Use of Anti-tspan8-Anti-CD3 bispecific antibody combined with PD-1 signal inhibitor for cancer treatment

Abstract

To provide an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treatment of a target cancer, or a pharmaceutical composition comprising the bispecific antibody; or to provide a cancer treatment method comprising administering the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor to the target.SOLUTION: A pharmaceutical composition comprises an anti-TSPAN8-anti-CD3 bispecific antibody including: (a) a Fab region of an anti-TSPAN8 antibody composed of a heavy chain fragment comprising a heavy chain variable region of the anti-TSPAN8 antibody and a light chain comprising a light chain variable region of the anti-TSPAN8 antibody; (b) an anti-CD3 scFv region comprising a heavy chain variable region of an anti-CD3 antibody and a light chain variable region of an anti-CD3 antibody; and (c) an Fc region composed of a first Fc polypeptide linked to the heavy chain fragment of the Fab region in (a), and a second Fc polypeptide linked to the anti-CD3 scFv region in (b).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of an anti-TSPAN8-anti-CD3 bispecific antibody in combination with a PD-1 signaling inhibitor in cancer treatment.

Background Art

[0002] Tetraspanin 8 (TSPAN8) is a four-transmembrane protein belonging to the tetraspanin family, having two extracellular loop regions, a small extracellular loop (SEL) and a large extracellular loop (LEL), and three cytoplasmic domains, and forms a molecular cluster having various transmembrane proteins and cytoplasmic proteins as scaffold proteins. TSPAN8 is known to be involved in cell adhesion, cell motility, cell activation, and proliferation, etc., and high expression is observed in gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, etc., and the association between enhanced TSPAN8 expression and cancer progression or metastasis has been reported (Non-Patent Document 1). Research aiming at cancer diagnosis and treatment using anti-TSPAN8 antibodies has been conducted (Patent Documents 1 to 2 and Non-Patent Documents 1 to 2).

[0003] Cluster of Differentiation 3 (CD3) is a protein that transmits an activation signal to T cells by forming a complex with a T cell receptor (TCR) on the T cell surface. CD3 is a complex composed of five subunits, gamma (γ), delta (δ), epsilon (ε), zeta (ζ), and eta (η) chains, and each subunit forms three types of dimers, εγ, εδ, and ζζ. Since CD3 is expressed in both normal T cells and neoplastic T cells, it is used as a marker for T cells, and various applications of bispecific antibodies containing various anti-cancer related antigen (Tumor Associate Antigen; TAA) antibodies and anti-CD3 antibodies as pharmaceuticals for cancer treatment have been reported (Non-Patent Document 3).

[0004] As an epoch-making method capable of obtaining cancer cell-selective cytotoxic activity at low antibody concentrations, bispecific T-cell-recruiting antibodies consisting of various antibody formats have been reported, and the effects of these antibodies on T cell-mediated immunotherapy are being investigated (Non-Patent Document 4). Bispecific T-cell-recruiting antibodies are bispecific antibodies containing an antibody against a TAA expressed on the surface of cancer cells and an antibody that binds to T cells. As the antibody that binds to T cells, anti-CD3 antibodies are often used. The bispecific T-cell-recruiting antibody of a molecule containing an anti-TAA and an anti-CD3 antibody brings the physical distance between the target cancer cells and cytotoxic T lymphocytes (CTLs) closer, activates CTLs with the anti-CD3 antibody, and kills cancer cells by the cytotoxic activity of CTLs (Redirected T Cell Cytotoxicity; RTCC). Catumaxomab, an anti-CD3-anti-epithelial cell adhesion molecule (EpCAM) bispecific antibody, and blinatumomab, an anti-CD3-anti-CD19 (Cluster of Differentiation 19) bispecific antibody, have already been confirmed to be effective clinically (Int. J. Cancer, 2010; Vol. 127: p. 2209-2221, N. Engl. J. Med., 2017; Vol. 376: p. 836-847), and currently, research and development of bispecific T-cell-recruiting antibodies against various TAAs are being carried out.

[0005] Programmed cell death-1 (PD-1; also known as PDCD1 or CD279) is a 50-55 kDa type I transmembrane protein belonging to the immunoglobulin superfamily (Int. Immunol., 1996; Vol. 8: p. 765-772). PD-1 is induced to express with the persistence of activation in T cells, but by binding to its ligands, Programmed death-ligand 1 (PD-L1; also known as PDCD1LG1, B7-H1 or CD274) or Programmed death-ligand 2 (PD-L2; also known as PDCD1LG2, B7-DC or CD273), it controls the activation of T cells in an inhibitory manner (Annu. Rev. Immunol., 2008; Vol. 26: p. 677-704). Generally, such a T cell activation control mechanism is called an immune checkpoint and is known as one of the negative feedback mechanisms to avoid causing excessive immune responses.

[0006] In the early stage of cancer development, immune cells such as T cells eliminate cancer through the anti-tumor immune response by the immune surveillance mechanism. On the other hand, cancer has acquired an immune escape mechanism by directly or indirectly suppressing immune cells in the cancer microenvironment. As direct T cell activation suppression mechanisms, immune checkpoint mechanisms such as the PD-1 / PD-L1 or PD-L2 (hereinafter referred to as the "PD-1 signal") pathway, the CTLA-4 / CD80 or CD86 pathway are known. In the tumor microenvironment of cancer, the expression of PD-1 in T cells and the expression of PD-L1 in tumors have been confirmed (Nat. Med., 2002; Vol. 8: p. 793-800), and it is considered that cancer shows immune escape by the activation of this PD-1 signal. It has been reported in multiple mouse cancer models that inhibition of PD-1 signaling leads to antitumor activity by releasing immune evasion mechanisms (Non-Patent Documents 5-7). Furthermore, PD-1 signal inhibitors such as anti-PD-1 antibodies, such as nivolumab and pembrolizumab, have been vigorously developed as PD-1 signaling drugs, and have achieved great results in melanoma, lung cancer, lymphoma, etc. Furthermore, research is being conducted on nucleic acid drugs and small molecule drugs as PD-1 signaling inhibitors in addition to antibodies (Non-Patent Document 8). Indirect mechanisms of suppressing activated T cells include the induction of regulatory T cells and bone marrow-derived regulatory cells by cancer cells and the production of immunosuppressive humoral factors (IL-10, TGF-β, indoleamine 2,3-dioxygenase (IDO), etc.), and the development of drugs targeting these humoral factors is underway (J. Hematol. Oncol., 2021; Vol. 14: p. 55-74, BioDrugs, 2018; Vol. 32: p. 311-317).

[0007] In order to improve the efficacy of treatment in cancer patients, combination therapy with multiple cancer immunotherapy drugs and combination studies of cancer immunotherapy drugs with existing anticancer drugs are being actively conducted (Non-Patent Documents 9 and 10). For example, combination studies of anti-PD-1 antibodies with other immune checkpoint inhibitor antibodies, anticancer drugs, molecular targeted drugs, radiation therapy, cancer vaccines, and oncolytic viruses are being conducted. However, to date, there have been no reports of a cancer treatment method using a combination of an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2012 / 010696 issue [Patent Document 2] WO2015 / 130115 issue [Non-patent literature]

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0010] The problem of the present invention is to provide an anti-TSPAN8-anti-CD3 bispecific antibody or a pharmaceutical composition containing the bispecific antibody for use in combination with a PD-1 signal inhibitor for the treatment of cancer in a subject, or to provide a method for treating cancer including administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor to a subject.

Means for Solving the Problems

[0011] The inventors aimed to create an antibody or pharmaceutical composition for treating TSPAN8-expressing cancers. Based on the sequence of 16B11.1, an anti-TSPAN8 antibody, they produced an anti-TSPAN8(16B11)-anti-CD3 bispecific antibody (Example 1). When this anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and an anti-PD-1 antibody were administered to mice transplanted with TSPAN8-expressing cancer cells, it was confirmed that they exhibited a significant antitumor effect compared to the case where the anti-PD-1 antibody was administered alone (Example 2). From these results, it was suggested that the combination of an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor is useful for treating TSPAN8-expressing cancers.

[0012] That is, the present invention relates to the following [1] to

[80] . [1] A pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating a subject's cancer, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: (a) A Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) An anti-CD3scFv region containing the heavy chain variable region of the anti-CD3 antibody and the light chain variable region of the anti-CD3 antibody, and (c) An Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3scFv region of (b), and the pharmaceutical composition. [2] The heavy chain variable region of the anti-TSPAN8 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, the pharmaceutical composition according to [1]. [3] The heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, the pharmaceutical composition according to [1] or [2]. [4] The Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6, the pharmaceutical composition according to any one of [1] to [3]. [5] The heavy chain variable region of the anti-CD3 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, the pharmaceutical composition according to any one of [1] to [4]. [6] The heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, the pharmaceutical composition according to any one of [1] to [5]. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO: 8. [8] The anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain fragment of an anti-TSPAN8 antibody in which a heavy chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4 is linked to a first Fc polypeptide; a light chain of an anti-TSPAN8 antibody containing a light chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6; and an anti-CD3 scFv region containing a heavy chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, and a polypeptide in which the anti-CD3 scFv region is linked to a second Fc polypeptide. The pharmaceutical composition according to any one of [1] to [7]. [9] The pharmaceutical composition according to any one of [1] to [8], comprising a polypeptide in which an anti-TSPAN8-anti-CD3 bispecific antibody is linked to a second Fc polypeptide, the anti-TSPAN8-anti-CD3 bispecific antibody comprising a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4 linked to a first Fc polypeptide, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, and an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8 and a light chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.

[10] The pharmaceutical composition according to any one of [1] to [9], comprising an Fc region comprising one or more mutations selected from the group consisting of LALA mutation, N297G mutation, or knobs-into-holes mutation.

[11] A pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signaling inhibitor for treating cancer in a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody consists of a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and a polypeptide in which an anti-CD3scFv region consisting of the amino acid sequence of SEQ ID NO: 8 is linked to a second Fc polypeptide.

[12] The pharmaceutical composition according to any one of [1] to

[11] , comprising a post-translationally modified anti-TSPAN8-anti-CD3 bispecific antibody as an active ingredient.

[13] The pharmaceutical composition according to [1] to

[12] , which is administered simultaneously, continuously, or sequentially with a PD-1 signaling inhibitor.

[14] The pharmaceutical composition according to [1] to

[12] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) contained in separate pharmaceutical compositions and administered simultaneously, continuously, or sequentially. The pharmaceutical composition according to any one of [1] to

[14] , wherein the cancer is a solid cancer of primary, metastatic or peritoneal seeding type. The pharmaceutical composition according to

[15] , wherein the cancer is selected from esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, and prostate cancer. The pharmaceutical composition according to any one of [1] to

[16] , wherein the PD-1 signal inhibitor is any one of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof. The pharmaceutical composition according to

[17] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody. The pharmaceutical composition according to

[18] , wherein the anti-PD-1 antibody is any one of nivolumab, pembrolizumab, pidilizumab, sparituzumab, or semiprimab. The pharmaceutical composition according to

[17] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody. An anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating cancer in a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: (a) A Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) An anti-CD3scFv region containing the heavy chain variable region and the light chain variable region of the anti-CD3 antibody, and (c) An Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3scFv region of (b). A bispecific antibody comprising the above.

[22] The heavy-chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and the light-chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, the bispecific antibody according to

[21] .

[23] The heavy-chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light-chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, the bispecific antibody according to any one of

[21] or

[22] .

[24] The Fab region of the anti-TSPAN8 antibody consists of a heavy-chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6, the bispecific antibody according to any one of

[21] to

[23] .

[25] The heavy-chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light-chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, the bispecific antibody according to any one of

[21] to

[24] .

[26] The heavy-chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light-chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, the bispecific antibody according to any one of

[21] to

[25] .

[27] The bispecific antibody according to any one of

[21] to

[26] , wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO: 8.

[28] The anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain fragment of an anti-TSPAN8 antibody in which a heavy chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4 is linked to a first Fc polypeptide, a light chain of an anti-TSPAN8 antibody containing a light chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, and an anti-CD3 scFv region containing a heavy chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, and a polypeptide in which a second Fc polypeptide is linked thereto, and is the bispecific antibody according to any one of

[21] to

[27] .

[29] The bispecific antibody of any one of

[21] to

[28] comprising a polypeptide in which an anti-TSPAN8-anti-CD3 bispecific antibody is linked to a second Fc polypeptide with an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence of SEQ ID NO: 8 from amino acid number 1 to 125 and a light chain variable region of an anti-CD3 antibody consisting of the amino acid sequence of SEQ ID NO: 8 from amino acid number 146 to 254, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 6 from amino acid number 1 to 107, and a heavy chain of an anti-TSPAN8 antibody in which a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid number 1 to 121 and a first Fc polypeptide are linked.

[30] The bispecific antibody of any one of

[21] to

[29] comprising an Fc region comprising one or more mutations selected from the group consisting of LALA mutation, N297G mutation, or knobs-into-holes mutation.

[31] An anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating cancer in a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody consists of a polypeptide in which a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an anti-CD3scFv region consisting of the amino acid sequence of SEQ ID NO: 8 are linked to a second Fc polypeptide.

[32] The bispecific antibody of any one of

[21] to

[31] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody is post-translationally modified.

[33] The bispecific antibody of

[21] to

[32] , which is administered simultaneously, continuously, or sequentially with a PD-1 signal inhibitor.

[34] The bispecific antibody of

[21] to

[32] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) contained in separate pharmaceutical compositions and administered simultaneously, continuously, or sequentially. The bispecific antibody according to any one of

[21] to

[34] , wherein the cancer is a solid cancer that is primary, metastatic, or peritoneal disseminated. The bispecific antibody according to

[35] , wherein the cancer is selected from esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, and prostate cancer. The bispecific antibody according to any one of

[21] to

[36] , wherein the PD-1 signal inhibitor is any one of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof. The bispecific antibody according to

[37] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody. The bispecific antibody according to

[38] , wherein the anti-PD-1 antibody is any one of nivolumab, pembrolizumab, pidilizumab, spartalizumab, or semiprimab. The bispecific antibody according to

[37] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody. A method for treating cancer, comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor to a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody is: (a) a Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3scFv region containing the heavy chain variable region and the light chain variable region of the anti-CD3 antibody, and (c) an Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3scFv region of (b), and the method comprises the above.

[42] The method according to

[41] , wherein the heavy chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6.

[43] The method according to

[41] or

[42] , wherein the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6.

[44] The method according to any one of

[41] to

[43] , wherein the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6.

[45] The method according to any one of

[41] to

[44] , wherein the heavy chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8.

[46] The method according to any one of

[41] to

[45] , wherein the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.

[47] The method according to any one of

[41] to

[46] , wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO: 8.

[48] The anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain variable region of an anti-TSPAN8 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, a heavy chain of the anti-TSPAN8 antibody in which the heavy chain fragment of the anti-TSPAN8 antibody is linked to the first Fc polypeptide, a light chain of the anti-TSPAN8 antibody containing a light chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, and an anti-CD3 scFv region containing a heavy chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, and a polypeptide in which the second Fc polypeptide is linked thereto, the method according to any one of

[41] to

[47] .

[49] The method according to any one of

[41] to

[48] , comprising a polypeptide in which an anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, a heavy chain of the anti-TSPAN8 antibody linked to a first Fc polypeptide, a light chain of the anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, and an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8 and a light chain variable region of the anti-CD3 antibody consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, and a second Fc polypeptide is linked thereto.

[50] The method according to any one of

[41] to

[49] , comprising an Fc region comprising one or more mutations selected from the group consisting of LALA mutation, N297G mutation, or knobs-into-holes mutation.

[51] A method for treating cancer, comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor to a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of the anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and a polypeptide in which an anti-CD3scFv region consisting of the amino acid sequence of SEQ ID NO: 8 and a second Fc polypeptide are linked.

[52] The method according to any one of

[41] to

[51] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody is post-translationally modified.

[53] The treatment method according to

[41] to

[52] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are administered simultaneously, continuously, or sequentially.

[54] The treatment method according to

[41] to

[52] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) contained in separate pharmaceutical compositions and administered simultaneously, continuously, or sequentially. The method according to any one of

[41] to

[54] , wherein the cancer is a solid cancer that is primary, metastatic, or peritoneal seeding cancer. The method according to

[55] , wherein the cancer is selected from esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, and prostate cancer. The method according to any one of

[41] to

[56] , wherein the PD-1 signal inhibitor is any one of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof. The method according to

[57] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody. The method according to

[58] , wherein the anti-PD-1 antibody is any one of nivolumab, pembrolizumab, pidilizumab, sparituzumab, or semiprimumab. The method according to

[58] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody. Use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signal inhibitor to treat cancer in a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: (a) a Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment comprising the heavy chain variable region of the anti-TSPAN8 antibody and a light chain comprising the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3scFv region comprising the heavy chain variable region of the anti-CD3 antibody and the light chain variable region of the anti-CD3 antibody, and (c) an Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3scFv region of (b), comprising the use.

[62] The use according to

[61] , wherein the heavy-chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and the light-chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6.

[63] The use according to either

[61] or

[62] , wherein the heavy-chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light-chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6.

[64] The use according to any one of

[61] to

[63] , wherein the Fab region of the anti-TSPAN8 antibody consists of a heavy-chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6.

[65] The use according to any one of

[61] to

[64] , wherein the heavy-chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light-chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8.

[66] The use according to any one of

[61] to

[65] , wherein the heavy-chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light-chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.

[67] Use according to any one of

[61] to

[66] , wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO: 8.

[68] Use according to any one of

[61] to

[67] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain fragment of an anti-TSPAN8 antibody in which a heavy chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4 is linked to a first Fc polypeptide, a light chain of an anti-TSPAN8 antibody containing a light chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, and an anti-CD3 scFv region containing a heavy chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, and a polypeptide in which the second Fc polypeptide is linked.

[69] Use according to any one of

[61] to

[68] , comprising a polypeptide in which an anti-TSPAN8-anti-CD3 bispecific antibody is linked to a heavy chain of an anti-TSPAN8 antibody in which a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4 is linked to a first Fc polypeptide, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, and an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8 and a light chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, and a second Fc polypeptide is linked thereto.

[70] Use according to any one of

[61] to

[69] , comprising an Fc region comprising one or more mutations selected from the group consisting of LALA mutation, N297G mutation, or knobs-into-holes mutation.

[71] Use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signaling inhibitor to treat cancer in a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody consists of a polypeptide in which a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an anti-CD3scFv region consisting of the amino acid sequence of SEQ ID NO: 8 are linked to a second Fc polypeptide.

[72] Use according to any one of

[61] to

[71] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody is post-translationally modified.

[73] Use of the anti-TSPAN8-anti-CD3 bispecific antibody according to

[61] to

[72] , wherein the pharmaceutical composition is administered simultaneously, continuously, or sequentially with a PD-1 signaling inhibitor.

[74] Use of the anti-TSPAN8-anti-CD3 bispecific antibody according to

[61] to

[72] , wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) contained in separate pharmaceutical compositions and administered simultaneously, continuously, or sequentially.

[75] Use according to any one of

[61] to

[74] , wherein the cancer is a solid cancer that is primary, metastatic, or peritoneal seeding cancer.

[76] Use according to

[75] , wherein the cancer is selected from esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, and prostate cancer.

[77] Use according to any one of

[61] to

[76] , wherein the PD-1 signal inhibitor is any one of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof.

[78] Use according to

[77] , wherein the PD-1 signal inhibitor is an anti-PD-1 antibody.

[79] Use according to

[78] , wherein the anti-PD-1 antibody is any one of nivolumab, pembrolizumab, pidilizumab, sparituzumab, or semiprimumab.

[80] Use according to

[77] , wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody. [Effect of the Invention]

[0013] The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention binds to both TSPAN8, which is a cancer antigen, and CD3, which is a T cell surface molecule, and enhances the cancer cell killing effect by T cells by bringing the physical distance between cancer cells and T cells closer. The combination of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signal inhibitor brings about a significant antitumor effect as compared with the single administration of the PD-1 signal inhibitor. Therefore, the present invention provides the use of an anti-TSPAN8-anti-CD3 bispecific antibody in combination with a PD-1 signal inhibitor in cancer treatment. [Brief Description of the Drawings]

[0014]

Figure 1

Mode for Carrying Out the Invention

[0015] The present invention will be described in detail below.

[0016] The terms in this specification are used in the meanings generally used by those skilled in the art in the relevant technical field, unless otherwise specifically defined below.

[0017] An antibody (or immunoglobulin) is a glycoprotein with a basic structure of a four-chain structure having a bilaterally symmetric Y-shaped structure consisting of two heavy chains having a single sequence and two light chains having a single sequence. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. The basic structure of the antibody molecule is common to each class, and two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000 are bound by disulfide bonds and non-covalent bonds to form an antibody molecule with a Y-shaped four-chain structure with a molecular weight of 150,000 to 190,000. The heavy chain consists of a polypeptide chain usually containing about 440 amino acids, has a characteristic structure for each class, and is called Igγ, Igμ, Igα, Igδ, Igε corresponding to IgG, IgM, IgA, IgD, IgE, respectively. Furthermore, IgG has subclasses of IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Igγ1, Igγ2, Igγ3, and Igγ4, respectively. The light chain consists of a polypeptide chain usually containing about 220 amino acids, and two types, λ type and κ type, are known, and are called Igλ and Igκ, respectively. The two types of light chains can pair with any type of heavy chain.

[0018] There are four disulfide bonds in the heavy chain (five in Igμ and Igε) and two in the light chain in the intra-chain disulfide bonds of the antibody molecule, forming one loop every 100 - 110 amino acid residues. Their three-dimensional structures are similar between each loop and are called structural units or domains. The domain located at the N-terminus of both the heavy chain and the light chain is called the variable region, and even antibodies produced from the same class (or subclass) of the same animal have diverse amino acid sequences and are known to be involved in the specific binding of the antibody to the antigen. The amino acid sequence of the domain on the C-terminal side downstream from the variable region is almost constant for each class or subclass and is called the constant region. The heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH) from the N-terminus to the C-terminus. CH is further divided into three domains: the CH1 domain, the CH2 domain, and the CH3 domain from the N-terminal side. The light chain has a light chain variable region (VL) and a light chain constant region (CL) from the N-terminus to the C-terminus.

[0019] The amino acid sequences of the three complementarity-determining regions (CDRs) present in VH and VL vary greatly and contribute to the variability of the variable region. The CDRs are regions consisting of approximately 5 to 10 amino acid residues that are present in the order of CDR1, CDR2, and CDR3 at the N-terminals of the heavy and light chains, respectively, and form the antigen-binding site. On the other hand, the portions other than the CDRs in the variable region are called framework regions (FRs), which consist of FR1 to 4 and have relatively few amino acid sequence changes.

[0020] When an antibody is treated with the proteolytic enzyme papain, three antibody fragments are obtained. The two N-terminal fragments are called Fab (Fragment, antigen binding) regions. In this specification, the "Fab region" refers to the region consisting of the VH and CH1 domains of the heavy chain and the light chain (VL and CL), and binds to an antigen at the antigen-binding site at the tip portion constituted by the Fab region. In this specification, the "heavy chain fragment" refers to the fragment consisting of the VH and CH1 domains of the heavy chain that constitutes the Fab region. Also, the C-terminal fragment is called the Fc (Fragment, crystallizable) region. In this specification, the "Fc polypeptide" refers to the polypeptide consisting of the CH2 and CH3 domains of the heavy chain, and the "Fc region" refers to the complex consisting of two Fc polypeptides. The heavy chain fragment and the Fc polypeptide are connected at a portion called the hinge region. Also, the two heavy chains of the antibody are disulfide-bonded at the hinge region.

[0021] In this specification, the term "antigen" is used in the generally used meaning, and in particular, is used as a term representing a molecule or a part of a molecule to which an antigen-binding protein such as an antibody or an antigen-binding fragment can specifically bind. An antigen can be a molecule such as a protein or a nucleic acid. One antigen may have one or more epitopes that can interact with different antibodies and the like.

[0022] As used herein, an "antigen-binding fragment" is a molecule comprising at least one polypeptide chain having antigen-binding activity derived from an antibody. Representative antigen-binding fragments include single-chain variable region fragments (scFv), Fab fragments, Fab' fragments, and F(ab')[ 2 [ fragments. An scFv is a monovalent antigen-binding fragment composed of VH and VL linked by a linker. A Fab fragment is a monovalent antigen-binding fragment composed of a light chain and a fragment containing the VH and CH1 domains of the heavy chain. A Fab' fragment is a monovalent antigen-binding fragment composed of a light chain and a fragment containing the VH, CH1 domain, and a part of the hinge region of the heavy chain, and the part of the hinge region contains cysteine residues that formed the inter-heavy chain disulfide bond. F(ab')[ 2 [ fragment is a divalent molecule in which Fab' fragments are linked by disulfide bonds. Monovalent means containing one antigen-binding site, and divalent means containing two antigen-binding sites. [

[0023] [ [ As used herein, an "scFv region" refers to a region containing a monovalent antigen-binding fragment comprising VH and VL linked by a linker. [

[0024] [ [ As used herein, a "bispecific antibody" refers to an antibody that can specifically bind to two different antigens. An "anti-TSPAN8-anti-CD3 bispecific antibody" means a bispecific antibody having binding activity against TSPAN8 and binding activity against CD3. [

[0025] [ [ As used herein, the term "antibody" is used as a term encompassing full-length antibodies, antigen-binding fragments, and bispecific antibodies of any structure, unless otherwise specifically limited in context. [

[0026] [ [As used herein, "human antibody" refers to an antibody having a human immunoglobulin amino acid sequence. As used herein, "humanized antibody" refers to an antibody in which some, most, or all of the amino acid residues other than the CDRs are replaced with amino acid residues derived from a human immunoglobulin molecule. The method of humanization is not particularly limited, and for example, a humanized antibody can be prepared with reference to US Patent No. 5,225,539, US Patent No. 6,180,370, etc.

[0027] The amino acid residue numbers of antibodies used in this specification can be defined according to their numbering systems by designating Kabat numbering or EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, 1991, NIH Publication No. 91-3242).

[0028] As used herein, "first" or "second" is used for convenience of distinction when there are two or more types of each part. The use of such terms is not intended to confer a specific order or meaning unless clearly stated.

[0029] As used herein, "linked" or "linked together" means that a plurality of components (for example, Fab region and Fc polypeptide) are bound directly or via one or more mediators (for example, peptide linker). As used herein, "peptide linker" means one or more arbitrary amino acid residues that can be introduced by genetic engineering for linking between variable regions. The length of the peptide linker used in the present invention is not particularly limited, and those skilled in the art can appropriately select it according to the purpose.

[0030] As used herein, "identity" means the value of Identity obtained by the parameters prepared by default using EMBOSS Needle (Nucleic Acids Res., 2015; Vol. 43: pW580-W584). The above parameters are as follows. Gap Open Penalty = 10 Gap Extend Penalty = 0.5 Matrix = EBLOSUM62 End Gap Penalty = false

[0031] As used herein, "subject" means a human or other animal in need of prevention or treatment of a disease. In one embodiment, the subject is a human in need of prevention or treatment of a disease. In one embodiment, the subject is a human having cancer.

[0032] As used herein, "treatment" means any therapeutic intervention, treatment, or administration of an active ingredient to a subject for the purpose of restoring, alleviating, improving, suppressing, or delaying the symptoms, conditions, progression, onset, exacerbation, or recurrence of a disease, biochemical signs associated with the disease.

[0033] As used herein, "active ingredient" means a substance contained in a pharmaceutical composition, drug, etc. used for the prevention or treatment of a disease that exhibits some physiological activity. In one embodiment, the active ingredient is an antibody, small molecule compound, nucleic acid, fusion protein, peptide. In one embodiment, the active ingredient is an antibody. In one embodiment, the active ingredient is a bispecific antibody.

[0034] As used herein, "pharmaceutical composition" means a drug formulated for the treatment of a subject, containing an active ingredient and a pharmaceutically acceptable excipient (including, but not limited to, excipients for drugs and carriers for drugs, etc.).

[0035] As used herein, "combined use", "combination", or "use in combination" means administering a plurality of active ingredients simultaneously, continuously, or sequentially to the same subject for the prevention or treatment of a disease. The plurality of active ingredients may be contained in the same pharmaceutical composition or separately in different pharmaceutical compositions. As used herein, "simultaneously" means administering a plurality of active ingredients in parallel within one administration period, "continuously" means administering another active ingredient without an intervening period after the administration of one active ingredient has ended, and "sequentially" means administering a plurality of active ingredients in order according to an administration schedule.

[0036] As used herein, the "effective amount" of a drug refers to the amount of the drug necessary to bring about a physiological change in the cells or tissues to which it is administered.

[0037] As used herein, the "PD-1 signaling inhibitor" means a drug that releases the suppression of immune cell activation by PD-1. The PD-1 signaling inhibitor can inhibit the function of the PD-1 immune checkpoint by binding to PD-1 or its ligands PD-L1 or PD-L2 and inhibiting the immunosuppressive signal. Any substance may be used as the PD-1 signaling inhibitor as long as it has the effect of blocking the PD-1 signal, and examples thereof include antibodies, low molecular weight compounds, nucleic acids (DNA or RNA, or may contain natural or artificial nucleic acids), fusion proteins, peptides, and the like. For example, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies can inhibit the PD-1 signal by inhibiting the binding of PD-1 to PD-L1 or PD-L2 (Expert Opinion on Therapeutic Patents, 2016; Vol. 26: p. 555-564).

[0038] The present invention relates to the following (1) to (4): (1) A pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signaling inhibitor for treating a target cancer (also referred to herein as "the pharmaceutical composition of the present invention"); (2) An anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signaling inhibitor for treating a target cancer; (3) A method for treating cancer comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject (also referred to herein as "the treatment method of the present invention"); or (4) Use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition used in combination with a PD-1 signaling inhibitor for treating a target cancer.

[0039] <The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention> The anti-TSPAN8-anti-CD3 bispecific antibody used in the present invention (also referred to herein as "the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention") has a structure consisting of the following (a) to (c): (a) A Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) An anti-CD3scFv region containing the heavy chain variable region and the light chain variable region of the anti-CD3 antibody, and (c) An Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3scFv region of (b), comprising a bispecific antibody.

[0040] The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention has a structure comprising one Fab region of a first antibody, an scFv region of a second antibody, and one Fc region. Antibodies with such a structure are also referred to as "bottle-opener type antibodies" (International Publication No. WO 2014 / 110601). In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is a human antibody or a humanized antibody.

[0041] The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, and includes the Fab region of the anti-TSPAN8 antibody.

[0042] In one embodiment, the heavy chain variable region of the anti-TSPAN8 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4. The light chain variable region of the anti-TSPAN8 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6.

[0043] In one embodiment, the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6.

[0044] As the heavy chain constant region from which the CH1 domain of the heavy chain fragment of the anti-TSPAN8 antibody is derived, any constant region of Igγ, Igμ, Igα, Igδ or Igε can be selected. As Igγ, for example, it is possible to select from Igγ1, Igγ2, Igγ3 or Igγ4. In one embodiment, the heavy chain fragment of the anti-TSPAN8 antibody comprises a CH1 domain derived from the human Igγ1 constant region.

[0045] As the CL of the light chain of the anti-TSPAN8 antibody, any constant region of Igλ or Igκ can be selected. In one embodiment, the light chain of the anti-TSPAN8 antibody comprises a CL that is the Igκ constant region. In one embodiment, the light chain of the anti-TSPAN8 antibody comprises a CL that is the human Igκ constant region.

[0046] In one embodiment, the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6.

[0047] The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an anti-CD3 scFv region containing the heavy chain variable region and the light chain variable region of the anti-CD3 antibody as the scFv region. In the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, an anti-CD3 scFv region known in the art or an anti-CD3 scFv region prepared based on the sequence information of the heavy chain variable region and the light chain variable region of an anti-CD3 antibody known in the art may be used. Known anti-CD3 antibodies include clones such as OKT3, UTCH1, L2K, and TR66, and their sequences have been used as bispecific antibodies (Pharmacol. Ther., 2018; Vol. 182: p. 161-175).

[0048] In one embodiment, the heavy chain variable region of the anti-CD3 antibody includes CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody includes CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8.

[0049] In one embodiment, the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.

[0050] In the anti-CD3 scFv region, the type and length of the peptide linker that connects the heavy-chain variable region and the light-chain variable region of the anti-CD3 antibody are not particularly limited and can be appropriately selected by those skilled in the art. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. As the peptide linker, for example, a glycine-serine linker (GS linker) or a glycine-lysine-proline-glycine-serine linker (GKPGS linker) can be used. Examples of such linkers include the following. Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser (SEQ ID NO: 9) Ser-Gly-Gly-Gly (SEQ ID NO: 10) Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 11) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 12) Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 13) Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 14) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 15) Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 16) (Gly-Gly-Gly-Gly-Ser) n (Ser-Gly-Gly-Gly-Gly) n Gly-Lys-Pro-Gly-Ser (SEQ ID NO: 17) (Gly-Lys-Pro-Gly-Ser) n The above n represents an integer of 1 or more. Those skilled in the art can appropriately select the length and sequence of the peptide linker according to the purpose.

[0051] In one embodiment, in the anti-CD3 scFv region, the peptide linker that connects the heavy chain variable region and the light chain variable region of the anti-CD3 antibody has the amino acid sequence of Gly-Lys-Pro-Gly-Ser (GKPGS; SEQ ID NO: 17). In one embodiment, the peptide linker that connects the heavy chain variable region and the light chain variable region of the anti-CD3 antibody has the amino acid sequence of (Gly-Lys-Pro-Gly-Ser)n. In one embodiment, the peptide linker that connects the heavy chain variable region and the light chain variable region of the anti-CD3 antibody has the amino acid sequence of (Gly-Lys-Pro-Gly-Ser) 4 and has the amino acid sequence of

[0052] In one embodiment, the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO: 8.

[0053] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain of an anti-TSPAN8 antibody in which a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4 is linked to a first Fc polypeptide, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region comprising a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, and a polypeptide comprising an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody comprising a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody comprising a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, which is linked to a second Fc polypeptide.

[0054] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain of an anti-TSPAN8 antibody in which a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4 is linked to a first Fc polypeptide, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 8, and a polypeptide comprising an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, which is linked to a second Fc polypeptide.

[0055] In the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, as the heavy chain constant region from which the first Fc polypeptide and the second Fc polypeptide constituting the Fc region are derived, any constant region of Igγ, Igμ, Igα, Igδ or Igε can be selected. As Igγ, for example, it can be selected from Igγ1, Igγ2, Igγ3 or Igγ4. In one embodiment, the first Fc polypeptide and the second Fc polypeptide are Fc polypeptides derived from the human Igγ1 constant region.

[0056] The Fc region in the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may contain mutations that reduce antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). L234A is a substitution of leucine at amino acid position 234 with alanine according to the EU index in the human Igγ1 constant region. L235A is a substitution of leucine at amino acid position 235 with alanine according to the EU index in the human Igγ1 constant region. The amino acid mutations of human Igγ1 constant region L234A and L235A are referred to as "LALA mutations". It is known that the mutations reduce the antibody-dependent cell cytotoxicity and complement-dependent cytotoxicity of the antibody (Mol. Immunol., 1992; Vol. 29: p. 633-639, J. Immunol., 2000; Vol. 164: p. 4178-4184).

[0057] The Fc region in the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may further contain mutations based on other known techniques. For example, the Fc region may contain an N297G mutation (Protein cell, 2018; Vol. 9: p. 63-73) or a mutation based on the Knobs into holes technology (hereinafter also referred to as the "Knobs into holes mutation"). The Knobs into holes technology promotes the heterodimerization of heavy chains by substituting the amino acid side chain present in the CH3 region of one heavy chain with a larger side chain (knob; protrusion) and substituting the amino acid side chain present in the CH3 region of the other heavy chain with a smaller side chain (hole; void), so that the protrusion is arranged in the void, and the desired heterodimeric antibody molecule can be efficiently obtained (Nature, 1994; Vol. 372: p. 379-383, Nature Biotech., 1998; Vol. 16: p. 677-681, J. Mol. Biol., 1997; Vol. 270: p. 26-35, Proc. Natl. Acad. Sci. USA, 2013; Vol. 110: p. E2987-E2996).

[0058] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc region containing amino acid mutations L234A and L235A (LALA mutation). In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc region containing the N297G mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc region containing the knobs-into-holes mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc region containing one or more mutations selected from the group consisting of the amino acid mutations L234A and L235A (LALA mutation), the N297G mutation, and the knobs-into-holes mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc region containing the amino acid mutations L234A and L235A (LALA mutation), the N297G mutation, and the knobs-into-holes mutation. In one embodiment, the knobs-into-holes mutation contained in the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is the T366W mutation in one Fc polypeptide forming the Fc region and the T366S, L368A, and Y407V mutations in another Fc polypeptide forming the Fc region (see International Publication No. WO 1998 / 050431).

[0059] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc region consisting of a first Fc polypeptide having an amino acid sequence from amino acid number 235 to 451 of SEQ ID NO: 4 and a second Fc polypeptide having an amino acid sequence from amino acid number 270 to 486 of SEQ ID NO: 8.

[0060] In this specification, the description of amino acid mutations such as the LALA mutation, the N297G mutation, and the knobs-into-holes mutation is based on the amino acid positions according to the EU index in the human Igγ1 constant region. For example, as described above, L234A is a substitution of leucine at amino acid position 234 with alanine according to the EU index in the human Igγ1 constant region.

[0061] In the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, the heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and the Fc polypeptide (first Fc polypeptide) are linked via a hinge region to constitute the heavy chain of the anti-TSPAN8 antibody. Also, in the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, the anti-CD3scFv region and the Fc polypeptide (second Fc polypeptide) may be linked via a hinge region.

[0062] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises the heavy chain of the anti-TSPAN8 antibody. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a polypeptide in which the anti-CD3scFv region and the second Fc polypeptide are linked via a hinge region. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises the heavy chain of the anti-TSPAN8 antibody and a polypeptide in which the anti-CD3scFv region and the second Fc polypeptide are linked via a hinge region.

[0063] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and a heavy chain of an anti-TSPAN8 antibody in which the first Fc polypeptide is linked via a hinge region, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region containing a CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, and an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody containing a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, and a polypeptide in which the second Fc polypeptide is linked via a hinge region.

[0064] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, a heavy chain of the anti-TSPAN8 antibody in which the first Fc polypeptide is linked via a hinge region, a light chain of the anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, and an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8 and a light chain variable region of the anti-CD3 antibody consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, and a polypeptide in which the second Fc polypeptide is linked via a hinge region.

[0065] In one embodiment, the heavy chain of the anti-TSPAN8 antibody consists of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the light chain of the anti-TSPAN8 antibody consists of the amino acid sequence of SEQ ID NO: 6. In one embodiment, the polypeptide in which the anti-CD3scFv region and the second Fc polypeptide are linked via a hinge region consists of the amino acid sequence of SEQ ID NO: 8.

[0066] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is a bispecific antibody consisting of a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of the anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and a polypeptide in which an anti-CD3scFv region consisting of the amino acid sequence of SEQ ID NO: 8 and a second Fc polypeptide are linked.

[0067] As used herein, "post-translational modification" refers to the modification of an antibody after translation when the antibody is expressed intracellularly. Examples of post-translational modifications include modifications such as pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy chain, glycosylation, oxidation, deamidation, glycation, etc., and lysine deletion by cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase. It is known that such post-translational modifications occur in various antibodies (J. Pharm. Sci., 2008; Vol. 97: p. 2426-2447).

[0068] In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamylation at the N-terminus of the heavy chain variable region and / or lysine deletion at the C-terminus of the heavy chain. It is known in the art that post-translational modification by N-terminal pyroglutamylation or C-terminal lysine deletion does not affect the activity of the antibody (Analytical Biochemistry, 2006; Vol. 348: p. 24-39).

[0069] The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention binds to human TSPAN8 (Gene ID: NM_004616.2) and human CD3εδ complex protein (CD3ε Gene ID: NM_000733.3, CD3δ Gene ID: NM_000732.4 or NM_001040651.1). Whether it binds to human TSPAN8 and human CD3εδ complex protein can be confirmed using known methods for measuring binding activity. Examples of methods for measuring binding activity include, for example, methods such as the Enzyme-Linked ImmunoSorbent Assay (ELISA) method and flow cytometry method.

[0070] The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention can be prepared by a method known in the art by those skilled in the art based on the sequence information of the heavy chain variable region and the light chain variable region of the anti-TSPAN8 antibody and the anti-CD3scFv region disclosed in this specification. Further, the anti-CD3scFv region of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention can be prepared by a method known in the art by those skilled in the art based on the sequence information of the heavy chain variable region and the light chain variable region of a known anti-CD3 antibody. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is a humanized antibody or a human antibody. In preparing a humanized antibody, appropriate back-mutations may be introduced using methods well known to those skilled in the art (Bioinformatics, 2015; Vol. 31: p. 434-435). The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is not particularly limited, but can be produced, for example, according to the method described in PCT / JP2021 / 41839. The method for preparing the anti-TSPAN8-anti-CD3 bispecific antibody described in PCT / JP2021 / 41839 (<Polynucleotide of the bispecific antibody of the present invention>, <Expression vector of the bispecific antibody of the present invention>, <Transformed host cell of the present invention>, <Transformed host cell of the present invention>, <Method for producing the bispecific antibody of the present invention> and examples, including but not limited to these) is incorporated herein by reference (Incorporation by Reference).

[0071] <Pharmaceutical composition of the present invention> The pharmaceutical composition of the present invention is manufactured using the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and comprises the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by a commonly used method using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. Examples of dosage forms of these pharmaceutical compositions include, for example, parenteral preparations such as injections and infusion preparations, and can be administered by intravenous administration, subcutaneous administration, intraperitoneal administration, etc. In formulating, within the pharmaceutically acceptable range, excipients, carriers, additives, etc. corresponding to these dosage forms can be used.

[0072] The pharmaceutical composition of the present invention may contain a post-translational modification product of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention. For example, pharmaceutical compositions containing antibodies that have undergone deletion of the C-terminal lysine and / or N-terminal pyroglutamylation are also included in the present invention.

[0073] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention described below and / or a post-translational modification product of the antibody: A heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region containing CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and a heavy chain of an anti-TSPAN8 antibody in which the first Fc polypeptide is linked thereto, CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6, a light chain of an anti-TSPAN8 antibody comprising a light chain variable region, and an anti-CD3scFv region comprising a heavy chain variable region of an anti-CD3 antibody containing CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody containing CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO: 8, and a polypeptide in which the second Fc polypeptide is linked thereto, a bispecific antibody.

[0074] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention described below and / or a post-translational modification product of the antibody: A bispecific antibody comprising a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, with a first Fc polypeptide linked thereto; a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6; and a polypeptide comprising an anti-CD3 scFv region comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8, with a second Fc polypeptide linked thereto.

[0075] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody comprising a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and a polypeptide comprising an anti-CD3 scFv region consisting of the amino acid sequence of SEQ ID NO: 8 with a second Fc polypeptide linked thereto, and / or a post-translational modification product of the antibody.

[0076] The dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention in formulation varies depending on the degree and age of the patient's symptoms, the dosage form of the formulation to be used, or the binding titer of the antibody, etc. For example, an anti-TSPAN8-anti-CD3 bispecific antibody in an amount of about 0.0001 mg / kg to 1000 mg / kg in terms of the dosage for human administration can be used in the formulation. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention in formulation is in the range of 0.0001 mg / kg to 1000 mg / kg in terms of the dosage for human administration. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention in formulation is in the range of 0.001 mg / kg to 100 mg / kg in terms of the dosage for human administration. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention in formulation is in the range of 0.01 mg / kg to 10 mg / kg in terms of the dosage for human administration. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention in formulation is preferably in the range of 0.01 mg / kg to 10 mg / kg in terms of the dosage for human administration.

[0077] <PD-1 signal inhibitor> In the present invention, the PD-1 signal inhibitor is used in combination with the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention or the pharmaceutical composition of the present invention for the treatment of cancer in a subject. In one embodiment, the pharmaceutical composition of the present invention contains the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signal inhibitor. In one embodiment, the pharmaceutical composition of the present invention is used in combination with another pharmaceutical composition containing a PD-1 signal inhibitor. The mechanism of action and treatment modality of the PD-1 signal inhibitor are not particularly limited as long as they block the PD-1 signal. Examples of the mechanism of action may include inhibition of the binding between molecules involved in the PD-1 signal, reduction in the expression level of PD-1 signal molecules (e.g., inhibition of protein production or induction of degradation, etc.). Examples of the treatment modality may include antibodies, small molecule compounds, nucleic acids (including DNA or RNA, natural or artificial nucleic acids), fusion proteins, peptides, and other treatment modalities.

[0078] A PD-1 signal inhibitor can be obtained by a method for measuring an inhibitory action on the binding of two proteins, PD-1 and PD-L1 or PD-L2, and an action of decreasing expression using, as an index, the expression level of PD-1 signal molecules and the like. For example, an inhibitor of the binding between PD-1 and PD-L1 or PD-L2 can be selected by the ability of the obtained inhibitor to inhibit the binding between PD-1 and PD-L1 or PD-L2 after obtaining an inhibitor that binds to any one of PD-1 and PD-L1 or PD-L2. The binding of the inhibitor to the protein can be evaluated using methods well known to those skilled in the art, such as flow cytometry (FCM), ELISA, surface plasmon resonance (SPR), thermal shift assay (TSA), isothermal titration calorimetry (ITC), and the like. Further, for example, an inhibitor that decreases the expression level of PD-1 signal molecules such as PD-1, PD-L1, or PD-L2 can be obtained using, as an index, the amount of proteins such as PD-1, PD-L1, or PD-L2 in cells. The inhibitory action of the PD-1 signal can be confirmed by actions such as T cell proliferation, IFN-γ release, and reporter assay. The action of an inhibitor that decreases the expression level of a certain protein can be confirmed using methods well known to those skilled in the art, such as ELISA, quantitative PCR, in situ hybridization, and live cell imaging.

[0079] Examples of PD-1 signal inhibitors include antibodies that inhibit PD-1 signals, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. Such antibodies may be humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and their antigen-binding fragments. Known anti-PD-1 antibodies include, but are not limited to, for example, those described in U.S. Patent No. 8,008,449, U.S. Patent No. 6,808,710, U.S. Patent No. 7,488,802, U.S. Patent No. 8,168,757, and U.S. Patent No. 8,354,509, as well as those described in International Publication No. 2006 / 121168 and International Publication No. 2012 / 145493. Known anti-PD-L1 antibodies include, but are not limited to, for example, those described in International Publication No. 2007 / 005874, International Publication No. 2010 / 077634, International Publication No. 2011 / 066389, International Publication No. 2013 / 079174, and U.S. Patent No. 8,217,149. In one embodiment, the PD-1 signal inhibitor used in the present invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof. In one embodiment, the PD-1 signal inhibitor used in the present invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. In one embodiment, the anti-PD-1 antibody may be an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, spartalizumab, or semiprimumab. In one embodiment, the anti-PD-L1 antibody may be an anti-PD-L1 antibody such as atezolizumab, durvalumab, or avelumab.

[0080] Examples of PD-1 signal inhibitors also include fusion proteins and small molecule compounds that inhibit the binding of PD-1 to PD-L1, such as AMP-224 (International Publication Nos. 2010 / 027827 and 2011 / 066342), BMS-1166 (Oncotarget, 2017; Vol. 8: p. 72167-72181), etc. Various PD-1 signal inhibitors are known in the art (Non-Patent Document 8).

[0081] <The treatment method of the present invention> The treatment method of the present invention involves administering the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention or the pharmaceutical composition of the present invention simultaneously, continuously, or sequentially with a PD-1 signaling inhibitor for cancer treatment in a subject, which is a method for treating cancer.

[0082] In one embodiment, the treatment method of the present invention is characterized in that the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and the PD-1 signaling inhibitor are (i) contained in the same pharmaceutical composition and administered to the subject simultaneously, or (ii) contained in separate pharmaceutical compositions and administered to the subject simultaneously, continuously, or sequentially.

[0083] In one embodiment, the treatment method of the present invention is characterized in that the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and the PD-1 signaling inhibitor are (i) contained in the same pharmaceutical composition and administered to the subject simultaneously, or (ii) contained in separate pharmaceutical compositions and administered to the subject on the same day.

[0084] In one embodiment, the treatment method of the present invention is a continuous use characterized by (a) starting the administration of the PD-1 signaling inhibitor after the completion of the administration of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention to the subject, or (b) starting the administration of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention after the completion of the administration of the PD-1 signaling inhibitor to the subject.

[0085] In one embodiment, the treatment method of the present invention is a sequential use in which the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signal inhibitor are administered to a subject according to an administration plan including an administration cycle. In one embodiment, the treatment method of the present invention is characterized in that, in at least one administration cycle or all administration cycles, the administration of the anti-TSPAN8-anti-CD3 bispecific antibody to the subject is started, and then the administration of the PD-1 signal inhibitor is started. In one embodiment, the treatment method of the present invention is characterized in that, in at least one administration cycle or all administration cycles, the administration of the PD-1 signal inhibitor to the subject is started, and then the administration of the anti-TSPAN8-anti-CD3 bispecific antibody or a pharmaceutical composition is started.

[0086] <Therapeutic use> The cancer treated by the pharmaceutical composition and treatment method of the present invention may be either a solid cancer or a blood cancer. The cancer treated by the present invention may be either primary, metastatic or peritoneal seeding. The cancer treated by the present invention is not particularly limited, and examples include gastric cancer, lung cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, multiple myeloma, T-cell lymphoma and other blood cancers, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, pancreatic cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, gastroesophageal junction cancer, esophageal cancer, testicular cancer, ovarian cancer, brain tumor and other solid cancers, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue, and in addition, sarcomas such as chondrosarcoma, Ewing sarcoma, malignant angioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, and blastomas such as glioblastoma, glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, etc.

[0087] In one embodiment, the cancers to be treated by the present invention are esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, and prostate cancer. In one embodiment, preferably, they are esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, and gastroesophageal junction cancer.

[0088] The dosage of the anti-TSPAN8-anti-CD3 bispecific antibody or PD-1 signal inhibitor of the present invention administered to a subject varies depending on the degree and age of the subject's symptoms, the antibody used, the dosage form of the pharmaceutical composition, inhibitor, etc., or the activity intensity of the active ingredient, etc. For example, about 0.0001 mg / kg to 1000 mg / kg can be used. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention administered to a subject is 0.0001 mg / kg to 1000 mg / kg. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention administered to a subject is 0.001 mg / kg to 100 mg / kg. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention administered to a subject is 0.01 mg / kg to 10 mg / kg.

[0089] Specific examples are provided herein for further understanding of the present invention, but these are for illustrative purposes only and do not limit the present invention.

Examples

[0090] [Example 1: Preparation of anti-TSPAN8-anti-CD3 bispecific antibody] [Example 1-1: Preparation of bispecific antibody vector of anti-TSPAN8 antibody] To be used for the bispecific antibody of the anti-TSPAN8 antibody, mutations were introduced into the heavy chain of 16B11.1 of SEQ ID NO: 2 to prepare 16B11.1_HC_H described in SEQ ID NO: 4. Specifically, three types of mutations were introduced into SEQ ID NO: 2. (1) The LALA mutation (L234A and L235A) in which leucine (L) at amino acid positions 238 and 239 (EU index: 234 and 235) was replaced with alanine (A); (2) The Knobs into holes mutation in which the amino acids at amino acid positions 370, 372, and 411 (EU index: 366, 368, and 407) were replaced with threonine (T) to serine (S), leucine (L) to alanine (A), and tyrosine (Y) to valine (V), respectively; (3) The mutation in which the amino acid at amino acid position 301 (EU index: 297) was replaced with asparagine (N) to glycine (G). The heavy chain amino acid sequence of the designed 16B11.1_HC_H is shown in SEQ ID NO: 4. The polynucleotide encoding 16B11.1_HC_H of SEQ ID NO: 4 was introduced into the pcDNA3.4-TOPO vector (Thermo Fisher scientific). The prepared vector was designated as pcDNA3.4-16B11.1_HC_H. In addition, a polypeptide was designed in which the C-terminal side of the variable region of the light chain of 16B11.1 was linked to the constant region amino acid sequence of the human κ chain (the sequence from amino acid position 108 to 213 of SEQ ID NO: 6) (SEQ ID NO: 6). The polynucleotide encoding the designed polypeptide was introduced into the pcDNA3.4 TOPO vector. The prepared light chain vector was designated as pcDNA3.4-16B11_LC.

[0091] [Example 1-2: Preparation of bispecific antibody vector of anti-human CD3 antibody] The sequence of the humanized anti-CD3 antibody was designed based on the sequences of the heavy-chain variable region and light-chain variable region of the mouse anti-CD3 antibody described in Japanese Patent No. 5686953 according to the method described in the literature (Front Biosci., 2008; Vol. 13: p. 1619-1633). The three-dimensional structure information (PDB Code: 5FCS) was analyzed using MOE, an integrated computational chemistry system provided by MOLSIS Inc., and backmutations were introduced into the framework region. Anti-CD3scFv-Fc was designed to be arranged in the order of the heavy-chain variable region (SEQ ID NO: 8, amino acid numbers 1 to 125), linker (SEQ ID NO: 8, amino acid numbers 126 to 145), light-chain variable region (SEQ ID NO: 8, amino acid numbers 146 to 254), hinge (SEQ ID NO: 8, amino acid numbers 255 to 269), CH2 domain (SEQ ID NO: 8, amino acid numbers 270 to 379), and CH3 domain (SEQ ID NO: 8, amino acid numbers 380 to 486). Furthermore, the following mutations were introduced into SEQ ID NO: 8: (1) a mutation substituting the amino acids corresponding to amino acid numbers 44 and 247 with cysteine (C); (2) a mutation substituting the amino acid at amino acid number 259 (EU index: 220) from C to S; (3) the LALA mutation substituting the amino acids at amino acid numbers 273 and 274 (EU indexes: 234 and 235) from L to A; (4) the knobs-into-holes mutation substituting the amino acid at amino acid number 405 (EU index: 366) from T to tryptophan (W); (5) a mutation substituting the amino acid at amino acid number 336 (EU index: 297) from N to G. To introduce these mutations, polynucleotides encoding the amino acid sequences containing each mutation point were synthesized and inserted into the pcDNA3.1(+) vector (Thermo Fisher scientific, V79020). The prepared vector was designated pcDNA3.1-m7_scFV_K. The nucleotide sequence of the prepared anti-CD3scFv-Fc is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8.

[0092] [Example 1-3: Preparation of Anti-TSPAN8-Anti-CD3 Bispecific Antibody] To prepare a bispecific antibody composed of the Fab region of an anti-TSPAN8 antibody, the anti-CD3scFv region, and the Fc region, pcDNA3.4-16B11.1_HC_H, pcDNA3.4-16B11_LC, and pcDNA3.1-m7_scFV_K were transfected into ExpiCHO-S cells according to a conventional method. The culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02), and further purified using a gel filtration column HiLoad® 26 / 600 Superdex® 200pg (Cytiva, 28-9893-36) to obtain a purified antibody with a purity of 95% or higher. The obtained antibody is referred to as an anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody.

[0093] [Example 2: In vivo combined effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody] The in vivo antitumor effect of the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and the anti-mouse PD-1 antibody was examined using B-hCD3E mice (human CD3ε extracellular domain knock-in mice) transplanted with human TSPAN8-expressing B16-F10 cells.

[0094] [Example 2-1: Construction of human TSPAN8-expressing B16-F10 cells] The B16-F10 cells, a mouse melanoma cell line, were obtained from the American Type Culture Collection (ATCC, CRL-6475). Dulbecco's modified Eagle's medium (SIGMA, D6429) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070-063) (the prepared medium is hereinafter referred to as "culture medium") was used at 37°C, 5% CO 2Cells were cultured under the following conditions. Human TSPAN8 was subcloned from TSPAN8 (Myc-DDK-tagged)-Human tetraspanin 8 (TSPAN8) (ORIGENE, RC202694) into the pMONO-blasti-mcs vector (InvivoGen, pmonob-mcs). This vector was introduced into B16-F10 cells by electroporation. A B16-F10 cell clone stably expressing human TSPAN8 (human TSPAN8-expressing B16-F10 cells) was obtained by selective culture in a culture medium supplemented with blasticidin (InvivoGen, ant-bl-1) at a final concentration of 10 μg / mL and sorting of expressing cells using a FACS Aria II cell sorter (Becton, Dickinson and Company).

[0095] [Example 2-2: Combined effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody in in vivo antitumor action] B-hCD3E female mice (C57BL / 6-CD3e tm2(CD3e) / Bcgen; Biocytogen, 110008) were obtained. Human TSPAN8-expressing B16-F10 cells were suspended in Matrigel (registered trademark) Basement Membrane Matrix (Corning Inc., 356237), and a cell suspension of 1×10 7 cells / mL was prepared. The cell suspension was inoculated subcutaneously on the side of 7- to 8-week-old mice at 1×10 6 cells / 100 μL each. Five days after cell inoculation, the tumor diameter was measured using calipers (Mitutoyo, CD-15AXR). The following formula was used to calculate the tumor volume [mm 3 . (Length of the major axis of the tumor [mm]) × (Length of the minor axis of the tumor [mm]) 2 × 0.5 Mice were grouped (n = 8) so that the tumor volumes were equal in each group, and administration of the test drug was started. The first day of administration was defined as day 0. Details of the test drugs, doses, administration schedules, etc. administered to the four groups in which the test was conducted are shown below. (1) Group 1: Phosphate Buffered Saline (PBS) administration group On days 0 and 7, PBS was intravenously administered at 10 mL / kg. (2) Group 2: Anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody administration group On days 0 and 7, the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody prepared at 0.1 mg / mL was intravenously administered at 1 mg / kg. (3) Group 3: Anti-mouse PD-1 antibody administration group On days 0, 3, 7, and 10, the anti-mouse PD-1 antibody (Bio X Cell, BE0146) prepared at 1 mg / mL was intraperitoneally administered at 10 mg / kg. (4) Group 4: Combined administration group of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody On days 0 and 7, the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody prepared at 0.1 mg / mL was intravenously administered at 1 mg / kg, and on days 0, 3, 7, and 10, the anti-mouse PD-1 antibody prepared at 1 mg / mL was intraperitoneally administered at 10 mg / kg. The tumor volumes were evaluated on days 3, 6, 10, and 13 for each group (Figure 1-1). On days 10 and 13 for Group 1, and on day 13 for Groups 2 and 3, the analysis was performed with n = 7 (due to the occurrence of one death case each). The tumor volumes on day 13 for Groups 2 and 3 were compared with those of Group 4 by unpaired Student's t-test (Figure 1-2). As shown in Figure 1-2, the tumor volumes of the combined group of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody were significantly smaller than those of the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody single-agent group and the anti-mouse PD-1 antibody single-agent group. This result suggests that in the treatment of human cancer, the combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody may obtain higher efficacy than treatment with a single agent.

Industrial Applicability

[0096] The method for treating cancer by combining the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention with a PD-1 signal inhibitor is expected to be useful for treating cancer.

Sequence Listing Free-Text

[0097] In the following numerical headings <223> of the Sequence Listing, the description of "Artificial Sequence" is described. Specifically, SEQ ID NO: 2 is the amino acid sequence of the heavy chain of 16B11.1, and the nucleotide sequence shown in SEQ ID NO: 1 is the nucleotide sequence encoding the amino acid sequence of the heavy chain of 16B11.1 shown in SEQ ID NO: 2. SEQ ID NO: 4 is the amino acid sequence of a polypeptide in which a heavy chain fragment containing the heavy chain variable region of 16B11.1 and a first Fc polypeptide are linked, and the nucleotide sequence shown in SEQ ID NO: 3 is the nucleotide sequence encoding the amino acid sequence of a polypeptide in which a heavy chain fragment containing the heavy chain variable region of 16B11.1 and a first Fc polypeptide are linked shown in SEQ ID NO: 4. The sequence of SEQ ID NO: 6 is the amino acid sequence of the light chain of 16B11.1, and the nucleotide sequence shown in SEQ ID NO: 5 is the nucleotide sequence encoding the amino acid sequence of the light chain of 16B11.1 shown in SEQ ID NO: 6. SEQ ID NO: 8 is the amino acid sequence of a polypeptide in which an anti-CD3scFv region and a second Fc polypeptide are linked, and the nucleotide sequence shown in SEQ ID NO: 7 is the nucleotide sequence encoding the amino acid sequence of a polypeptide in which an anti-CD3scFv region and a second Fc polypeptide are linked shown in SEQ ID NO: 8. SEQ ID NOs: 9 to 17 are the amino acid sequences of various linkers described in the detailed description of the invention.

Claims

**Claim 1** A pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating a target cancer, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: (a) a Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region containing the heavy chain variable region and the light chain variable region of the anti-CD3 antibody, and (c) an Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3 scFv region of (b). A pharmaceutical composition as described above. **Claim 2** The pharmaceutical composition according to claim 1, wherein the heavy chain variable region of the anti-TSPAN8 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO:

6. **Claim 3** The pharmaceutical composition according to claim 1 or 2, wherein the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO:

6. **Claim 4** The pharmaceutical composition according to any one of claims 1 to 3, wherein the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO:

6. **Claim 5** The heavy-chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light-chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO:

8. The pharmaceutical composition according to any one of claims 1 to 4.

6. The heavy-chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light-chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO:

8. The pharmaceutical composition according to any one of claims 1 to 5.

7. The anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO:

8. The pharmaceutical composition according to any one of claims 1 to 6.

8. The pharmaceutical composition according to any one of claims 1 to 7, comprising a polypeptide in which an anti-TSPAN8-anti-CD3 bispecific antibody is linked to a second Fc polypeptide, the anti-TSPAN8-anti-CD3 bispecific antibody comprising: a heavy chain of an anti-TSPAN8 antibody in which a heavy chain fragment of the anti-TSPAN8 antibody containing a heavy chain variable region comprising CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4 is linked to a first Fc polypeptide; a light chain of the anti-TSPAN8 antibody containing a light chain variable region comprising CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6; and an anti-CD3 scFv region containing a heavy chain variable region of an anti-CD3 antibody comprising CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and a light chain variable region of the anti-CD3 antibody comprising CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 236 to 244 of SEQ ID NO:

8.

9. The pharmaceutical composition according to any one of claims 1 to 8, comprising a polypeptide in which an anti-TSPAN8-anti-CD3 bispecific antibody is linked to a second Fc polypeptide, the anti-TSPAN8-anti-CD3 bispecific antibody comprising: a heavy chain of an anti-TSPAN8 antibody in which a heavy chain fragment of the anti-TSPAN8 antibody containing a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4 is linked to a first Fc polypeptide; a light chain of the anti-TSPAN8 antibody containing a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6; and an anti-CD3 scFv region containing a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and a light chain variable region of the anti-CD3 antibody consisting of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO:

8.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising an Fc region containing one or more mutations selected from the group consisting of the LALA mutation, the N297G mutation, or the Knobs-into-Holes mutation.

11. A pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating cancer in a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody consists of a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and a polypeptide in which an anti-CD3 scFv region consisting of the amino acid sequence of SEQ ID NO: 8 is linked to a second Fc polypeptide.

12. The pharmaceutical composition according to any one of claims 1 to 11, comprising a translationally modified anti-TSPAN8-anti-CD3 bispecific antibody as an active ingredient.

13. The pharmaceutical composition according to claims 1 to 12, which is administered simultaneously, continuously, or sequentially with a PD-1 signal inhibitor.

14. The pharmaceutical composition according to claims 1 to 12, wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered simultaneously, or (ii) contained in separate pharmaceutical compositions and administered simultaneously, continuously, or sequentially.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the cancer is a primary, metastatic, or peritoneal disseminated solid cancer.

16. The pharmaceutical composition according to claim 15, wherein the cancer is selected from esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, and prostate cancer.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the PD-1 signal inhibitor is any one of an anti-PD-1 antibody, an anti-PD-L1 antibody, or a PD-L2 antibody, or an antigen-binding fragment thereof.

18. The pharmaceutical composition according to claim 17, wherein the PD-1 signal inhibitor is an anti-PD-1 antibody.

19. The pharmaceutical composition according to claim 18, wherein the anti-PD-1 antibody is any one of nivolumab, pembrolizumab, pidilizumab, spartalizumab, or semaprilumab.

20. The pharmaceutical composition according to claim 17, wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody.

21. An anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating a target cancer, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: (a) a Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody; (b) an anti-CD3 scFv region containing the heavy chain variable region of the anti-CD3 antibody and the light chain variable region of the anti-CD3 antibody; and (c) an Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3 scFv region of (b). A bispecific antibody comprising the same.

22. An anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor for treating a target cancer, wherein the anti-TSPAN8-anti-CD3 bispecific antibody is composed of a polypeptide consisting of a heavy chain of an anti-TSPAN8 antibody having the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody having the amino acid sequence of SEQ ID NO: 6, and an anti-CD3 scFv region having the amino acid sequence of SEQ ID NO: 8 linked to a second Fc polypeptide.

23. A method for treating cancer, comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor to a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: (a) a Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody; (b) an anti-CD3 scFv region containing the heavy chain variable region of the anti-CD3 antibody and the light chain variable region of the anti-CD3 antibody; and (c) an Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3 scFv region of (b). A method comprising the same.

24. A method for treating cancer, comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor to a subject, wherein the anti-TSPAN8-anti-CD3 bispecific antibody is composed of a polypeptide consisting of a heavy chain of an anti-TSPAN8 antibody having the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody having the amino acid sequence of SEQ ID NO: 6, and an anti-CD3 scFv region having the amino acid sequence of SEQ ID NO: 8 linked to a second Fc polypeptide.

25. Use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signal inhibitor to treat a target cancer, wherein the anti-TSPAN8-anti-CD3 bispecific antibody comprises: a) a Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment comprising the heavy chain variable region of the anti-TSPAN8 antibody and a light chain comprising the light chain variable region of the anti-TSPAN8 antibody, b) an anti-CD3 scFv region comprising the heavy chain variable region and the light chain variable region of the anti-CD3 antibody, and c) an Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a) and a second Fc polypeptide linked to the anti-CD3 scFv region of (b), the use comprising.

26. Use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signal inhibitor to treat a target cancer, wherein the anti-TSPAN8-anti-CD3 bispecific antibody consists of a polypeptide comprising the heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, the light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an anti-CD3 scFv region consisting of the amino acid sequence of SEQ ID NO: 8 linked to a second Fc polypeptide.

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