TW202404638A - Use of anti-tspan8-anti-cd3 bispecific antibody in combination with pd-1 signal inhibitors in cancer treatment - Google Patents

Abstract

The present invention addresses the problem of providing: 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 containing said bispecific antibody; and a cancer treatment method including administering, to a target, the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor. The combination of the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and the PD-1 signal inhibitor is effective for treatment of TSPAN8-expressing cancer.

Description

The use of combination of anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signaling inhibitor in cancer treatment

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

Tetraspanin-8 (TSPAN8) is a 4-pass membrane penetrating protein belonging to the tetraspanin family. It has a small extracellular loop (SEL) and a large extracellular loop (SEL). loop; LEL), two extracellular loop domains and three cytoplasmic domains serve as scaffolding proteins to form molecular clusters with various membrane-penetrating proteins and cytoplasmic proteins. TSPAN8 is known to be related to cell adhesion, cell motility, cell activation and proliferation, etc. High expression has been observed in gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, etc. It has been reported that increased expression of TSPAN8 is related to cancer progression or metastasis. properties, etc. (Non-Patent Document 1). Research aimed at diagnosing or treating cancer using anti-TSPAN8 antibodies is ongoing (Patent Documents 1 and 2 and Non-Patent Documents 1 and 2).

Cluster of Differentiation 3 (CD3) is a protein that conveys activation messages to T cells by forming a complex with the T cell receptor (TCR) on the surface of T cells. CD3 is a complex composed of five subunits of gamma (γ), delta (δ), epsilon (ε), zeta (ζ) and eta (η) chains. Each subunit forms 3 of εγ, εδ and ζζ. kind of dimer. CD3 is expressed on both normal T cells and tumor T cells, so it is used as a marker of T cells. In addition, there are reports of various anti-cancer-associated antigen (Tumor Associate Antigen; TAA) antibodies and anti-cancer drugs for the purpose of cancer treatment. Application of CD3 antibody bispecific antibodies in pharmaceuticals (Non-Patent Document 3).

As an innovative method that can obtain cancer cell-selective cytotoxic activity at low antibody concentrations, bispecific T-cell-recruiting antibodies composed of various antibody formats have been reported. The effect of T cell-mediated immunotherapy is being continuously studied (Non-Patent Document 4). Bispecific T cell recruiting antibodies are bispecific antibodies that include antibodies against TAA expressed on the surface of cancer cells and antibodies that bind to T cells. Antibodies that bind to T cells, mostly anti-CD3 antibodies. Bispecific T cell recruiting antibodies containing molecules of anti-TAA and anti-CD3 antibodies will bring the target cancer cells into physical proximity with cytotoxic T lymphocytes (CTLs), activate CTLs through anti-CD3 antibodies, and Kill cancer cells through the cytotoxic activity of CTL (Redirected T Cell Cytotoxicity; RTCC). Catumaxomab as an anti-CD3-anti-Epithelial Cell Adhesion Molecule (EpCAM) bispecific antibody or Baili as an anti-CD3-anti-CD19 (Cluster of Differentiation 19) bispecific antibody Blinatumomab has been confirmed to have clinical effects (Int. J. Cancer, 2010; Vol. 127: p. 2209-2221, N. Engl. J. Med., 2017; Vol. 376: p. 836 -847), currently, the research and development of various anti-TAA bispecific T cell recruiting antibodies is ongoing.

Programmed cell death-1 (PD-1; also known as PDCD1 or CD279) is a 50~55kDa type I membrane-penetrating protein belonging to the immunoglobulin superfamily (Int. Immunol., 1996; Vol. 8: p. 765- 772). PD-1 induces expression in T cells with sustained activation, but by binding to 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) controls the activation of T cells to be inhibitory (Annu. Rev. Immunol., 2008; Vol. 26: p. 677-704). Generally speaking, such a T cell activation control mechanism is called an immune checkpoint and is known to be one of the negative feedback mechanisms used to prevent excessive immune responses.

In the early stages of cancer, immune cells such as T cells eliminate cancer through anti-tumor immune responses through immune surveillance mechanisms. On the other hand, cancer cells directly or indirectly suppress immune cells in the cancer microenvironment to acquire immune evasion mechanisms. As direct activated T cell inhibitory mechanisms, immune checkpoint mechanisms such as the PD-1/PD-L1 or PD-L2 (hereinafter referred to as "PD-1 message") pathway, CTLA-4/CD80 or CD86 pathway are known. In the tumor microenvironment of cancer, PD-1 expression in T cells and PD-L1 expression in tumors have been confirmed (Nat. Med., 2002; Vol. 8: p. 793-800), and it is believed that the cancer system Immune evasion is demonstrated through activation of this PD-1 message. In multiple mouse cancer-bearing models, it has been reported that it exhibits anti-tumor effects by blocking PD-1 signaling to disable the immune evasion mechanism (Non-patent Documents 5 to 7). Furthermore, as PD-1 signaling agents, the development of PD-1 signaling inhibitors such as anti-PD-1 antibodies such as nivolumab and pembrolizumab is being vigorously pursued. , achieved great results in melanoma, lung cancer, lymphoma, etc. Furthermore, in addition to antibodies, research on nucleic acid drugs/low molecular drugs, etc., as PD-1 signaling inhibitors has also been carried out (Non-Patent Document 8). As indirect mechanisms for suppressing activated T cells, induction of control T cells by cancer cells or bone marrow-derived control cells, and immunosuppressive liquid factors (IL-10, TGF-β, indinoline) are known. (J. Hematol. Oncol., 2021; Vol. 14: p. 55-74) , BioDrugs, 2018; Vol.32: p.311-317).

In order to improve the effectiveness of treatment for cancer patients, experiments are being conducted on the combined use of multiple cancer immune drugs or the combined use of cancer immune drugs and existing anticancer drugs (Non-Patent Documents 9 and 10). For example, trials of the combination of anti-PD-1 antibodies with other immune checkpoint-blocking antibodies, anticancer agents, molecular target drugs, radiation therapy, cancer vaccines, and tumor-lytic viruses are being conducted. However, so far, a cancer treatment method using the combined use of an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor has not been reported. [Prior technical literature] [Patent Document]

[Patent Document 1] International Publication No. 2012/010696 [Patent Document 2] International Publication No. 2015/130115 [Non-patent literature]

[Non-patent document 1] Biomolecules, 2020; 10: p.383 [Non-patent document 2] Cancers, 2019; 11: p.179 [Non-patent document 3] Pharmacology and Therapeutics, 2018; 182: p.161-175 [Non-patent document 4] mAbs, 2017; 9: p.182-212 [Non-patent document 5] Scientific Reports, 2021; 11: p.21087-21099 [Non-patent document 6] Nature Communications, 2017; 8: p.14572-14582 [Non-patent document 7] Journal for Immunotherapy of Cancer, 2019; 7: p.37-52 [Non-patent document 8] Molecules, 2019; 24: p.2071-2100 [Non-patent document 9] Cancer Discovery, 2021; 11: p.1368-1397 [Non-patent document 10] Moledular Medicine Reports, 2021; 23: p.362-377

[Problem to be solved by the invention]

An object of the present invention is to provide an anti-TSPAN8-anti-CD3 bispecific antibody or a pharmaceutical composition containing the bispecific antibody that is used in combination with a PD-1 signaling inhibitor to treat a target cancer, or to provide a pharmaceutical composition containing the bispecific antibody. A treatment method for administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject's cancer. [Means used to solve problems]

The present inventors produced an anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody based on the sequence of 16B11.1 of the anti-TSPAN8 antibody for the purpose of creating an antibody or pharmaceutical composition for the treatment of TSPAN8-expressing cancer. (Example 1). When the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and the anti-PD-1 antibody were administered to mice transplanted with TSPAN8-expressing cancer cells, it was confirmed that compared with the administration of the anti-PD-1 antibody alone, Showed significant anti-tumor effect (Example 3). These results suggest that the combination of anti-TSPAN8-anti-CD3 bispecific antibodies and PD-1 signaling inhibitors may be useful in the treatment of TSPAN8-expressing cancers. Furthermore, the combined use of this anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody is more effective than anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and anti-PD-1 Antibodies or anti-PD-L1 antibodies alone promote the activation of T cells and the production of interferon-γ by T cells in vitro, and show cytotoxic activity (Example 2).

That is, the present invention relates to the following [1]~[92]. [1] A pharmaceutical composition for treating cancer of a subject containing an anti-TSPAN8-anti-CD3 bispecific antibody, the anti-TSPAN8-anti-CD3 bispecific antibody comprising: (a) consisting of The Fab region of the anti-TSPAN8 antibody, which is composed of a heavy chain fragment of the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) includes the heavy chain variable region of the anti-CD3 antibody and an anti-CD3scFv region of the light chain variable region of an anti-CD3 antibody, and (c) a first Fc polypeptide consisting of a heavy chain fragment linked to the Fab region of (a) and an anti-CD3scFv region linked to the anti-CD3scFv region of (b) The Fc region composed of the second Fc polypeptide, the pharmaceutical composition is used in combination with a PD-1 signaling inhibitor. [2] The pharmaceutical composition of [1], wherein the heavy chain variable region of the anti-TSPAN8 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, The CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66, and the CDR3 composed of the amino acid sequence of amino acid numbers 99 to 110 of sequence number 4; the light chain variable region of the anti-TSPAN8 antibody , including CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR2 composed of the amino acid sequence of SEQ ID NO: 6 CDR3 consisting of the amino acid sequence of amino acid numbers 89 to 96 of 6. [3] The pharmaceutical composition of [1] or [2], wherein the heavy chain variable region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4, and the anti-TSPAN8 antibody The light chain variable region is composed of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. [4] The pharmaceutical composition according to any one of [1] to [3], wherein the Fab region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 It consists of a heavy chain fragment and a light chain composed of the amino acid sequence of SEQ ID NO: 6. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the heavy chain variable region of the anti-CD3 antibody includes the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8. The CDR1 composed of the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8, and the CDR3 composed of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8; anti- The light chain variable region of the CD3 antibody includes CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181 of SEQ ID NO: 8, and CDR1 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8. The CDR2 composed of SEQ ID NO: 8, and the CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the heavy chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8 , the light chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the anti-CD3 scFv region is composed of the amino acid sequence of amino acid numbers 1 to 254 of SEQ ID NO: 8. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the anti-TSPAN8-anti-CD3 bispecific antibody contains: an amino group containing amino acid numbers 31 to 35 of SEQ ID NO: 4 CDR1 composed of the amino acid sequence of SEQ ID NO: 4, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4, and CDR2 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the heavy chain variable region of CDR3 to the first Fc polypeptide; contains an amine containing amino acid numbers 24 to 34 of SEQ ID NO: 6 CDR1 composed of the amino acid sequence, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR2 composed of the amino acid sequence of amino acid numbers 89 to 96 of SEQ ID NO: 6 The light chain of the anti-TSPAN8 antibody of the light chain variable region of the CDR3, and the CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, the amino acid number of SEQ ID NO: 8 The heavy chain variable region of an anti-CD3 antibody consisting of CDR2 consisting of amino acid sequences 50 to 68, and CDR3 consisting of amino acid sequences 101 to 114 of SEQ ID NO: 8, and a heavy chain variable region consisting of the sequence The CDR1 composed of the amino acid sequence of SEQ ID NO: 8 from 168 to 181, the CDR2 composed of the amino acid sequence of SEQ ID NO: 8 from 197 to 203, and the CDR2 composed of the amino acid sequence of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody of the CDR3 composed of the amino acid sequence of acid numbers 236 to 244 and the second Fc polypeptide. [9] The pharmaceutical composition according to any one of [1] to [8], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: amino acids containing amino acid numbers 1 to 121 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the heavy chain variable region of the anti-TSPAN8 antibody to the first Fc polypeptide, and contains amino acid numbers 1 to 107 of SEQ ID NO: 6 The light chain of an anti-TSPAN8 antibody having a light chain variable region composed of amino acid sequences, and the heavy chain variable region of an anti-CD3 antibody having an amino acid sequence composed of amino acid numbers 1 to 125 of SEQ ID NO: 8 region, and the polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody consisting of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8 and the second Fc polypeptide. Peptides. [10] The pharmaceutical composition according to any one of [1] to [9], which contains a compound selected from the group consisting of LALA mutations (L234A and L235A), N297G mutations, and Knobs into holes (Knobs into holes) mutations (here, The aforementioned mutation positions are one or two or more mutated Fc regions consisting of a group of amino acid positions (amino acid positions according to the EU index) in the human Igγ1 constant region. [11] The pharmaceutical composition according to any one of [1] to [9], which includes an Fc region containing LALA mutation, N297G mutation, and Knobs into holes mutation. [12] The pharmaceutical composition of [10] or [11], wherein the Knobs into holes mutation is the T366W mutation in one Fc polypeptide forming the Fc region, and another Fc polypeptide forming the Fc region T366S, L368A and Y407V mutations (here, the aforementioned mutation positions are amino acid positions that comply with the EU index in the human Igγ1 constant region). [13] A pharmaceutical composition for treating cancer of a subject containing an anti-TSPAN8-anti-CD3 bispecific antibody. The anti-TSPAN8-anti-CD3 bispecific antibody is composed of: SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6, the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6, and the anti-CD3 scFv region composed of the amino acid sequence of SEQ ID NO: 8 The pharmaceutical composition is composed of a polypeptide linked to a second Fc polypeptide, and is used in combination with a PD-1 signaling inhibitor. [14] The pharmaceutical composition according to any one of [1] to [13], which contains a post-translationally modified anti-TSPAN8-anti-CD3 bispecific antibody as an active ingredient. [15] The pharmaceutical composition according to any one of [1] to [14], which is administered to the subject simultaneously, continuously, or sequentially with the PD-1 signaling inhibitor. [16] The pharmaceutical composition according to any one of [1] to [14], which contains anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signaling inhibitor (i) in the same pharmaceutical composition. , and administered to the subject simultaneously, or (ii) contained in different pharmaceutical compositions, and administered to the subject simultaneously, continuously, or sequentially. [17] The pharmaceutical composition according to any one of [1] to [16], wherein the cancer is primary, metastatic or peritoneal disseminated solid cancer. [18] The pharmaceutical composition of [17], wherein the cancer is esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer. [19] The pharmaceutical composition according to any one of [1] to [18], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or these The antigen-binding fragment. [20] The pharmaceutical composition of [19], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody. [21] The pharmaceutical composition of [20], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, or spartalizumab, Or cemiplimab. [22] The pharmaceutical composition of [19], wherein the PD-1 signaling inhibitor is an anti-PD-L1 antibody. [23] The pharmaceutical composition of [22], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab. [24] A bispecific antibody, which is an anti-TSPAN8-anti-CD3 bispecific antibody used to treat cancer of a subject, the anti-TSPAN8-anti-CD3 bispecific antibody comprising: (a) consisting of an anti-TSPAN8 antibody The Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment of the heavy chain variable region and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) the heavy chain variable region of the anti-CD3 antibody and the anti-CD3 antibody The anti-CD3 scFv region of the light chain variable region, and (c) 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 Fc region composed of peptides, the anti-TSPAN8-anti-CD3 bispecific antibody system is used in combination with a PD-1 signaling inhibitor. [25] The bispecific antibody of [24], wherein the heavy chain variable region of the anti-TSPAN8 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, and SEQ ID NO: The CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4, and the CDR3 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4; the light chain variable of the anti-TSPAN8 antibody The region includes CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and the CDR2 composed of the amino acid sequence of SEQ ID NO: 6 CDR3 composed of amino acid number 6 and amino acid sequence 89 to 96. [26] The bispecific antibody of [24] or [25], wherein the heavy chain variable region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4, and the anti-TSPAN8 The light chain variable region of the antibody is composed of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. [27] The bispecific antibody according to any one of [24] to [26], wherein the Fab region of the anti-TSPAN8 antibody is composed of: the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 It consists of a heavy chain fragment and a light chain composed of the amino acid sequence of SEQ ID NO: 6. [28] The bispecific antibody according to any one of [24] to [27], wherein the heavy chain variable region of the anti-CD3 antibody includes the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8 The CDR1 formed, the CDR2 composed of the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8, and the CDR3 composed of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8; The light chain variable region of the anti-CD3 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 168 to 181 of SEQ ID NO: 8, and the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8 The CDR2 constituted, and the CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8. [29] The bispecific antibody according to any one of [24] to [28], wherein the heavy chain variable region of the anti-CD3 antibody is represented by the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8 The light chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8. [30] The bispecific antibody according to any one of [24] to [29], wherein the anti-CD3 scFv region is composed of the amino acid sequence of amino acid numbers 1 to 254 of SEQ ID NO: 8. [31] The bispecific antibody according to any one of [24] to [30], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: an amine including amino acid numbers 31 to 35 of SEQ ID NO: 4 CDR1 composed of the amino acid sequence, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4, and CDR2 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the heavy chain variable region of the CDR3 to the first Fc polypeptide; containing amino acid numbers 24 to 34 of SEQ ID NO: 6 CDR1 composed of the amino acid sequence of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR2 composed of the amino acid sequence of amino acid numbers 89 to 96 of SEQ ID NO: 6 The light chain of the anti-TSPAN8 antibody that constitutes the light chain variable region of CDR3, and the CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and the amino acid sequence of SEQ ID NO: 8 The heavy chain variable region of the anti-CD3 antibody of CDR2 consisting of the amino acid sequence of SEQ ID NO: 50 to 68, and the CDR3 consisting of the amino acid sequence of SEQ ID NO: 8 from 101 to 114, and the heavy chain variable region of the anti-CD3 antibody consisting of The CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181 of SEQ ID NO: 8, the CDR2 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8, and the amine of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody of the CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 and the second Fc polypeptide. [32] The bispecific antibody according to any one of [24] to [31], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: an amino group containing amino acid numbers 1 to 121 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the heavy chain variable region composed of the acid sequence and the first Fc polypeptide, contains amino acid numbers 1 to 107 of SEQ ID NO: 6 The light chain of the anti-TSPAN8 antibody with a light chain variable region composed of the amino acid sequence of SEQ ID NO: 8, and the heavy chain of the anti-CD3 antibody composed of the amino acid sequence of SEQ ID NO: 8 can be variable region, and the polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody consisting of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8 and the second Fc polypeptide. Peptides. [33] The bispecific antibody according to any one of [24] to [32], which contains a mutation selected from the group consisting of LALA mutations (L234A and L235A), N297G mutations, and Knobs into holes (Knobs into holes) mutation (here , the aforementioned mutation position is one or more than two kinds of mutated Fc region consisting of a group of amino acid positions (amino acid positions according to the EU index in the human Igγ1 constant region). [34] The bispecific antibody according to any one of [24] to [32], which includes an Fc region containing LALA mutation, N297G mutation, and Knobs into holes mutation. [35] The bispecific antibody of [33] or [34], wherein the Knobs into holes mutation is the T366W mutation in one Fc polypeptide forming the Fc region, and another Fc polypeptide forming the Fc region T366S, L368A and Y407V mutations in the peptide (here, the aforementioned mutation positions are amino acid positions following the EU index in the human Igγ1 constant region). [36] A bispecific antibody, which is an anti-TSPAN8-anti-CD3 bispecific antibody used to treat cancer of a subject. The anti-TSPAN8-anti-CD3 bispecific antibody is composed of: the amino acid of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody composed of the sequence, the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6, and the anti-CD3 scFv region and the second Fc composed of the amino acid sequence of SEQ ID NO: 8 The anti-TSPAN8-anti-CD3 bispecific antibody system is composed of linked polypeptides and is used in combination with a PD-1 signaling inhibitor. [37] The bispecific antibody according to any one of [24] to [36], wherein the anti-TSPAN8-anti-CD3 bispecific antibody system is post-translationally modified. [38] The bispecific antibody according to any one of [24] to [37], which is administered to the subject simultaneously, continuously or sequentially with the PD-1 signaling inhibitor. [39] The bispecific antibody according to any one of [24] to [37], which combines anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signaling inhibitor (i) in the same pharmaceutical composition Contained and administered to the subject simultaneously, or (ii) contained in different pharmaceutical compositions and administered to the subject simultaneously, continuously or sequentially. [40] The bispecific antibody according to any one of [24] to [39], wherein the cancer is primary, metastatic or peritoneal disseminated solid cancer. [41] The bispecific antibody of [40], wherein the cancer is esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer. [42] The bispecific antibody of any one of [24]~[41], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or this Antigen-binding fragments. [43] The bispecific antibody of [42], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody. [44] The bispecific antibody of [43], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pitilizumab, spatalizumab, or cimepril monoclonal antibodies. [45] The bispecific antibody of [42], wherein the PD-1 signaling inhibitor is an anti-PD-L1 antibody. [46] The bispecific antibody of [45], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab. [47] A treatment method comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject's cancer, the anti-TSPAN8-anti-CD3 bispecific antibody comprising: (a) consisting of The Fab region of the anti-TSPAN8 antibody, which is composed of a heavy chain fragment of the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) includes the heavy chain variable region of the anti-CD3 antibody and an anti-CD3scFv region of the light chain variable region of an anti-CD3 antibody, and (c) a first Fc polypeptide consisting of a heavy chain fragment linked to the Fab region of (a) and an anti-CD3scFv region linked to the anti-CD3scFv region of (b) The Fc region composed of the second Fc polypeptide. [48] The method of [47], wherein the heavy chain variable region of the anti-TSPAN8 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, and the amine of SEQ ID NO: 4 CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66, and CDR3 consisting of the amino acid sequence of amino acid numbers 99 to 110 of sequence number 4; the light chain variable region of the anti-TSPAN8 antibody, including CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR2 composed of the amino acid sequence of SEQ ID NO: 6 CDR3 composed of amino acid sequences from amino acid numbers 89 to 96. [49] The method of [47] or [48], wherein the heavy chain variable region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody The chain variable region is composed of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. [50] The method according to any one of [47] to [49], wherein the Fab region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4. It consists of a chain fragment and a light chain composed of the amino acid sequence of SEQ ID NO: 6. [51] The method according to any one of [47] to [50], wherein the heavy chain variable region of the anti-CD3 antibody includes the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8 CDR1, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8; anti-CD3 antibody The light chain variable region includes CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181 of SEQ ID NO: 8, and CDR1 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8 CDR2, and CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8. [52] The method according to any one of [47] to [51], wherein the heavy chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the anti-CD3 antibody is The light chain variable region of the CD3 antibody is composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8. [53] The method according to any one of [47] to [52], wherein the anti-CD3 scFv region is composed of the amino acid sequence of amino acid numbers 1 to 254 of SEQ ID NO: 8. [54] The method according to any one of [47] to [53], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: an amino acid sequence including amino acid numbers 31 to 35 of SEQ ID NO: 4 The CDR1 formed, the CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4, and the CDR3 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the anti-TSPAN8 antibody in the heavy chain variable region to the first Fc polypeptide; contains amino acids including amino acid numbers 24 to 34 of SEQ ID NO: 6 CDR1 composed of the sequence of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR3 composed of the amino acid sequence of SEQ ID NO: 6 of amino acid numbers 89 to 96 The light chain of the anti-TSPAN8 antibody of the light chain variable region, and a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and the CDR1 consisting of the amino acid numbers 50 to 35 of SEQ ID NO: 8 CDR2 composed of amino acid sequence 68, and the heavy chain variable region of an anti-CD3 antibody composed of CDR3 composed of amino acid sequence 101 to 114 of SEQ ID NO: 8, and a heavy chain variable region of an anti-CD3 antibody composed of SEQ ID NO: 8 CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181, CDR2 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8, and CDR2 composed of the amino acid sequence of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody of the CDR3 consisting of amino acid sequences from 236 to 244 and the second Fc polypeptide. [55] The method according to any one of [47] to [54], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: an amino acid sequence consisting of amino acid numbers 1 to 121 of SEQ ID NO: 4. The heavy chain of the anti-TSPAN8 antibody formed by linking the heavy chain fragment of the heavy chain variable region of the anti-TSPAN8 antibody to the first Fc polypeptide contains the amino groups of amino acid numbers 1 to 107 of SEQ ID NO: 6 The light chain of an anti-TSPAN8 antibody having a light chain variable region composed of the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable region of an anti-CD3 antibody having an amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, And a polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8 and the second Fc polypeptide. [56] The method according to any one of [47] to [55], which includes a mutation selected from the group consisting of LALA mutations (L234A and L235A), N297G mutations, and Knobs into holes (Knobs into holes) mutation (here, the aforementioned mutation The position is a group of 1 or 2 or more mutated Fc regions consisting of amino acid positions that comply with the EU index in the human Igγ1 constant region. [57] The method according to any one of [47] to [55], which includes an Fc region containing LALA mutation, N297G mutation, and Knobs into holes mutation. [58] The method of [56] or [57], wherein the Knobs into holes mutation is the T366W mutation in one Fc polypeptide that forms the Fc region, and the T366W mutation in another Fc polypeptide that forms the Fc region. T366S, L368A and Y407V mutations (here, the aforementioned mutation positions are amino acid positions that comply with the EU index in the human Igγ1 constant region). [59] A treatment method comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject's cancer, the anti-TSPAN8-anti-CD3 bispecific antibody is composed of: SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6, the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6, and the anti-CD3 scFv region composed of the amino acid sequence of SEQ ID NO: 8 It consists of a polypeptide linked to a second Fc polypeptide. [60] The method according to any one of [47] to [59], wherein the anti-TSPAN8-anti-CD3 bispecific antibody system is post-translationally modified. [61] The method of any one of [47] to [60], wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor are administered to the subject simultaneously, continuously, or sequentially. [62] The method according to any one of [47] to [60], wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor (i) are contained in the same pharmaceutical composition, and at the same time administered to the subject, or (ii) contained in different pharmaceutical compositions and administered to the subject simultaneously, continuously, or sequentially. [63] The method according to any one of [47] to [62], wherein the cancer is primary, metastatic or peritoneal disseminated solid cancer. [64] The method of [63], wherein the cancer is esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer. [65] The method according to any one of [47]~[64], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an antigen thereof Combine fragments. [66] The method of [65], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody. [67] The method of [66], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spatumalizumab, or cimepilumab. [68] The method of [65], wherein the PD-1 signaling inhibitor is an anti-PD-L1 antibody. [69] The method of [68], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab or avelumab. [70] 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 of a target, the anti-TSPAN8-anti-CD3 bispecific antibody Anti-TSPAN8 antibody, comprising: (a) the Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment including the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody; (b) An anti-CD3 scFv region comprising the heavy chain variable region of an anti-CD3 antibody and the light chain variable region of an anti-CD3 antibody, and (c) a first Fc polypeptide consisting of a heavy chain fragment linked to the Fab region of (a) and An Fc region consisting of a second Fc polypeptide linked to the anti-CD3 scFv region of (b). [71] The use of [70], wherein the heavy chain variable region of the anti-TSPAN8 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, and the amine of SEQ ID NO: 4 CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66, and CDR3 consisting of the amino acid sequence of amino acid numbers 99 to 110 of sequence number 4; the light chain variable region of the anti-TSPAN8 antibody, including CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR2 composed of the amino acid sequence of SEQ ID NO: 6 CDR3 composed of amino acid sequences from amino acid numbers 89 to 96. [72] The use of [70] or [71], wherein the heavy chain variable region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody The chain variable region is composed of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. [73] The use according to any one of [70] to [72], wherein the Fab region of the anti-TSPAN8 antibody is composed of: a heavy chain composed of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 It consists of a fragment and a light chain composed of the amino acid sequence of SEQ ID NO: 6. [74] The use according to any one of [70] to [73], wherein the heavy chain variable region of the anti-CD3 antibody includes the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8 CDR1, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8; anti-CD3 antibody The light chain variable region includes CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181 of SEQ ID NO: 8, and CDR1 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8 CDR2, and CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8. [75] The use according to any one of [70] to [74], wherein the heavy chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the anti-CD3 antibody is The light chain variable region of the CD3 antibody is composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8. [76] The use according to any one of [70] to [75], wherein the anti-CD3 scFv region is composed of the amino acid sequence of amino acid numbers 1 to 254 of SEQ ID NO: 8. [77] The use of any one of [70] to [76], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: an amino acid sequence including amino acid numbers 31 to 35 of SEQ ID NO: 4 The CDR1 formed, the CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4, and the CDR3 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the anti-TSPAN8 antibody in the heavy chain variable region to the first Fc polypeptide; contains amino acids including amino acid numbers 24 to 34 of SEQ ID NO: 6 CDR1 composed of the sequence of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and CDR3 composed of the amino acid sequence of SEQ ID NO: 6 of amino acid numbers 89 to 96 The light chain of the anti-TSPAN8 antibody of the light chain variable region, and a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and the CDR1 consisting of the amino acid numbers 50 to 35 of SEQ ID NO: 8 CDR2 composed of amino acid sequence 68, and the heavy chain variable region of an anti-CD3 antibody composed of CDR3 composed of amino acid sequence 101 to 114 of SEQ ID NO: 8, and a heavy chain variable region of an anti-CD3 antibody composed of SEQ ID NO: 8 CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181, CDR2 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8, and CDR2 composed of the amino acid sequence of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody of the CDR3 consisting of amino acid sequences from 236 to 244 and the second Fc polypeptide. [78] The use of any one of [70] to [77], wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: an amino acid sequence consisting of amino acid numbers 1 to 121 of SEQ ID NO: 4. The heavy chain of the anti-TSPAN8 antibody formed by linking the heavy chain fragment of the heavy chain variable region of the anti-TSPAN8 antibody to the first Fc polypeptide contains the amino groups of amino acid numbers 1 to 107 of SEQ ID NO: 6 The light chain of an anti-TSPAN8 antibody having a light chain variable region composed of the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable region of an anti-CD3 antibody having an amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, And a polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8 and the second Fc polypeptide. [79] The use of any one of [70]~[78], which includes a mutation selected from the group consisting of LALA mutations (L234A and L235A), N297G mutations, and Knobs into holes (Knobs into holes) mutation (here, the aforementioned mutation The position is a group of 1 or 2 or more mutated Fc regions consisting of amino acid positions that comply with the EU index in the human Igγ1 constant region. [80] The use of any one of [70]~[78], which includes an Fc region containing LALA mutation, N297G mutation, and Knobs into holes mutation. [81] As used in [79] or [80], where the Knobs into holes mutation is the T366W mutation in one Fc polypeptide that forms the Fc region, and the T366W mutation in another Fc polypeptide that forms the Fc region. T366S, L368A and Y407V mutations (here, the aforementioned mutation positions are amino acid positions that comply with the EU index in the human Igγ1 constant region). [82] 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 for treating cancer, the anti-TSPAN8-anti-CD3 bispecific antibody The antibody is composed of: the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6, and the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 8 The anti-CD3 scFv region composed of amino acid sequences is composed of a polypeptide obtained by linking the second Fc polypeptide. [83] The use of any one of [70] to [82], wherein the anti-TSPAN8-anti-CD3 bispecific antibody system is post-translationally modified. [84] The use of any one of [70] to [83], wherein the anti-TSPAN8-anti-CD3 bispecific antibody system and the PD-1 signaling inhibitor are administered to the subject simultaneously, continuously, or sequentially. [85] The use of any one of [70]~[83], wherein anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signaling inhibitor (i) are contained in the same pharmaceutical composition, and at the same time administered to the subject, or (ii) contained in different pharmaceutical compositions and administered to the subject simultaneously, continuously, or sequentially. [86] The use according to any one of [70] to [85], wherein the cancer is primary, metastatic or peritoneal disseminated solid cancer. [87] The use of [86], wherein the cancer is esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer. [88] The use of any one of [70]~[87], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an antigen thereof Combine fragments. [89] The use of [88], wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody. [90] The use of [89], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spatumalizumab, or cimepilimab. [91] As used in [88], wherein the PD-1 signaling inhibitor is an anti-PD-L1 antibody. [92] The use of [91], wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab. [Effects of the invention]

The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention binds to both TSPAN8, a cancer antigen, and CD3, a T cell surface molecule, and enhances the resistance of cancer cells caused by T cells by bringing the cancer cells and T cells closer in physical distance. Killing effect. The combination of the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor of the present invention results in a significant anti-tumor effect compared to the single administration of the PD-1 signaling inhibitor. Therefore, the present invention provides the use of an anti-TSPAN8-anti-CD3 bispecific antibody in combination with a PD-1 signaling inhibitor in cancer treatment.

The present invention is described in detail below.

Unless otherwise defined, the terms used in this specification will be used in the meaning generally used by those with ordinary knowledge in the technical field.

Antibodies (or immunoglobulins) refer to sugars with a 4-chain structure with a symmetrical Y-shaped structure as the basic structure, consisting of 2 heavy chains with a single sequence and 2 light chains with a single sequence. protein. There are 5 types of antibodies: IgG, IgM, IgA, IgD and IgE. The basic structure of antibody molecules is common to all types. It is formed by 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 bonded by disulfide bonds and non-covalent bonds. An antibody molecule composed of four Y-shaped chains with a molecular weight of 150,000 to 190,000. Heavy chains are usually composed of polypeptide chains containing about 440 amino acids. Each type has a characteristic structure, corresponding to IgG, IgM, IgA, IgD, and IgE, respectively called Igγ, Igμ, Igα, and Igδ. ,Igε. Furthermore, there are subtypes of IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chain systems are called Igγ1, Igγ2, IgG3, and IgG4. The light chain is usually composed of a polypeptide chain containing about 220 amino acids. There are two known types, lambda type and kappa type, called Igλ and Igκ respectively. The above two types of light chains can be paired with any kind of heavy chain.

There are 4 intra-chain disulfide bonds in the antibody molecule (5 in the heavy chain (5 in Igμ and Igε)) and 2 in the light chain. Every 100 to 110 amino acid residues form a ring. state domain. These three-dimensional structures are similar between each ring domain and are called structural units or domains. The N-terminal domains of both heavy and light chains are called variable regions. Even antibodies produced by the same type (or subtype) of the same animal also have diverse amino acid sequences. It is known that they are similar to antibodies and The binding specificity of the antigen is related to the binding. The amino acid sequence of the domain on the C-terminal side downstream of the variable region is approximately constant in each type or subtype, and is called a 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 from the N-terminal side: CH1 domain, CH2 domain, and CH3 domain. 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.

The amino acid sequences present in the three complementarity determining regions (CDRs) of VH and VL vary greatly, contributing to the variability of the variable regions. CDR is a region composed of approximately 5 to 10 amino acid residues present in the order of CDR1, CDR2, and CDR3 at the N-termini of the heavy chain and light chain, forming an antigen-binding site. On the other hand, the part other than the CDR of the variable region is called the framework region (FR), consisting of FR1 to 4, and has less changes in the amino acid sequence.

If the antibody is treated with the proteolytic enzyme papain, three antibody fragments can be obtained. The two fragments on the N-terminal side are called Fab (antigen binding fragment, Fragment, antigen binding) regions. In this specification, the "Fab region" refers to the region composed of the VH and CH1 domains of the heavy chain and the light chain (VL and CL), and the antigen-binding site of the front end portion composed of the Fab region binds to the antigen. In this specification, the "heavy chain fragment" refers to a fragment consisting of the VH and CH1 domains of the heavy chain constituting the Fab region. In addition, the fragment on the C-terminal side is called an Fc (crystallizable fragment, crystallizable) region. In this specification, "Fc polypeptide" refers to a polypeptide composed of the CH2 domain and CH3 domain of the heavy chain, and "Fc region" refers to a complex composed of two Fc polypeptides. In this specification, the "hinge region" or "hinge" refers to the highly mobile peptide region that exists between the CH1 domain and the CH2 domain of the heavy chain. The heavy chain fragment and the Fc polypeptide are called It is a partial connection of the hinge area. In addition, the two heavy chains of the antibody are disulfide-bonded in the hinge region.

In this specification, "antigen" is used in a commonly used sense, and is particularly used as a term indicating a molecule or a part of a molecule to which an antigen-binding protein such as an antibody or antigen-binding fragment can specifically bind. Antigens can be molecules such as proteins, nucleic acids, etc. One antigen may have one or more epitopes that can interact with different antibodies, etc.

In this specification, "antigen-binding fragment" refers to a molecule containing 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. scFv is a monovalent antigen-binding fragment composed of VH and VL linked by a linker. Fab fragments are monovalent antigen-binding fragments composed of fragments containing the light chain and the VH and CH1 domains of the heavy chain. Fab' fragment is a monovalent antigen-binding fragment composed of a fragment containing a light chain, a VH and CH1 domain of a heavy chain, and a portion of the hinge region, which contains the disulfide bond that constitutes the inter-heavy chain. of cysteine residues. F(ab') ₂ fragment is a bivalent molecule in which Fab' fragments are linked by disulfide bonds. "Monovalent" means containing one antigen-binding site, and "bivalent" means containing two antigen-binding sites.

In this specification, the "scFv region" refers to a region containing a monovalent antigen-binding fragment including VH and VL connected via a linker.

In this specification, "bispecific antibody" refers to an antibody that can specifically bind to two different antigens. "Anti-TSPAN8-anti-CD3 bispecific antibody" means a bispecific antibody that has binding activity to TSPAN8 and binding activity to CD3.

In this specification, unless otherwise limited by context, "antibody" is used as a term including full-length antibodies, antigen-binding fragments, and bispecific antibodies of all structures.

In this specification, "human antibody" means an antibody having the amino acid sequence of human immunoglobulin. In this specification, "humanized antibody" refers to an antibody in which part, most, or all of the amino acid residues other than CDRs are substituted with amino acid residues derived from human immunoglobulin molecules. The method of humanization is not particularly limited. For example, humanized antibodies can be prepared by referring to U.S. Patent No. 5225539, U.S. Patent No. 6180370, etc.

The amino acid residue number of the antibody used in this specification can be determined by specifying the Kabat number or EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, 1991, NIH Publication No. 91-3242). Follow these numbering systems.

In this specification, "first" or "second" are used for convenience of distinction when there are two or more types of a part. The use of such terms is not intended to confer a specific order or meaning unless explicitly recited.

In this specification, "linked" or "linked" means that multiple components (such as Fab regions and Fc polypeptides) are combined directly or through one or more mediators (such as peptide linkers). In this specification, "peptide linker" or "linker" means one or more arbitrary amino acid residues used to connect variable regions that can be introduced through genetic engineering. The length of the peptide linker used in the present invention is not particularly limited, and those with ordinary knowledge in the art can appropriately select it according to the purpose.

"Identity" in this manual means the Identity value obtained by presetting the prepared parameters using EMBOSS Needle (Nucleic Acids Res., 2015; Vol. 43: pW580-W584). The aforementioned parameters are as follows. Gap Open Penalty=10 Gap Extend Penalty=0.5 Matrix=EBLOSUM62 End Gap Penalty=false

In this specification, "analogue" means an antibody that has the same amino acid sequence in the antibody and Fab regions and can specifically bind to the antigen, but contains different amino acid sequences in the Fc region.

In this specification, "subject" means humans or other animals necessary for the prevention or treatment of diseases. In one embodiment, the target is a human being for whom prevention or treatment of disease is necessary. In one embodiment, the subject is a human with cancer.

In this specification, "treatment" means treatment performed on a subject with the purpose of restoring, alleviating, improving, inhibiting or delaying the progression, onset, severity or recurrence of symptoms, symptoms, and biochemical signs related to the disease. A certain therapeutic intervention, treatment, or administration of an active ingredient to a subject.

In this specification, "active ingredient" means a substance contained in pharmaceutical compositions, medicines, etc. used for the prevention or treatment of diseases that exhibits some physiological activity. In one embodiment, the active ingredients are antibodies, low molecular compounds, nucleic acids, fusion proteins, and peptides. In one embodiment, the active ingredient is an antibody. In one embodiment, the active ingredient is a bispecific antibody.

In this specification, "pharmaceutical composition" refers to a compound containing active ingredients and pharmaceutically acceptable excipients (for example, pharmaceutical excipients or pharmaceutical carriers, but not limited to these) and prescribed for the purpose of treating a subject. of medicine.

In this specification, "combined use", "combination" or "combined use" means that for the prevention or treatment of diseases, multiple types of active ingredients are administered to the same subject simultaneously, continuously or sequentially. The plurality of active ingredients may be contained in the same pharmaceutical composition, or may be contained in different pharmaceutical compositions respectively. In this manual, "simultaneously" means to administer multiple active ingredients in parallel during the administration period of one of them, and "continuously" means that the administration of one of the active ingredients is completed immediately after the administration of the other. The administration of ingredients "sequentially" means that multiple active ingredients are administered sequentially according to the administration schedule.

In this specification, the "effective dose" of a drug refers to the dose necessary to cause physiological changes in the cells or tissues to which it is administered.

In this specification, "PD-1 signaling inhibitor" means an agent that relieves the inhibition of immune cell activation caused by PD-1. PD-1 signaling inhibitors can block the immunosuppressive signaling by binding to PD-1 or its ligands PD-L1 or PD-L2, thereby blocking the immune checkpoint function caused by PD-1. PD-1 signal inhibitors can be any substance as long as they have the effect of blocking PD-1 signals. For example, they can be antibodies, low molecular compounds, nucleic acids (which can include DNA or RNA, or natural or artificial nucleic acids). , fusion proteins, peptides, etc. For example, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies can block the PD-1 message by blocking the binding of PD-1 to PD-L1 or PD-L2 (Expert Opinion on Therapeutic Patents ,2016;Vol.26:p.555-564).

The present invention relates to the following (1)~(4): (1) A pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signaling inhibitor for the purpose of treating cancer (also referred to as "the pharmaceutical composition of the present invention" in this specification) "); (2) Anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signaling inhibitor in order to treat the target cancer (also referred to as "bispecific antibody used in combination in the present invention" in this specification) ; (3) A treatment method comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to the subject's cancer (also referred to as the "treatment method of the present invention" in this specification); or (4) 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 for the treatment of cancer (also referred to as "the present invention" in this specification) Uses of the Bispecific Antibodies of the Invention").

<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 as the "anti-TSPAN8-anti-CD3 bispecific antibody of the present invention" in this specification) is composed of the following structures (a) to (c) Composition: A bispecific antibody containing (a) The Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment including the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including 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 an 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 anti-TSPAN8-anti-CD3 bispecific antibody of the present invention has a structure including a Fab region of the first antibody, a scFv region of the second antibody, and an Fc region. Antibodies with such a structure are also called "bottle-opener type" or "triple F type" (International Publication No. 2017/218707).

The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an anti-TSPAN8 antibody composed 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. Fab area.

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

In one embodiment, the heavy chain variable region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody is composed of SEQ ID NO: 4 6 consists of amino acid sequences numbered 1 to 107.

The heavy chain constant region from which the CH1 domain of the heavy chain fragment of the anti-TSPAN8 antibody is derived can be selected from any type of Igγ, Igμ, Igα, Igδ or Igε constant region. Igγ can be selected from, for example, Igγ1, Igγ2, Igγ3, or Igγ4. In one embodiment, the heavy chain fragment of the anti-TSPAN8 antibody includes a CH1 domain derived from the human Igγ1 constant region.

The CL of the light chain of the anti-TSPAN8 antibody can be selected regardless of the constant region of Igλ or Igκ. In one embodiment, the light chain of the anti-TSPAN8 antibody includes the CL of the human Igκ constant region.

In one embodiment, the Fab region of the anti-TSPAN8 antibody is composed of: a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 6 Made up of light chains.

The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes, as the scFv region, an anti-CD3 scFv region including the heavy chain variable region and the light chain variable region of the anti-CD3 antibody. In the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, it is also possible to use the anti-CD3 scFv region known in the field or the sequence information based on the heavy chain variable region and light chain variable region of the anti-CD3 antibody known in the field. Anti-CD3scFv region produced. Well-known anti-CD3 antibodies include clones of OKT3, UTCH1, L2K, TR66, etc., and their sequences are used as bispecific antibodies (Pharmacol. Ther., 2018; Vol. 182: p. 161-175) .

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

In one embodiment, the heavy chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody is composed of SEQ ID NO: 8 8 consists of the amino acid sequence of amino acid numbers 146 to 254.

In the anti-CD3 scFv region, the type and length of the peptide linker connecting 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 with ordinary knowledge in the art. In one embodiment, the length of the peptide linker is 5 amino acids or more. The upper limit is not particularly limited, but is usually 30 amino acids or less. The length of the peptide linker is not less than 5 amino acids and not more than 30 amino acids, preferably not less than 5 amino acids and not more than 20 amino acids, particularly preferably not less than 15 amino acids and not more than 20 amino acids. Below acid. 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 (Sequence Number 9) Ser-Gly-Gly-Gly (Sequence Number 10) Gly-Gly-Gly-Gly-Ser (Sequence Number 11) Ser-Gly-Gly-Gly-Gly (Sequence Number 12) Gly-Gly-Gly-Gly-Gly-Ser (Sequence Number 13) Ser-Gly-Gly-Gly-Gly-Gly (Sequence Number 14) Gly- Gly-Gly-Gly-Gly-Gly-Ser (Sequence Number 15) Ser-Gly-Gly-Gly-Gly-Gly-Gly (Sequence Number 16) (Gly-Gly-Gly-Gly-Ser) _n (Ser-Gly -Gly-Gly-Gly) _n Gly-Lys-Pro-Gly-Ser (Serial Number 17) (Gly-Lys-Pro-Gly-Ser) _n The above n represents an integer of 1 or more. The length or sequence of the peptide linker can be appropriately selected according to the purpose by those with ordinary knowledge in the art.

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

In one embodiment, the anti-CD3 scFv region is composed of the amino acid sequence of amino acid numbers 1 to 254 of SEQ ID NO: 8.

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes: a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, and The anti-TSPAN8 antibody of the heavy chain variable region of CDR3 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4 and the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment with the first Fc polypeptide; contains a CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, consisting of SEQ ID NO: 6 Anti-TSPAN8 of the light chain variable region of the CDR3 consisting of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6 and the CDR3 consisting of the amino acid sequence of amino acid numbers 89 to 96 of SEQ ID NO: 6 The light chain of an antibody, and a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8 CDR2, and the heavy chain variable region of the anti-CD3 antibody of the CDR3 consisting of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8, and the heavy chain variable region of the anti-CD3 antibody consisting of amino acid numbers 168 to 181 of SEQ ID NO: 8 CDR1 composed of the amino acid sequence of SEQ ID NO: 8, CDR2 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8, and CDR2 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody constituting CDR3 with the second Fc polypeptide.

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes: an antibody containing a heavy chain variable region composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4. The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the TSPAN8 antibody to the first Fc polypeptide contains a light chain variable consisting of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6 The light chain of the anti-TSPAN8 antibody in the region, and the heavy chain variable region of the anti-CD3 antibody consisting of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the heavy chain variable region of the amino acid sequence of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of an anti-CD3 antibody and the second Fc polypeptide consisting of amino acid sequences numbered 146 to 254.

In the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, the heavy chain constant region from which the first Fc polypeptide and the second Fc polypeptide constituting the Fc region are derived, whether Igγ, Igμ, Igα, Igδ or Igε Any constant area can be selected. Igγ can be selected from, for example, 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.

The Fc region of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may also include mutations that reduce antibody-dependent cytotoxicity (ADCC) or complement-dependent toxicity (CDC). L234A refers to the replacement of leucine with alanine at position 234 of the amino acid in the constant region of human Igγ1 that complies with EU guidelines. L235A refers to the replacement of leucine with alanine at position 235 of the amino acid in the constant region of human Igγ1 that complies with EU guidelines. The amino acid variations in the human Igγ1 constant region L234A and L235A are called "LALA mutations". This mutation is known to reduce the antibody-dependent cytotoxic activity or complement-dependent toxic activity of the antibody (Mol. Immunol., 1992; Vol. 29: p. 633-639, J. Immunol., 2000; Vol. 164: p .4178-4184).

The Fc region of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may further include mutations based on other known technologies. For example, the Fc region may also include the N297G mutation in the human Igγ1 constant region that complies with EU guidelines (Protein cell, 2018; Vol.9: p.63-73) or the mutation based on Knobs into holes technology (hereinafter also referred to as "Knobs into holes"). variation"). Knobs into holes technology is to replace the amino acid side chains present in the CH3 region of one heavy chain with larger side chains (knobs; protrusions), and replace the amino acid side chains present in the CH3 region of the other heavy chain. The amino acid side chain is replaced with a smaller side chain (hole; gap), and the protrusions are arranged in the gap to promote the heterodimerization of the heavy chain, and the target heterodimer can be obtained efficiently Technology of oxidizing antibody molecules (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).

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an Fc region containing a LALA mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an Fc region containing the N297G mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an Fc region containing Knobs into holes mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an Fc region containing one or more mutations selected from the group consisting of LALA mutation, N297G mutation, and Knobs into holes mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes an Fc region containing LALA mutation, N297G mutation, and 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 an Fc polypeptide that forms the Fc region, and the Knobs into holes mutation that forms the Fc region. T366S, L368A and Y407V mutations in another Fc polypeptide (refer to International Publication No. 1998/050431).

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes: a first Fc polypeptide composed of the amino acid sequence of amino acid numbers 235 to 451 of SEQ ID NO: 4 and The Fc region is composed of the second Fc polypeptide composed of the amino acid sequence of amino acid numbers 270 to 486 of SEQ ID NO: 8.

In addition, in this specification, the description of amino acid mutations such as LALA mutation, N297G mutation, Knobs into holes mutation, etc. are based on the amino acid positions in the human Igγ1 constant region that comply with EU guidelines. For example, as mentioned above, L234A refers to the replacement of leucine with alanine at position 234 of the amino acid in the constant region of human Igγ1 that complies with EU guidelines.

In the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, the "heavy chain of the anti-TSPAN8 antibody" means the heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and the Fc polypeptide (the first Fc polypeptide). Polypeptides obtained by linking polypeptides) also include polypeptides obtained by linking a heavy chain fragment containing the heavy chain variable region of an anti-TSPAN8 antibody to a first Fc polypeptide through a hinge region. Furthermore, in the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, the anti-CD3 scFv region and the Fc polypeptide (second Fc polypeptide) are linked, especially through the hinge region.

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a first Fc polypeptide linked through a hinge region. Heavy chain of anti-TSPAN8 antibody. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes a polypeptide in which an anti-CD3 scFv region and a second Fc polypeptide are linked through a hinge region. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a first Fc polypeptide linked through a hinge region. The heavy chain of the anti-TSPAN8 antibody, the light chain of the anti-TSPAN8 antibody, and the polypeptide obtained by linking the anti-CD3scFv region and the second Fc polypeptide through the hinge region.

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes: a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4; Anti-TSPAN8 antibody of the heavy chain variable region of CDR3 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4 and consisting of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment and the first Fc polypeptide through the hinge region; contains CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and the light chain variable region of CDR3 composed of the amino acid sequence of amino acid numbers 89 to 96 of SEQ ID NO: 6 The light chain of the anti-TSPAN8 antibody, and a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8 The CDR2 constituted, and the heavy chain variable region of the anti-CD3 antibody of the CDR3 composed of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8, and the heavy chain variable region of the anti-CD3 antibody consisting of amino acid number 168 of SEQ ID NO: 8 CDR1 composed of the amino acid sequence of SEQ ID NO: 8 to 181, CDR2 composed of the amino acid sequence of SEQ ID NO: 8 from 197 to 203, and CDR2 composed of the amino acid sequence of SEQ ID NO: 8 from 236 to 244. A polypeptide obtained by linking the anti-CD3scFv region of the light chain variable region of the anti-CD3 antibody of the CDR3 acid sequence to the second Fc polypeptide through the hinge region.

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes: an antibody containing a heavy chain variable region composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4. The heavy chain of the anti-TSPAN8 antibody, which is obtained by linking the heavy chain fragment of the TSPAN8 antibody to the first Fc polypeptide through a hinge region, contains a light chain consisting of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. The light chain of the anti-TSPAN8 antibody in the chain variable region, and the heavy chain variable region of the anti-CD3 antibody consisting of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the heavy chain variable region of the anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody and the second Fc polypeptide through the hinge region, consisting of the amino acid sequence of amino acid numbers 146 to 254.

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is composed of: the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, and the heavy chain of the amino acid sequence of SEQ ID NO: 6. The bispecific antibody consists of the light chain of the anti-TSPAN8 antibody composed of the sequence, and the polypeptide obtained by linking the anti-CD3 scFv region composed of the amino acid sequence of Sequence No. 8 and the second Fc polypeptide.

In this specification, "post-translational modification" means that the antibody is modified after translation when the antibody is expressed in cells. Examples of post-translational modifications include modifications such as pyroglutamylation, glycosylation, oxidation, deamidation, and glycation of glutamine or glutamic acid at the N-terminal end of the heavy chain, or The deletion of lysine is caused by cleaving the lysine at the C-terminus of the heavy chain with carboxypeptidase. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci., 2008; Vol. 97: p. 2426-2447).

In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention can also be post-translationally modified. In one embodiment, the post-translational modification is pyroglutamine chelation 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 modifications such as N-terminal pyroglutamine chelation or C-terminal lysine deletion will not affect the activity of antibodies (Analytical Biochemistry, 2006; Vol. 348: p. 24- 39).

The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention binds to human TSPAN8 (gene number: NM_004616.2) and human CD3εδ complex protein (CD3ε gene number: NM_000733.3, CD3δ gene number: NM_000732.4 or NM_001040651. 1). Whether it binds to human TSPAN8 and human CD3εδ complex protein can be confirmed using a known binding activity measurement method. Examples of methods for measuring binding activity include Enzyme-Linked Immuno Sorbent Assay (ELISA), flow cytometric analysis, and the like.

The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention can be based on the heavy chain variable region and light chain variable region of the anti-TSPAN8 antibody and anti-CD3 scFv region disclosed in this specification, as long as those with ordinary knowledge in the technical field The sequence information of the region and the like are produced by methods known in this field. In addition, the anti-CD3 scFv region of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention can be based on the heavy chain variable region and light chain variable region of known anti-CD3 antibodies as long as those with ordinary knowledge in the technical field The sequence information, etc., are produced by methods known in this field. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is a humanized antibody or a human antibody. When producing humanized antibodies, methods well known to those with ordinary knowledge in the technical field can also be used to appropriately introduce reverse mutations (Bioinformatics, 2015; Vol. 31: p. 434-435). The anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is not particularly limited, and can be produced according to the method described in International Publication No. 2022/102768, for example. Method for producing an anti-TSPAN8-anti-CD3 bispecific antibody described in International Publication No. 2022/102768 (including <polynucleotide of the bispecific antibody of the present invention>, <expression vector of the bispecific antibody of the present invention>, <The transformed host cell of the present invention>, <The method of producing bispecific antibodies of the present invention> and Examples (but not limited thereto) are incorporated by reference in this specification.

<Pharmaceutical composition of the present invention> The present invention provides a pharmaceutical composition (pharmaceutical composition of the present invention) containing an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signaling inhibitor for treating cancer. The pharmaceutical composition of the present invention is a pharmaceutical composition containing the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention for treating cancer of a subject, and is used in combination with a PD-1 signaling inhibitor. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition used in a method for treating cancer in a subject. The treatment method includes administering the pharmaceutical composition and a PD-1 signaling inhibitor to the subject. . The pharmaceutical composition of the present invention is produced using the aforementioned anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, and contains the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and pharmaceutically acceptable excipients. The anti-TSPAN8-anti-CD3 bispecific antibody contained in the pharmaceutical composition of the present invention may have the molecular structure and peptide linker described in the item "Anti-TSPAN8-anti-CD3 bispecific antibody of the present invention", Mutation, post-translational modification, etc. The pharmaceutical composition of the present invention can be prepared by a commonly used method using excipients commonly used in this field, that is, pharmaceutical excipients or pharmaceutical carriers. Examples of dosage forms of such pharmaceutical compositions include parenteral preparations such as injections and intravenous drips, and can be administered by intravenous administration, subcutaneous administration, intraperitoneal administration, etc. During formulation, excipients, carriers, additives, etc. that are suitable for these dosage forms can be used within the pharmaceutically acceptable range.

The pharmaceutical composition of the present invention may contain a post-translationally modified form of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention. For example, pharmaceutical compositions containing antibodies or the like that have been subjected to deletion of C-terminal lysine or N-terminal pyroglutamine chelation are also included in the pharmaceutical compositions of the present invention.

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 and/or a post-translationally modified form of the antibody described below: A bispecific antibody comprising: a CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, and an amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4 The heavy chain fragment of the anti-TSPAN8 antibody of the heavy chain variable region of the CDR2 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 is linked to the first Fc polypeptide. The resulting heavy chain of the anti-TSPAN8 antibody contains CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, and amino acids composed of amino acid numbers 50 to 56 of SEQ ID NO: 6 The CDR2 composed of the sequence, and the light chain of the anti-TSPAN8 antibody of the light chain variable region of the CDR3 composed of the amino acid sequence of amino acid numbers 89 to 96 of SEQ ID NO: 6, and the light chain of the anti-TSPAN8 antibody containing the amino acid sequence of SEQ ID NO: 8 CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8, and CDR2 consisting of amino acid number 101 of SEQ ID NO: 8 The heavy chain variable region of the anti-CD3 antibody of the CDR3 composed of the amino acid sequence of SEQ ID NO: 8 to 114, and the CDR1 composed of the amino acid sequence of SEQ ID NO: 8 from 168 to 181, SEQ ID NO: 8 The light chain variable region of the anti-CD3 antibody of the CDR2 composed of the amino acid sequence of amino acid numbers 197 to 203, and the CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region to a second Fc polypeptide.

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 and/or a post-translationally modified form of the antibody described below: A bispecific antibody comprising: a heavy chain fragment of an anti-TSPAN8 antibody containing a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4 and a first Fc polypeptide The heavy chain of the anti-TSPAN8 antibody obtained by concatenation, the light chain of the anti-TSPAN8 antibody containing the light chain variable region consisting of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6, and the light chain of the anti-TSPAN8 antibody containing the sequence The heavy chain variable region composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8 and the light chain variable region composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8 A polypeptide obtained by linking the anti-CD3 scFv region to a second Fc polypeptide.

In one embodiment, the pharmaceutical composition of the present invention contains a heavy chain of an anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, and an anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6. The light chain of the antibody, and the anti-TSPAN8-anti-CD3 bispecific antibody and/or the polypeptide obtained by linking the anti-CD3 scFv region composed of the amino acid sequence of SEQ ID NO: 8 and the second Fc polypeptide. Pharmaceutical compositions of post-translationally modified antibodies.

When the pharmaceutical composition of the present invention is formulated, the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention added depends on the degree of symptoms or age of the patient, the dosage form of the preparation used, or the binding capacity of the antibody. It varies. For example, based on the dosage for human administration, about 0.0001 mg/kg to 1000 mg/kg of anti-TSPAN8-anti-CD3 bispecific antibody can be used in the preparation. In one embodiment, the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention added during formulation is in the range of 0.0001 mg/kg to 1000 mg/kg in terms of human dosage. In one embodiment, the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention added during formulation is in the range of 0.001 mg/kg to 100 mg/kg in terms of the dosage converted to human administration. In one embodiment, the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention added during formulation is in the range of 0.01 mg/kg to 10 mg/kg based on the dosage converted to human administration. In one embodiment, the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention added during formulation is preferably in the range of 0.01 mg/kg to 10 mg/kg based on the dosage converted to human administration. .

＜PD-1 signaling inhibitor＞ In the present invention, the PD-1 signaling 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 in order to treat the target cancer.

The mechanism of action and treatment modality of the PD-1 signaling inhibitor are not particularly limited as long as they block PD-1 signaling. As an action mechanism, for example, it can be an action mechanism that blocks the binding between molecules related to the PD-1 message, reduces the expression amount of the PD-1 message molecule (for example, hinders the production of proteins or induces decomposition, etc.). As therapeutic tools, they may be, for example, antibodies, low molecular compounds, nucleic acids (which may include DNA or RNA, natural or artificial nucleic acids), fusion proteins, peptides, and other therapeutic tools.

PD-1 signaling inhibitors can be measured by measuring the binding inhibitory effect of two proteins, PD-1 and PD-L1 or PD-L2, and the performance reducing effect using the expression amount of PD-1 signaling molecules as an indicator. to obtain. For example, an inhibitor that binds to PD-1 and PD-L1 or PD-L2 can be used to inhibit PD-1 and PD after obtaining an inhibitor that binds to either PD-1 and PD-L1 or PD-L2. The resulting inhibitors are screened for their ability to bind to -L1 or PD-L2. Inhibitors can be bound to proteins by using, for example, flow cytometry (FCM), ELISA, surface plasmon resonance (SPR), thermal shift analysis (TSA), isothermal titration calorimetry (ITC), etc. It shall be evaluated by methods well known to those with ordinary knowledge in the technical field. Also, for example, inhibitors that reduce the expression of PD-1 signaling molecules such as PD-1, PD-L1 or PD-L2 can be based on the protein levels of PD-1, PD-L1 or PD-L2 in cells. To obtain the indicator. The blocking effect of PD-1 message can be confirmed by the effects of interferon-γ release on T cell proliferation and reporter gene analysis. The effect of an inhibitor that reduces 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.

PD-1 signaling inhibitors include, for example, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, and other antibodies that block PD-1 signaling. Such antibodies can be humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antigen-binding fragments thereof. Known anti-PD-1 antibodies are not limited, and include, for example, U.S. Patent No. 8008449, U.S. Patent No. 6808710, U.S. Patent No. 7488802, U.S. Patent No. 8168757, and U.S. Patent No. 8354509, and International Publication No. 2006/ Antibodies described in No. 121168 and International Publication No. 2012/145493. Known anti-PD-L1 antibodies are not limited, and include, for example, 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. 8217149. No. recorded antibodies. In one embodiment, the PD-1 signaling inhibitor used in the present invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an antigen-binding fragment thereof. In one embodiment, the PD-1 signaling 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 can be an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, spatalizumab, cimepilimab, etc. 1 antibody. In one embodiment, the anti-PD-L1 antibody may be an anti-PD-L1 antibody such as atezolizumab, durvalumab, avelumab, or the like.

Examples of PD-1 signaling inhibitors include AMP-224 (International Publication No. 2010/027827 and International Publication No. 2011/066342) and BMS-1166 (Oncotarget, 2017; Vol.8: p.72167-72181 ) and other fusion proteins or low molecular compounds that hinder the binding of PD-1 to PD-L1. Various PD-1 signaling inhibitors are known in this technical field (Non-Patent Document 8).

In one embodiment of the present invention, the anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signaling inhibitor of the present invention can be (i) contained in the same pharmaceutical composition and administered to the subject at the same time, or they can also be administered to the subject at the same time. Can (ii) be contained in different pharmaceutical compositions and administered to the subject simultaneously, continuously or sequentially.

In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating cancer of a subject, and contains the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signaling inhibitor. In one embodiment, the pharmaceutical composition of the present invention is used in combination with another pharmaceutical composition containing a PD-1 signaling inhibitor. In one embodiment, the pharmaceutical composition of the present invention is a combination of pharmaceutical compositions for treating cancer of the subject (also referred to as the "combination medicine of the present invention" in this specification), and the combination includes the pharmaceutical composition of the present invention. Pharmaceutical compositions of anti-TSPAN8-anti-CD3 bispecific antibodies and pharmaceutical compositions containing PD-1 signaling inhibitors. In one embodiment, the combination medicine of the present invention is a combination of pharmaceutical compositions used in a method of treating cancer in a subject. The treatment method includes combining a medicine containing the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention. The composition and a pharmaceutical composition containing a PD-1 signaling inhibitor are administered to the subject.

<Bispecific antibodies for combined use of the present invention and uses of the bispecific antibodies of the present invention> Furthermore, the present invention provides an anti-TSPAN8-anti-CD3 bispecific antibody (bispecific antibody used in combination in the present invention) used in combination with a PD-1 signaling inhibitor for treating cancer. In one embodiment, the bispecific antibody used in combination in the present invention is an anti-TSPAN8-anti-CD3 bispecific antibody used in a method for treating cancer in a subject, and the treatment method includes using the bispecific antibody. A PD-1 signaling inhibitor is administered to the subject. Furthermore, the present invention provides the use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition (the bispecific antibody of the present invention) to be used in combination with a PD-1 signaling inhibitor for the treatment of cancer. uses of antibodies). The use of the bispecific antibody of the present invention is the use of an anti-TSPAN8-anti-CD3 bispecific antibody, which is used to manufacture a pharmaceutical composition for treating cancer of a subject. The pharmaceutical composition is for treating cancer of the subject. cancer, and used in combination with PD-1 signaling inhibitors. In one embodiment, the use of the bispecific antibody of the present invention is the use of an anti-TSPAN8-anti-CD3 bispecific antibody for manufacturing a pharmaceutical composition for use in a method of treating cancer in a subject, the The treatment method includes administering the pharmaceutical composition and the PD-1 signaling inhibitor to the subject. The anti-TSPAN8-anti-CD3 bispecific antibody used in the combined use of the bispecific antibody of the present invention and the use of the bispecific antibody of the present invention may have the aforementioned <Anti-TSPAN8-anti-CD3 bispecific antibody of the present invention ＞Molecular structure, peptide linker, mutation, post-translational modification, etc. recorded in the project.

＜The treatment method of the present invention＞ The present invention provides a therapeutic method comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject's cancer (the therapeutic method of the present invention). The anti-TSPAN8-anti-CD3 bispecific antibody used in the treatment method of the present invention may have the molecular structure, peptide linkers, mutations, and Post-translation modification, etc.

In one embodiment, in the treatment method of the present invention, the anti-TSPAN8-anti-CD3 bispecific antibody system of the present invention and the PD-1 signaling inhibitor are administered to the subject simultaneously, continuously, or sequentially.

In one embodiment, in the treatment method of the present invention, the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor of the present invention are (i) contained in the same pharmaceutical composition and administered to the patient at the same time. The subject, or (ii) contained in different pharmaceutical compositions and administered to the subject simultaneously, continuously or sequentially.

In one embodiment, in the treatment method of the present invention, the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor of the present invention are (i) contained in the same pharmaceutical composition and administered to the patient at the same time. subject, or (ii) contained in different pharmaceutical compositions and administered to the subject on the same day.

In one embodiment, in the treatment method of the present invention, (a) after the administration of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention to the subject is completed, administration of a PD-1 signaling inhibitor is started, or (b) ) After the administration of the PD-1 signaling inhibitor to the subject is completed, administration of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is started.

In one embodiment, the treatment method of the present invention is to administer the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and the PD-1 signaling inhibitor to a subject according to an administration plan including administration cycles. Use it sequentially. In one embodiment, the treatment method of the present invention starts administration of the anti-TSPAN8-anti-CD3 bispecific antibody to the subject in at least one administration cycle or all administration cycles, and then starts PD- 1Administration of message inhibitors. In one embodiment, the treatment method of the present invention starts administering the PD-1 signaling inhibitor to the subject in at least one administration cycle or all administration cycles, and then starts anti-TSPAN8-anti-CD3 Administration of bispecific antibodies or pharmaceutical compositions.

＜Therapeutic use＞ The cancer treated by the pharmaceutical composition or treatment method of the present invention may be either solid cancer or blood cancer. The cancer treated by the present invention can be any of primary, metastatic or peritoneal dissemination. The cancer treated by the present invention is not particularly limited, and examples include gastric cancer, lung cancer; acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. , B-cell lymphoma, multiple myeloma, T-cell lymphoma and other blood cancers; myelodysplasia syndrome, adenocarcinoma, squamous cell carcinoma, adenocarcinoma, 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, neck cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, gallbladder cancer, Solid cancers such as cholangiocarcinoma, biliary tract cancer, kidney cancer, pancreatic cancer, colon cancer, large intestine cancer, rectal cancer, small intestine cancer, gastroesophageal junction cancer, esophageal cancer, testicular cancer, ovarian cancer, brain tumors, etc., as well as bone Cancers of tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue, as well as sarcomas such as chondrosarcoma, Evans sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, etc., or glioblastoma glioblastoma multiforme, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatic blastoma, pleuropulmonary blastoma, omentoblastoma, etc. blastoma etc.

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, esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, and gastroesophageal junction cancer are preferred.

The amount of the anti-TSPAN8-anti-CD3 bispecific antibody or PD-1 signaling inhibitor of the present invention administered to the subject depends on the degree of symptoms or age of the subject, the dosage form of the antibody, pharmaceutical composition, inhibitor, etc. used , or the activity intensity of the active ingredients varies. For example, about 0.0001mg/kg~1000mg/kg can be used. In one embodiment, the dosage of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention administered to the 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 the 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 the subject is 0.01 mg/kg to 10 mg/kg.

Specific examples are provided here for reference in order to better understand the present invention, but these are for the purpose of illustration and do not limit the present invention. [Example]

[Example 1: Preparation of anti-TSPAN8-anti-CD3 bispecific antibody] [Example 1-1: Preparation of bispecific antibody vector for anti-TSPAN8 antibody] In order to use the bispecific antibody as an anti-TSPAN8 antibody, a mutation was introduced into the heavy chain (16B11.1_HC) of 16B11.1 of SEQ ID NO: 2 to produce 16B11.1_HC_H described in SEQ ID NO: 4. Specifically, the following three mutations were introduced into sequence number 2. (1) LALA mutations (L234A and L235A) in which leucine (L) is replaced by alanine (A) in amino acid numbers 238 and 239 (EU indicators: 234 and 235), (2) Amino acid number 370, The amino acids of 372 and 411 (EU indicators: 366, 368 and 407) are respectively replaced by threonine (T) to serine (S), L to A, and tyrosine (Y) to valerine. Knobs into holes (Knobs into holes) mutation of amino acid (V), (3) amino acid number 301 (EU index: 297) is replaced by asparagine (N) to glycine (G) of variation. The designed heavy chain amino acid sequence of 16B11.1_HC_H is shown in SEQ ID NO: 4. The polynucleotide encoding 16B11.1_HC_H of sequence number 4 was introduced into the pcDNA3.4-TOPO vector (Thermo Fisher scientific company). The vector produced was called pcDNA3.4-16B11.1_HC_H. In addition, a polypeptide (sequence) designed with the amino acid sequence of the constant region of the human kappa chain (sequence of amino acid numbers 108 to 213 of SEQ ID NO. 6) linked to the C-terminus of the light chain variable region of 16B11.1 No. 6). The polynucleotide encoding the designed polypeptide was introduced into the pcDNA3.4 TOPO vector. The light chain vector produced was called pcDNA3.4-16B11_LC.

[Example 1-2: Preparation of bispecific antibody vector for anti-human CD3 antibody] 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, a method described in the literature (Front Biosci., 2008; Vol. 13: p. 1619-1633) Design the sequence of humanized anti-CD3 antibodies. Use the integrated computational chemistry system MOE provided by MOLSIS Inc. to analyze the three-dimensional structure information (PDB Code: 5FCS) and introduce back mutations into the framework region. The anti-CD3scFv-Fc was designed to sequentially configure the heavy chain variable region (SEQ ID NO: 8, amino acid number 1 to 125), the linker (SEQ ID NO: 8, amino acid number 126 to 145), and the light chain variable region (SEQ ID NO: 8 amino acid number 146 to 254), hinge (SEQ ID NO: 8 amino acid number 255 to 269), CH2 domain (SEQ ID NO: 8 amino acid number 270 to 379) and CH3 domain (SEQ ID NO: 8 amino acid group acid numbers 380 to 486). Furthermore, for Sequence No. 8, (1) a mutation was introduced in which the amino acid corresponding to amino acid No. 44 and amino acid No. 247 was replaced with cysteine (C), (2) the amino acid No. The mutation in which the amino acid number 259 (EU index: 220) is replaced from C to S, (3) the LALA mutation in which the amino acid number 273 and 274 (EU index: 234 and 235) is replaced from L to A , (4) Knobs into holes mutation of amino acid number 405 (EU index: 366) substituted from T to tryptophan (W), (5) Amino acid number 336 (EU index: 297) ) amino acid is substituted from N to G. In order to introduce these mutations, polynucleotides encoding amino acid sequences containing respective mutation points were synthesized and inserted into the pcDNA3.1(+) vector (Thermo Fisher Scientific, V79020). The vector produced was called pcDNA3.1-m7_scFV_K. The base 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.

[Example 1-3: Preparation of anti-TSPAN8-anti-CD3 bispecific antibody] In order to produce a bispecific antibody composed of the Fab region, the anti-CD3scFv region and the Fc region of the anti-TSPAN8 antibody, pcDNA3.4-16B11.1_HC_H, pcDNA3.4-16B11_LC, and pcDNA3.1-m7_scFV_K were subjected to ExpiCHO in accordance with conventional methods. -S cells (Thermo Fischer Scientific, A29127) transfection. The culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02), and further used a gel filtration column HiLoad (registered trademark) 26/600 Superdex (registered trademark) 200pg (Cytiva, 28-9893-36 ) purification to obtain purified antibodies with a purity of more than 95%. The resulting antibody was called anti-TSPAN8(16B11)-anti-CD3 bispecific antibody.

[Example 2: In vitro effects of combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody] [Example 2-1: Preparation of Expanded PanT cells] will be RPMI 1640 (Thermo Fisher Scientific, 11875-119) was added with a final concentration of 10% FBS (Cytiva, SH30084.03), 1% penicillin/streptomycin (Thermo Fisher Scientific, 15070-063), 1% MEM non-essential amino acids (Merck, M7145), 1% sodium pyruvate (Merck, S8636), 1% GlutaMAX I (Thermo Fisher Scientific, 35050-061), 1% HEPES ( Thermo Fisher Scientific, 15630-080) is called "effector medium". Anti-CD3 antibody (BioLegend, 317315) diluted with phosphate buffer saline (PBS) to a final concentration of 3 μg/mL was added to a 24-well tissue culture microplate (IWAKI, 3820-024) (hereinafter referred to as "24-well plate"). "), the anti-CD3 antibody is solid-phased. PanT cell isolation kit-human (Miltenyi Biotec, 130-096-535) was used, and PanT was isolated from human peripheral blood mononuclear cells (LONZA, CC-2702) according to the manufacturer's experimental instructions. Cells (including both CD4 T cells and CD8 T cells, hereinafter referred to as "PanT cells") are isolated. The isolated PanT cells were centrifuged, the supernatant was removed, and then suspended in the effector medium. In the aforementioned 24-well plate obtained by solidifying the anti-CD3 antibody, the suspended PanT cells were seeded at 1.5x10 ⁶ cells/well. Human IL-2 (PeproTech, 200-02) at a final concentration of 10 ng/mL and anti-CD28 antibody (BioLegend, 302923) at a final concentration of 1 μg/mL were further added, and the total amount was adjusted to 1 mL/well with effector medium. Cultivate in a 37°C, 5% _CO2 incubator. PanT cells were recovered 2 days later, and the recovered PanT cells were suspended in the effector medium and seeded in 6 wells of a tissue culture microplate (IWAKI, 3810-006) (hereinafter referred to as "6-well plate"). Human IL-2 at a final concentration of 10 ng/mL was further added, and cultured in a 37°C, 5% CO ₂ incubator. Human IL-2 with a final concentration of 10ng/mL was added every 2 or 3 days and passage was repeated. Ten days after the start of culture, all cells were recovered, centrifuged, and the supernatant was removed, resuspended in Bambanker (GC Lymphotec, CS-02-001), aliquoted into tubes, and stored frozen at -80°C. . The cryopreserved PanT cells are referred to as “Expanded PanT cells” in this specification.

[Example 2-2: Preparation of RKO cells stably expressing human TSPAN8-GFP] RKO cells of human colorectal cancer cell lines were obtained from American Type Culture Collection (ATCC, CRL-2577). RKO cells were cultured in RPMI-1640 medium (Merck, R8758) containing 10% FBS and 1% penicillin/streptomycin (hereinafter referred to as "medium") at 37°C and 5% CO. cultured under conditions ₂ . In order to produce cells expressing the fusion protein of TSPAN8 and GFP, TSPAN8 (Myc-DDK-tagged)-human tetraspanin 8 (TSPAN8) (ORIGENE, RC202694) was cleaved using restriction enzymes and encoded human The polynucleotide of the polypeptide of TSPAN8 was cloned into pCMV6-AC-GFP vector (ORIGENE Company, PS100010) (hereinafter referred to as "human TSPAN8-GFP expression vector"). The prepared human TSPAN8-GFP expression vector was used to perform lipid transfection on RKO cells using Lipofectamine 3000 (Thermo Fisher Scientific, L3000008) according to the experimental guidelines recommended by the manufacturer. Selective culture was performed in a medium supplemented with Geneticin selective antibiotic (Thermo Fisher Scientific, 10131-027) at a final concentration of 600 μg/mL. Cells were separated using MACS Cell Separation (Miltenyi Biotec) to obtain an RKO cell line that stably expresses human TSPAN8-GFP (hereinafter referred to as "RKO_hTSPAN8-GFP cells").

[Example 2-3: Combination effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody on in vitro cancer cell proliferation inhibition] Using RKO_hTSPAN8-GFP cells The co-culture system with Expanded PanT cells was used to evaluate the combined effect of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody on cancer cell proliferation. In a half area 96-well microculture plate (Greiner Bio-One, 675090) (hereinafter referred to as "half area culture plate"), 10 ⁴ cells/well of RKO_hTSPAN8-GFP cells prepared in the culture medium were used. Sow seeds and culture in a 37°C, 5% _CO2 incubator. After 3 hours, the Expanded PanT cells prepared in the culture medium were seeded at 3×10 ⁴ cells/well in a half-mass culture plate. Use the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody obtained in Examples 1-3, and the anti-human PD-1 antibody nivolumab, pembrolizumab or anti-human PD-L1 antibody. Tecilizumab analogs and durvalumab analogs were used as test antibodies. Nivolumab is designed based on the amino acid sequences of the full-length heavy chain and full-length light chain of nivolumab (sequence numbers 17 and 18) recorded in International Publication No. 2014/055648. Pembrolizumab is designed based on the amino acid sequences of the complete heavy chain and the complete light chain of pembrolizumab (Sequence Numbers 31 and 36) recorded in International Publication No. 2008/156712. From the designed sequence, nivolumab and pembrolizumab were obtained according to the method described in Example 11 of International Publication No. 2021/241616. As the isotype control group (isotype control) of nivolumab and pembrolizumab, RecombiMAb human IgG4 (S228P) isotype control group, anti-egg lysozyme (Bio X Cell Company, BE0349) was used. As atezolizumab analogs, durvalumab analogs and their isotype control groups, human PD-L1/B7-H1 (Research Grade Atezolizumab Biosimilar) antibodies (R & D Systems, Inc., MAB10348), human PD-L1/B7-H1 (Research Grade Durvalumab Biosimilar) antibody (R & D systems, MAB10355) and Ultra-LEAF purified human IgG1 isotype control recombinant antibody (BioLegend, 403502). Using the culture medium, the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody was prepared at a final concentration of 0 or 10 μg/mL, and the isotype control group, nivolumab, pembrolizumab, and A were prepared at a final concentration of 1 μg/mL. Tecilizumab analog or durvalumab analog, added to a half volume culture plate. After addition, culture the half-volume culture plate in a 37°C, 5% _CO2 incubator. Three days later, a part of the supernatant was recovered and stored at -80°C for measurement of interferon-γ production. The suspended cells were collected in PP culture plate 96V (Sumitomo Bakelite Co., Ltd., MS-3396P) (hereinafter referred to as "V-bottom micro culture plate") in PBS containing 10% FBS, and the adherent cells remaining in the half volume culture plate were After fixing with BD Phosflow Fix Buffer I (Becton, Dickinson and Company, 557870), the fluorescence intensity of GFP was measured with Infinite M200PRO (TECAN), and the background-subtracted value was used as an indicator of cell proliferation. The results of four independent tests are shown in Figures 1 and 2. As shown in Figure 1 and Figure 2, anti-TSPAN8(16B11)-anti-CD3 bispecific antibody inhibits the proliferation of RKO_hTSPAN8-GFP cells in the co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells. The inhibitory effect on RKO_hTSPAN8-GFP cell proliferation caused by the combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab is greater than that of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody. A single dose of antibody, nivolumab, or pembrolizumab was more potent (Figure 1). Similarly, the inhibitory effect on RKO_hTSPAN8-GFP cell proliferation caused by the combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and atezolizumab analogs or durvalumab analogs was stronger than that of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody. 16B11)-anti-CD3 bispecific antibody, atezolizumab analog, or imrvalumab analog as a single dose (Fig. 2).

[Example 2-4. Effect of the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody for in vitro T cell activation] The suspended cells recovered from the V-bottom microculture plate in Example 2-3 were centrifuged at 540×g for 5 minutes, and the supernatant was removed and added to autoMACS electrophoresis buffer (Miltenyi Biotec, 130- 091-221) with 1/40 of human BD Fc Block (Becton, Dickinson and Company, 564220) added. Add 10 μL/well of PE anti-human CD4 antibody (BioLegend, 317410), APC-H7 mouse anti-human CD8 (Becton, Dickinson and Company, 560179), and BV421 small diluted in autoMACS electrophoresis buffer to each well. Mouse anti-human CD45 (Becton, Dickinson and Company, 563879), APC anti-human CD25 (BioLegend, 302610), let stand at 4°C for 1 hour. After washing once with autoMACS electrophoresis buffer, resuspend in autoMACS electrophoresis buffer containing 1/200 of 7-AAD solution (Becton, Dickinson and Company, 559925), and use CytoFLEX S (Beckman Coulter) to Flow cytometric analysis measures the binding of various antibodies. Data analysis was performed using FlowJo (Becton, Dickinson and Company). Analyze CD4 or CD8 positive cells that differentiate between CD45 positive (an indicator of immune cells) and 7-AAD negative (an indicator of viable cells). The expression amount of activation marker CD25 on CD4 or CD8 positive cells was expressed as the average fluorescence intensity of APC anti-human CD25 of fluorescently labeled antibodies against CD25. The results of three independent tests are shown in Figures 3-1, 3-2, 4-1, and 4-2. Anti-TSPAN8(16B11)-anti-CD3 bispecific antibody can induce CD4 T cells (Figure 3-1, Figure 4-1) and CD8 T cells (Figure 3-2) in the co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells. , Figure 4-2) CD25 performance. Changes in CD25 expression in CD4 T cells (Figure 3-1) and CD8 T cells (Figure 3-2) caused by the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab Performance was higher than a single dose of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody, nivolumab, or pembrolizumab. Similarly, the combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and atezolizumab analogs or durvalumab analogs resulted in CD4 T cells (Figure 4-1) and CD8 T cells (Figure 4-1). The performance of CD25 in 4-2) is higher than that of a single dose of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody, atezolizumab analog, or durvalumab analog. The results show that the combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody activates CD4 T cells and CD8 T cells more significantly than each agent alone.

[Example 2-5. Effect of the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody in vitro on interferon-γ production] For the supernatant recovered in Example 2-3, an AlphaLISA interferon-γ assay kit (Perkin Elmer, AL217F) was used to measure the amount of interferon-γ produced in the supernatant according to the experimental guidelines recommended by the manufacturer. Figure 5 shows the combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab. Figure 6 shows the combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and atezolizumab. The combined use of monoclonal antibody analogs or durvalumab analogs. As shown in Figures 5 and 6, by adding anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody, the production of interferon-γ was detected in the co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells. The combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab resulted in a higher production of interferon-γ than the anti-TSPAN8(16B11)-anti-CD3 bispecific antibody. antibody, nivolumab, or pembrolizumab as a single dose (Figure 5). Furthermore, the production of interferon-γ caused by a single dose of nivolumab or pembrolizumab in the absence of the anti-TSPAN8(16B11)-anti-CD3 bispecific antibody was below the detection limit. Similarly, coadministration of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody with atezolizumab analogs or durvalumab analogs resulted in higher levels of interferon-γ production than anti-TSPAN8(16B11)-anti-CD3 bispecific antibodies. 16B11)-anti-CD3 bispecific antibody, atezolizumab analog, or durvalumab analog single dose more (Figure 6). Furthermore, the production of interferon-γ caused by a single dose of atezolizumab analog or durvalumab analog in the absence of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody is the detection limit. the following. The results show that the combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody significantly promotes interference in T cells compared with each agent alone. Production of hormone-gamma.

[Example 3: In vivo effects of combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody] B-hCD3E mice (human CD3ε extracellular domain knock-in mice) transplanted with B16-F10 cells expressing human TSPAN8 were used to conduct experiments with anti-TSPAN8 (16B11)-anti-CD3 bispecific antibodies and anti-small cell Study on the in vivo anti-tumor effect of mouse PD-1 antibody.

[Example 3-1: Construction of human TSPAN8 expressing B16-F10 cells] The B16-F10 cells of the mouse melanoma cell line were obtained from the American Type Culture Collection (ATCC, CRL-6475). Use Dulbecco's modified Eagle medium (SIGMA, D6429) supplemented with a final concentration of 10% FBS and a final concentration of 1% penicillin/streptomycin (hereinafter the prepared medium is referred to as "Eagle medium") at 37°C, 5% Cultured under CO ₂ conditions. From TSPAN8 (Myc-DDK-tagged)-human tetraspanin 8 (TSPAN8) (ORIGENE Company, RC202694), polynucleotides encoding polypeptides containing human TSPAN8 were subclone into pMONO-blasti -mcs vector (InvivoGen, pmonob-mcs). The vector was introduced into B16-F10 cells by electroporation. Selective culture was performed using Eagle's medium supplemented with blasticidin (InvivoGen, ant-bl-1) at a final concentration of 10 μg/mL and using a FACS Aria II cell sorter (Becton, Dickinson and Company Company) to obtain stable B16-F10 cell lines expressing human TSPAN8 (B16-F10 cells expressing human TSPAN8).

[Example 3-2: Combination effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody on in vivo anti-tumor effect] B-hCD3E female mice (C57BL/6- CD3e ^{tm2 (CD3e)} /Bcgen; Biocytogen, 110008). B16-F10 cells expressing human TSPAN8 were suspended in Matrigel (registered trademark) Basement Membrane Matrix (Corning Inc., 356237) to prepare a cell suspension of 1×10 ⁷ cells/mL. The cell suspension was inoculated subcutaneously into the flanks of 7- to 8-week-old mice at 1×10 ⁶ cells/100 μL. Five days after cell inoculation, the tumor diameter was measured using a caliper (Mitutoyo, CD-15AXR). Tumor volume [mm ³ ] was calculated using the following formula. (Length of the long axis of the tumor [mm]) × (Length of the short axis of the tumor [mm]) ² × 0.5 The mice were divided into groups (n = 8) so that the tumor volume became equal in each group, and administration of the test drug began. The initial day of investment is defined as day 0. The following describes the details of the test chemicals administered to the four groups that were tested, the dosage, and the administration schedule. (1) Group 1: In the PBS administration group, PBS was intravenously administered at 10 mL/kg on the 0th and 7th days. (2) Group 2: Anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody is administered to the group on days 0 and 7. Anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody will be prepared at 0.1 mg/mL. Administer intravenously at 1 mg/kg. (3) Group 3: Anti-mouse PD-1 antibody administration group: On days 0, 3, 7, and 10, anti-mouse PD-1 antibody (Bio /kg for intraperitoneal administration. (4) Group 4: Combination administration of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody. On days 0 and 7, anti-TSPAN8 was prepared at 0.1 mg/mL. (16B11)-anti-CD3 bispecific antibody was administered intravenously at 1 mg/kg, and anti-mouse PD-1 antibody prepared at 1 mg/mL was administered intraperitoneally at 10 mg/kg on days 0, 3, 7, and 10. Internal investment. Tumor volume was evaluated on days 3, 6, 10, and 13 of each group (Figure 7-1). The 10th and 13th days of group 1, the 13th day of group 2 and group 3 are analyzed with n=7 (due to one death each). The tumor volume on day 13 of cohort 2 and cohort 3 was compared with the tumor volume on day 13 of cohort 4 by unpaired Student's t test (Figure 7-2). As shown in Figure 7-2, the tumor volume of the group treated with anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody was larger than that of the anti-TSPAN8(16B11)-anti-CD3 bispecific antibody alone. The tumor volume was significantly smaller in the group and the anti-mouse PD-1 antibody single-dose group. This result shows that in the treatment of human cancer, the combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody has the possibility of achieving higher efficacy than single-agent treatment. [Industrial utilization possibility]

The method of treating cancer by combining the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signaling inhibitor of the present invention is expected to be useful in the treatment of cancer. [Sequence listing non-keyword text]

SEQ ID NO: 2 is the amino acid sequence of the heavy chain of 16B11.1, and the base sequence shown in SEQ ID NO: 1 is the base 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 obtained by linking the heavy chain fragment containing the heavy chain variable region of 16B11.1 and the first Fc polypeptide. The base sequence shown in SEQ ID NO: 3 is The base sequence encoding the amino acid sequence of the polypeptide obtained by linking the heavy chain fragment containing the heavy chain variable region of 16B11.1 and the first Fc polypeptide shown in Sequence Number 4. The sequence of SEQ ID NO: 6 is the amino acid sequence of the light chain of 16B11.1, and the base sequence represented by SEQ ID NO: 5 is the base 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 obtained by linking the anti-CD3scFv region and the second Fc polypeptide. The base sequence shown in SEQ ID NO: 7 encodes the anti-CD3scFv region shown in SEQ ID NO: 8 and the amino acid sequence of the polypeptide. The base sequence of the amino acid sequence of the polypeptide obtained by linking the second Fc polypeptide. Sequence numbers 9 to 17 are the amino acid sequences of various linkers described in the detailed description of the invention.

[Figure 1] Figure 1 shows the inhibition of cancer cell proliferation in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells, anti-TSPAN8 (16B11)-anti-CD3 The effect of combined use of bispecific antibodies and anti-human PD-1 antibodies. The vertical axis of the figure represents the test antibody, and the horizontal axis represents the fluorescence intensity of GFP, which is an indicator of cell proliferation. Each bar represents the average fluorescence intensity. Error bars represent standard errors. [Figure 2] Figure 2 shows the inhibition of cancer cell proliferation in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells, anti-TSPAN8 (16B11)-anti-CD3 The effect of combined use of bispecific antibodies and anti-human PD-L1 antibodies. The vertical axis of the figure represents the test antibody, and the horizontal axis represents the fluorescence intensity of GFP, which is an indicator of cell proliferation. Each bar represents the average fluorescence intensity. Error bars represent standard errors. [Figure 3-1] Figure 3-1 shows anti-TSPAN8 (16B11)-anti-CD3 bispecificity in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells. Activation of CD4 T cells caused by the combined use of antibodies and anti-human PD-1 antibodies. The vertical axis of the figure represents the test antibody, and the horizontal axis represents the average fluorescence intensity of the fluorescently labeled antibody for the activation marker CD25 of CD4 T cells. Each bar represents the average of the average fluorescence intensity. Error bars represent standard errors. [Figure 3-2] Figure 3-2 shows anti-TSPAN8 (16B11)-anti-CD3 bispecificity in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells. Activation of CD8 T cells caused by the combined use of antibodies and anti-human PD-1 antibodies. The vertical axis of the figure represents the test antibody, and the horizontal axis represents the average fluorescence intensity of the fluorescently labeled antibody for the activation marker CD25 of CD8 T cells. Each bar represents the average of the average fluorescence intensity. Error bars represent standard errors. [Figure 4-1] Figure 4-1 shows anti-TSPAN8 (16B11)-anti-CD3 bispecificity in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells. Activation of CD4 T cells caused by the combined use of antibodies and anti-human PD-L1 antibodies. The vertical axis of the figure represents the test antibody, and the horizontal axis represents the average fluorescence intensity of the fluorescently labeled antibody for the activation marker CD25 of CD4 T cells. Each bar represents the average of the average fluorescence intensity. Error bars represent standard errors. [Figure 4-2] Figure 4-2 shows anti-TSPAN8 (16B11)-anti-CD3 bispecificity in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells. Activation of CD8 T cells caused by the combined use of antibodies and anti-human PD-L1 antibodies. The vertical axis of the figure represents the test antibody, and the horizontal axis represents the average fluorescence intensity of the fluorescently labeled antibody for the activation marker CD25 of CD8 T cells. Each bar represents the average of the average fluorescence intensity. Error bars represent standard errors. [Figure 5] Figure 5 shows the amount of interferon-γ produced in a co-culture system of RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells, anti-TSPAN8(16B11)- The effect of combined use of anti-CD3 bispecific antibody and anti-human PD-1 antibody. The vertical axis of the graph represents the test antibody, and the horizontal axis represents the average amount of interferon-γ produced. Error bars represent standard errors. [Figure 6] Figure 6 shows the amount of interferon-γ produced in a co-culture system of human RKO_hTSPAN8-GFP cells stably expressing TSPAN8-GFP in human colorectal cancer cell lines and Expanded PanT cells, anti-TSPAN8 (16B11) -The effect of combined use of anti-CD3 bispecific antibody and anti-human PD-L1 antibody. The vertical axis of the graph represents the test antibody, and the horizontal axis represents the average amount of interferon-γ produced. Error bars represent standard errors. [Figure 7-1] Figure 7-1 shows the tumor proliferation of B16-F10 cells expressing human TSPAN8 in the B-hCD3E mouse model as the average of the tumor volumes on each day after the start of antibody administration. In summary, the effect of combined use of anti-TSPAN8(16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody. Error bars represent the standard error of tumor volume. The vertical axis of the graph represents the tumor volume, and the horizontal axis represents the number of days since the start of administration of the test antibody. [Figure 7-2] Figure 7-2 shows the tumor volume of each individual 13 days after the start of administration. Regarding the tumor proliferation of human TSPAN8-expressing B16-F10 cells in the B-hCD3E mouse model, anti-TSPAN8 ( 16B11)-The combined effect of anti-CD3 bispecific antibody and anti-mouse PD-1 antibody. Error bars represent the standard error of tumor volume. The vertical axis of the figure represents the tumor volume, and the horizontal axis represents the tested antibody. Horizontal lines represent the mean and standard error. Significance value P value is determined by unpaired Student's t test and by comparing the tumor volume of the combined group with the group administered with a single dose of anti-TSPAN8(16B11)-anti-CD3 or the group administered with a single dose of anti-mouse PD-1 Tumor volume was compared. * and ** in the figure represent groups with P values less than the significant level of 0.05 and 0.01 respectively.

TW202404638A_112113569_SEQL.xml

Claims (29)

A pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody for treating cancer of a subject, This anti-TSPAN8-anti-CD3 bispecific antibody contains: (a) The Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment including the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising the heavy chain variable region of an anti-CD3 antibody and the light chain variable region of an 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 pharmaceutical composition is used in combination with a PD-1 signaling inhibitor. The pharmaceutical composition of claim 1, wherein the heavy chain variable region of the anti-TSPAN8 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, and the amino group of SEQ ID NO: 4 The CDR2 composed of the amino acid sequence of sequence number 50 to 66, and the CDR3 composed of the amino acid sequence of sequence number 4 from amino acid sequence 99 to 110; the light chain variable region of the anti-TSPAN8 antibody includes The CDR1 composed of the amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 6, the CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and the amine of SEQ ID NO: 6 CDR3 composed of amino acid sequences with amino acid numbers 89 to 96. For example, the pharmaceutical composition of claim 1 or 2, wherein the heavy chain variable region of the anti-TSPAN8 antibody is composed of the amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody The region is composed of the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the Fab region of the anti-TSPAN8 antibody is composed of: a heavy chain fragment composed of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 and It consists of a light chain composed of the amino acid sequence of SEQ ID NO: 6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the heavy chain variable region of the anti-CD3 antibody includes CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and CDR2 composed of the amino acid sequence of amino acid numbers 50 to 68 of SEQ ID NO: 8, and CDR3 composed of the amino acid sequence of amino acid numbers 101 to 114 of SEQ ID NO: 8; light chain of anti-CD3 antibody The variable region includes CDR1 composed of the amino acid sequence of amino acid numbers 168 to 181 of SEQ ID NO: 8, CDR2 composed of the amino acid sequence of amino acid numbers 197 to 203 of SEQ ID NO: 8, and CDR3 composed of the amino acid sequence of amino acid numbers 236 to 244 of SEQ ID NO: 8. For example, the pharmaceutical composition according to any one of claims 1 to 5, wherein the heavy chain variable region of the anti-CD3 antibody is composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the anti-CD3 antibody The light chain variable region is composed of the amino acid sequence of amino acid numbers 146 to 254 of SEQ ID NO: 8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the anti-CD3 scFv region is composed of the amino acid sequence of amino acid numbers 1 to 254 of SEQ ID NO: 8. Such as the pharmaceutical composition of any one of claims 1 to 7, wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: containing the amino acid sequence consisting of amino acid numbers 31 to 35 of SEQ ID NO: 4 The heavy chain of CDR1, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4, and CDR3 composed of the amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 4 can be The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the anti-TSPAN8 antibody in the variable region to the first Fc polypeptide; contains the amino acid sequence consisting of amino acid numbers 24 to 34 of SEQ ID NO: 6 CDR1, CDR2 composed of the amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 6, and the light chain of CDR3 composed of the amino acid sequence of amino acid numbers 89 to 96 of SEQ ID NO: 6 The light chain of the anti-TSPAN8 antibody in the variable region, and a CDR1 composed of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 8, and an amine composed of amino acid numbers 50 to 68 of SEQ ID NO: 8 The CDR2 composed of the amino acid sequence of SEQ ID NO: 8, and the heavy chain variable region of the anti-CD3 antibody of the CDR3 composed of the amino acid sequence of SEQ ID NO: 8, and the amino group of SEQ ID NO: 8 CDR1 composed of the amino acid sequence of SEQ ID NO: 168 to 181, CDR2 composed of the amino acid sequence of SEQ ID NO: 8 of amino acid numbers 197 to 203, and CDR2 composed of amino acid sequence of SEQ ID NO: 8 of 236 to 244 A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody composed of the amino acid sequence of the CDR3 with the second Fc polypeptide. The pharmaceutical composition according to any one of claims 1 to 8, wherein the anti-TSPAN8-anti-CD3 bispecific antibody includes: a complex consisting of amino acid sequences of amino acid numbers 1 to 121 of SEQ ID NO: 4. The heavy chain of the anti-TSPAN8 antibody obtained by linking the heavy chain fragment of the anti-TSPAN8 antibody in the chain variable region to the first Fc polypeptide contains the amino acid sequence of amino acid numbers 1 to 107 of SEQ ID NO: 6. The light chain of the anti-TSPAN8 antibody composed of a light chain variable region, and the heavy chain variable region of the anti-CD3 antibody composed of the amino acid sequence of amino acid numbers 1 to 125 of SEQ ID NO: 8, and the sequence A polypeptide obtained by linking the anti-CD3 scFv region of the light chain variable region of the anti-CD3 antibody and the second Fc polypeptide composed of the amino acid sequence of amino acid numbers 146 to 254 of No. 8. For example, the pharmaceutical composition according to any one of claims 1 to 9, which contains a compound selected from the group consisting of LALA mutations (L234A and L235A), N297G mutations, and Knobs into holes (Knobs into holes) mutations (here, the aforementioned mutation positions are in accordance with The Fc region is a group of one or more mutated Fc regions consisting of the amino acid position of the EU index in the human Igγ1 constant region. For example, the pharmaceutical composition according to any one of claims 1 to 9, which includes an Fc region containing LALA mutation, N297G mutation, and Knobs into holes mutation. For example, the pharmaceutical composition of claim 10 or 11, wherein the Knobs into holes mutation is the T366W mutation in one Fc polypeptide forming the Fc region, and the T366S and L368A in another Fc polypeptide forming the Fc region. and Y407V mutation (here, the aforementioned mutation position is an amino acid position that complies with the EU index in the human Igγ1 constant region). A pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody for treating cancer of a subject, The anti-TSPAN8-anti-CD3 bispecific antibody is composed of: the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, and the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6 chain, and a polypeptide obtained by linking the anti-CD3scFv region composed of the amino acid sequence of SEQ ID NO: 8 and the second Fc polypeptide, The pharmaceutical composition is used in combination with a PD-1 signaling inhibitor. For example, the pharmaceutical composition according to any one of claims 1 to 13, which contains a post-translationally modified anti-TSPAN8-anti-CD3 bispecific antibody as an active ingredient. For example, the pharmaceutical composition according to any one of claims 1 to 14 is administered to the subject simultaneously, continuously or sequentially with the PD-1 signaling inhibitor. For example, the pharmaceutical composition according to any one of claims 1 to 14, which contains anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signaling inhibitor (i) in the same pharmaceutical composition and is administered at the same time to the subject, or (ii) contained in different pharmaceutical compositions and administered to the subject simultaneously, continuously or sequentially. The pharmaceutical composition according to any one of claims 1 to 16, wherein the cancer is primary, metastatic or peritoneal disseminated solid cancer. For example, the pharmaceutical composition of claim 17, wherein the cancer is esophageal cancer, colorectal cancer, pancreatic cancer, gastric cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer. For example, the pharmaceutical composition according to any one of claims 1 to 18, wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or an antigen-binding fragment thereof. For example, the pharmaceutical composition of claim 19, wherein the PD-1 signaling inhibitor is an anti-PD-1 antibody. For example, the pharmaceutical composition of claim 20, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spartalizumab ( spartalizumab), or cemiplimab. For example, the pharmaceutical composition of claim 19, wherein the PD-1 signaling inhibitor is an anti-PD-L1 antibody. For example, the pharmaceutical composition of claim 22, wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab. A bispecific antibody that is an anti-TSPAN8-anti-CD3 bispecific antibody for treating cancer in a subject, This anti-TSPAN8-anti-CD3 bispecific antibody contains: (a) The Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment including the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising the heavy chain variable region of an anti-CD3 antibody and the light chain variable region of an 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 anti-TSPAN8-anti-CD3 bispecific antibody system is used in combination with a PD-1 signaling inhibitor. A bispecific antibody that is an anti-TSPAN8-anti-CD3 bispecific antibody for treating cancer in a subject, The anti-TSPAN8-anti-CD3 bispecific antibody is composed of: the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, and the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6 chain, and a polypeptide obtained by linking the anti-CD3scFv region composed of the amino acid sequence of SEQ ID NO: 8 and the second Fc polypeptide, The anti-TSPAN8-anti-CD3 bispecific antibody system is used in combination with a PD-1 signaling inhibitor. A treatment method comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject's cancer, This anti-TSPAN8-anti-CD3 bispecific antibody contains: (a) The Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment including the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising the heavy chain variable region of an anti-CD3 antibody and the light chain variable region of an 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 treatment method comprising administering an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signaling inhibitor to a subject's cancer, The anti-TSPAN8-anti-CD3 bispecific antibody is composed of: the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, and the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6 chain, and is composed of a polypeptide obtained by linking the anti-CD3 scFv region composed of the amino acid sequence of SEQ ID NO: 8 and the second Fc polypeptide. The use of an anti-TSPAN8-anti-CD3 bispecific antibody, which is used to manufacture a pharmaceutical composition used in combination with a PD-1 signaling inhibitor for the treatment of cancer, This anti-TSPAN8-anti-CD3 bispecific antibody contains: (a) The Fab region of the anti-TSPAN8 antibody composed of a heavy chain fragment including the heavy chain variable region of the anti-TSPAN8 antibody and a light chain including the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising the heavy chain variable region of an anti-CD3 antibody and the light chain variable region of an 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 of an anti-TSPAN8-anti-CD3 bispecific antibody, which is used to manufacture a pharmaceutical composition used in combination with a PD-1 signaling inhibitor for the treatment of cancer, The anti-TSPAN8-anti-CD3 bispecific antibody is composed of: the heavy chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 4, and the light chain of the anti-TSPAN8 antibody composed of the amino acid sequence of SEQ ID NO: 6 chain, and is composed of a polypeptide obtained by linking the anti-CD3 scFv region composed of the amino acid sequence of SEQ ID NO: 8 and the second Fc polypeptide.