[0202] 19 and the table below show the results of adding T-LAK to U266 cells, a multiple myeloma-derived cell line that expresses BCMA, and measuring the cytotoxic activity of the bispecific antibody BCMA0001. The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-BCMA and anti-TRBC1 bispecific antibody BCMA0001 was thought to exhibit very potent cytotoxic activity against BCMA-expressing cells.

[0203] Example 9 Cytotoxic Activity of Anti-CD33 / Anti-TRBC1 Bispecific Antibody In this Example, to evaluate the in vitro efficacy of the anti-CD33 / anti-TRBC1 bispecific antibody prepared in Example 1, MOLM13 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. The MOLM13 target cells are a cell line that expresses CD33. Cytotoxic activity was measured using a method similar to that described in Example 3.

[0204] Figure 20 and the table below show the results of measuring the cytotoxic activity of each bispecific antibody when T-LAK was added to MOLM13 cells, a cell line derived from acute myeloid leukemia that expresses CD33. The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-CD33 and anti-TRBC1 bispecific antibody was thought to exhibit very potent cytotoxic activity against antigen-expressing cells.

[0205] Example 10: Cytotoxic activity of anti-CD123 / anti-TRBC1 bispecific antibody In this example, to evaluate the in vitro efficacy of the anti-CD123 / anti-TRBC1 bispecific antibody prepared in Example 1, MOLM13 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. The MOLM13 target cells are a cell line that expresses CD123. Cytotoxic activity was measured using a method similar to that described in Example 3.

[0206] 21 and the table below show the results of measuring the cytotoxic activity of each bispecific antibody when T-LAK was added to MOLM13 cells, a cell line derived from acute myeloid leukemia that expresses CD123. The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-CD123 and anti-TRBC1 bispecific antibody was thought to exhibit very potent cytotoxic activity against antigen-expressing cells.

[0207] Example 11: Cytotoxic activity of anti-HER2 / anti-TRBC1 bispecific antibody In this example, to evaluate the in vitro efficacy of the anti-HER2 / anti-TRBC1 bispecific antibody prepared in Example 1, MKN7 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. Method: MKN7 target cells are a cell line that expresses HER2. Two days before activity measurement, the target cells were seeded into a 96-well culture plate and incubated in 5% CO ２ , and incubated at 37°C. On the day of measurement, the plate seeded with target cells was washed three times with HBSS and incubated with 10 μM Calcein-AM supplemented with 5 mM Probenecid at 37°C for 30 minutes to label the target cells with calcein. After incubation, the plate was washed three times with HBSS, and after the final wash, bispecific antibodies adjusted to various concentrations were added to the 96-well culture plate. The required amount of effector cells was recovered from the culture medium, washed once with assay medium, counted, and then added to the 96-well culture plate containing the bispecific antibodies. The effector cells and labeled target cells were added at a ratio of 10:1. The plate containing the bispecific antibodies, labeled target cells, and effector cells was incubated at 37°C for 30 minutes at 5% CO ２ The cells were incubated at 37°C for 2 hours. The 96-well 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. The cytotoxicity was calculated in the same manner as described in Example 3.

[0208] Figure 22 and the table below show the results of adding T-LAK to MKN7 cells, a HER2-expressing tubular adenocarcinoma-derived cell line, and measuring the cytotoxic activity of each bispecific antibody. The EC50 and Emax values ​​(N=3) are shown in the table below. The anti-HER2 and anti-TRBC1 bispecific antibody was thought to exhibit very potent cytotoxic activity against antigen-expressing cells. The anti-HER2 and anti-TRBC1 bispecific antibody was thought to exhibit effective cytotoxic activity against antigen-expressing solid tumors.

Claims

1. A tumor therapeutic agent (excluding T-cell tumors) 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 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 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 binding of the bispecific antigen-binding molecule to the target antigen on normal T cells activates 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 tumor treatment.

2. The therapeutic agent according to claim 1, wherein the target antigen on normal T cells and having a subtype is TRBC (T cell receptor beta constant region), and the subtype of the target antigen on normal T cells is TRBC1 or TRBC2.

3. 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:5, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

10.

4. The therapeutic agent according to claim 3, 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:1 and a light chain variable region (VL) that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2, or a heavy chain variable region (VH) that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:90 and a light chain variable region (VL) that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

91.

5. 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: 11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VL domain comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

16.

6. The therapeutic agent according to claim 5, 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:3, and a light chain variable region VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

4.

7. The therapeutic agent according to claim 1 or 2, wherein the tumor is a hematological malignant tumor excluding T-cell tumors.

8. The therapeutic agent according to claim 7, wherein the hematological malignancy is a myeloid tumor or a B-cell tumor.

9. The therapeutic agent according to claim 1 or 2, wherein the tumor is a solid cancer.

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