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WO2023199927A1 - Utilisation d'un anticorps bispécifique anti-tspan8-anti-cd3 associé à un inhibiteur de signal pd-1 pour le traitement du cancer - Google Patents

Utilisation d'un anticorps bispécifique anti-tspan8-anti-cd3 associé à un inhibiteur de signal pd-1 pour le traitement du cancer Download PDF

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WO2023199927A1
WO2023199927A1 PCT/JP2023/014792 JP2023014792W WO2023199927A1 WO 2023199927 A1 WO2023199927 A1 WO 2023199927A1 JP 2023014792 W JP2023014792 W JP 2023014792W WO 2023199927 A1 WO2023199927 A1 WO 2023199927A1
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amino acid
antibody
tspan8
seq
acid sequence
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Japanese (ja)
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由幸 天田
貴志 茶圓
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Astellas Pharma Inc
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Astellas Pharma Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to the use of anti-TSPAN8-anti-CD3 bispecific antibodies in combination with PD-1 signal inhibitors in cancer therapy.
  • Tetraspanin-8 is a four-transmembrane protein that belongs to the tetraspanin family, and consists of a small extracellular loop (SEL) and a large extracellular loop (LEL). Two It has an extracellular loop region and three cytoplasmic domains, and forms a molecular cluster with a wide variety of transmembrane and cytoplasmic proteins as scaffolding proteins.
  • TSPAN8 is known to be involved in cell adhesion, cell motility, cell activation and proliferation, etc., and is highly expressed in gastric cancer, pancreatic cancer, colon cancer, liver cancer, etc. A relationship between increased expression and cancer progression or metastasis has been reported (Non-Patent Document 1). Research aimed at diagnosis and treatment of cancer using anti-TSPAN8 antibodies is being conducted (Patent Documents 1 and 2, and Non-Patent Documents 1 and 2).
  • CD3 Cluster of Differentiation 3
  • TCR T cell receptor
  • CD3 is a complex consisting of five types of subunits: gamma ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), zeta ( ⁇ ), and eta ( ⁇ ) chains, and each subunit consists of ⁇ , ⁇ , and ⁇ chains. It forms three types of dimers. Since CD3 is expressed on both normal T cells and tumorous T cells, it is used as a marker for T cells, and is also used as a marker for various anticancer-related antigens (Tumor Associate Antigen) for the purpose of cancer treatment.
  • TAA tumor associated Antigen
  • Bispecific T-cell-recruiting antibodies consisting of various antibody formats have been reported as an innovative method that can obtain cancer cell-selective cytotoxic activity at low antibody concentrations. The effects of these antibodies on T cell-mediated immunotherapy are being investigated (Non-Patent Document 4).
  • a bispecific T cell recruiting antibody is a bispecific antibody that includes an antibody against TAA expressed on the surface of cancer cells and an antibody that binds to T cells.
  • Anti-CD3 antibodies are often used as antibodies that bind to T cells.
  • Bispecific T cell recruitment antibodies, molecules containing anti-TAA and anti-CD3 antibodies bring target cancer cells and cytotoxic T lymphocytes (CTLs) closer to each other physically, and the anti-CD3 antibodies target CTLs.
  • CTLs cytotoxic T lymphocytes
  • Programmed cell death-1 (PD-1; also called PDCD1 or CD279) is a 50-55 kDa type I transmembrane protein that belongs to the immunoglobulin superfamily (Int. Immunol., 1996; Vol. 8: p. 765- 772). Expression of PD-1 is induced in T cells as activation continues, and the ligand Programmed death-ligand 1 (PD-L1; also called PDCD1LG1, B7-H1 or CD274) or Programmed death-ligand 2 (PD-L1; also called PDCD1LG1, B7-H1 or CD274) L2; PDCD1LG2, also called B7-DC or CD273) suppressively controls the activation of T cells (Annu. Rev. Immunol., 2008; Vol. 26: p. 677-704). Generally, such a T cell activation control mechanism is called an immune checkpoint, and is known as one of the negative feedback mechanisms to prevent excessive immune responses.
  • immune cells such as T cells eliminate cancer by antitumor immune responses by immune surveillance mechanisms.
  • cancer has acquired an immune escape mechanism by directly or indirectly suppressing immune cells in the cancer microenvironment.
  • Immune checkpoint mechanisms such as the PD-1/PD-L1 or PD-L2 (hereinafter referred to as "PD-1 signal") pathway, CTLA-4/CD80 or CD86 pathway are known as direct activated T cell suppression mechanisms.
  • PD-1 signal the PD-1/PD-L1 or PD-L2 pathway
  • CTLA-4/CD80 or CD86 pathway are known as direct activated T cell suppression mechanisms.
  • PD-1 signal the PD-1/PD-L1 or PD-L2 pathway
  • CTLA-4/CD80 or CD86 pathway are known as direct activated T cell suppression mechanisms.
  • Non-Patent Documents 5 to 7 It has been reported in multiple mouse tumor-bearing models that release of the immune escape mechanism through inhibition of PD-1 signals results in antitumor effects. Furthermore, as PD-1 signal drugs, PD-1 signal inhibitors such as anti-PD-1 antibodies such as nivolumab and pembrolizumab are being actively developed, and great results have been achieved in melanoma, lung cancer, lymphoma, etc. Furthermore, as PD-1 signal inhibitors, in addition to antibodies, research is also being conducted on nucleic acid medicines, low-molecular medicines, etc. (Non-Patent Document 8).
  • Indirect activated T cell suppression mechanisms include the induction of regulatory T cells and bone marrow-derived regulatory cells by cancer cells, and immunosuppressive humoral factors (IL-10, TGF- ⁇ , indoleamine 2,3-diamine). Oxygenase (IDO), etc.) is known to be produced, and drugs targeting these humoral factors are being developed (J. Hematol. Oncol., 2021; Vol. 14: p. 55-74, BioDrugs, 2018; Vol. 32: p. 311-317).
  • Non-Patent Documents 9 and 10 In order to increase the effectiveness of treatment for cancer patients, combination therapy with multiple cancer immunotherapy drugs and combination trials of cancer immunotherapy drugs and existing anticancer drugs are being actively pursued (Non-Patent Documents 9 and 10). ). For example, combination tests are being conducted on anti-PD-1 antibodies and other immune checkpoint inhibitory antibodies, anticancer drugs, molecular target drugs, radiotherapy, cancer vaccines, and oncolytic viruses. However, to date, a method for treating cancer using a combination of an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor has not been reported.
  • An object of the present invention is to provide an anti-TSPAN8-anti-CD3 bispecific antibody or a pharmaceutical composition comprising the bispecific antibody, which is used in combination with a PD-1 signal inhibitor for the treatment of cancer in a target.
  • the present invention also provides a method for treating cancer, which comprises administering to a subject an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor.
  • the present inventors aimed to create an antibody or pharmaceutical composition for use in the treatment of TSPAN8-expressing cancer, and based on the sequence of the anti-TSPAN8 antibody 16B11.1, we developed an anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody. An antibody was produced (Example 1). When this anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody were administered to mice transplanted with TSPAN8-expressing cancer cells, the results showed that compared to when anti-PD-1 antibody was administered alone, It was confirmed that it exhibited a significant antitumor effect (Example 3).
  • anti-TSPAN8-anti-CD3 bispecific antibody and PD-1 signal inhibitor is useful for treating TSPAN8-expressing cancer.
  • the combination of this anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody can be used together with anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody, anti-PD- 1 antibody or anti-PD-L1 antibody alone, it promoted activation of T cells and production of interferon- ⁇ by T cells in vitro, and exhibited cytotoxic activity (Example 2).
  • a pharmaceutical composition for treating cancer in a target comprising an anti-TSPAN8-anti-CD3 bispecific antibody
  • the anti-TSPAN8-anti-CD3 bispecific antibody : (a) Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, 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); including; A pharmaceutical composition, wherein said pharmaceutical composition is used in combination with a PD-1 signal inhibitor.
  • the heavy chain variable region of the anti-TSPAN8 antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and SEQ ID NO:
  • the light chain variable region of the anti-TSPAN8 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6.
  • the pharmaceutical composition according to [1] comprising CDR2 consisting of an amino acid sequence from 50 to 56, and CDR3 consisting of an amino acid sequence from amino acid numbers 89 to 96 of SEQ ID NO: 6.
  • the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence of amino acid number 1 to 107 of SEQ ID NO: 6.
  • the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6 [1] to [3]
  • the heavy chain variable region of the anti-CD3 antibody has CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 68 of SEQ ID NO: 8, and SEQ ID NO:
  • the light chain variable region of the anti-CD3 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO.
  • the pharmaceutical composition according to any one of [1] to [4], comprising CDR2 consisting of the amino acid sequence from 197 to 203 and CDR3 consisting of the amino acid sequence from amino acid numbers 236 to 244 of SEQ ID NO: 8.
  • the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.
  • the pharmaceutical composition according to any one of [1] to [6], wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid numbers 1 to 254 of SEQ ID NO: 8.
  • the anti-TSPAN8-anti-CD3 bispecific antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and A heavy chain of an anti-TSPAN8 antibody in which a first Fc polypeptide is linked to a heavy chain fragment of an anti-TSPAN8 antibody that includes a heavy chain variable region including CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, SEQ ID NO: CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO.
  • CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8
  • CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8
  • the heavy chain variable region of an anti-CD3 antibody including CDR3 consisting of the amino acid sequence from amino acid numbers 101 to 114 of SEQ ID NO: 8, and CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO: 8
  • the pharmaceutical composition according to any one of [1] to [7], which comprises a polypeptide linked to a peptide.
  • the anti-TSPAN8-anti-CD3 bispecific antibody consists of a heavy chain fragment of an anti-TSPAN8 antibody containing the heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the first Fc polypeptide.
  • [10] The group consisting of LALA mutations (L234A and L235A), N297G mutation, and Knobs into holes mutation (wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region)
  • the pharmaceutical composition according to any one of [1] to [9] comprising an Fc region containing the LALA mutation, the N297G mutation, and the knobs into holes mutation.
  • Knobs into holes mutations are the T366W mutation in one Fc polypeptide forming the Fc region, and the T366S, L368A and Y407V mutations in another Fc polypeptide forming the Fc region (wherein The mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region), [10] or the pharmaceutical composition according to [11].
  • a pharmaceutical composition for treating cancer in a target comprising an anti-TSPAN8-anti-CD3 bispecific antibody
  • the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an amino acid sequence of SEQ ID NO: 8. consisting of a polypeptide in which an anti-CD3 scFv region consisting of A pharmaceutical composition, wherein said pharmaceutical composition is used in combination with a PD-1 signal inhibitor.
  • the pharmaceutical composition according to any one of [1] to [14], wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are (i) in the same pharmaceutical composition. (ii) contained in separate pharmaceutical compositions and administered to a subject simultaneously, sequentially or sequentially.
  • the pharmaceutical composition according to [19], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody.
  • the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spartalizumab, or cemiplimab.
  • the PD-1 signal inhibitor is an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab.
  • An anti-TSPAN8-anti-CD3 bispecific antibody for treating cancer of interest comprising: The anti-TSPAN8-anti-CD3 bispecific antibody: (a) Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, 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); including; A bispecific antibody, wherein the anti-TSPAN8-anti-CD3 bispecific antibody is used in combination with a PD-1 signal inhibitor.
  • the heavy chain variable region of the anti-TSPAN8 antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and SEQ ID NO:
  • the light chain variable region of the anti-TSPAN8 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6.
  • the bispecific antibody according to [24] comprising CDR2 consisting of the amino acid sequence from 50 to 56, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6.
  • the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence of amino acid number 1 to 107 of SEQ ID NO: 6.
  • the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6 [24] to [26]
  • the bispecific antibody according to any one of.
  • the heavy chain variable region of the anti-CD3 antibody has CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 68 of SEQ ID NO: 8, and SEQ ID NO:
  • the light chain variable region of the anti-CD3 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO.
  • the bispecific antibody according to any one of [24] to [27], comprising CDR2 consisting of the amino acid sequence from 197 to 203, and CDR3 consisting of the amino acid sequence from amino acid numbers 236 to 244 of SEQ ID NO: 8.
  • the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.
  • the bispecific antibody according to any one of [24] to [29], wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO:8.
  • the anti-TSPAN8-anti-CD3 bispecific antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and A heavy chain of an anti-TSPAN8 antibody in which a first Fc polypeptide is linked to a heavy chain fragment of an anti-TSPAN8 antibody that includes a heavy chain variable region including CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, SEQ ID NO: CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO.
  • CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8
  • CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8
  • the heavy chain variable region of an anti-CD3 antibody including CDR3 consisting of the amino acid sequence from amino acid numbers 101 to 114 of SEQ ID NO: 8, and CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO: 8
  • the bispecific antibody according to any one of [24] to [30], which comprises a polypeptide linked to a peptide.
  • the anti-TSPAN8-anti-CD3 bispecific antibody consists of a heavy chain fragment of an anti-TSPAN8 antibody containing the heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the first Fc polypeptide.
  • [33] The group consisting of LALA mutations (L234A and L235A), N297G mutation, and Knobs into holes mutation (wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region)
  • the bispecific antibody according to any one of [24] to [32] which comprises an Fc region containing the LALA mutation, the N297G mutation, and the knobs into holes mutation.
  • Knobs into holes mutations are the T366W mutation in one Fc polypeptide forming the Fc region, and the T366S, L368A and Y407V mutations in another Fc polypeptide forming the Fc region (wherein The bispecific antibody according to [33] or [34], wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region.
  • An anti-TSPAN8-anti-CD3 bispecific antibody for treating cancer of interest comprising:
  • the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an amino acid sequence of SEQ ID NO: 8. consisting of a polypeptide in which an anti-CD3 scFv region consisting of A bispecific antibody, wherein the anti-TSPAN8-anti-CD3 bispecific antibody is used in combination with a PD-1 signal inhibitor.
  • the bispecific antibody according to any one of [24] to [37], wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor (i) have the same pharmaceutical composition; (ii) in separate pharmaceutical compositions and administered to a subject simultaneously, sequentially or sequentially.
  • the bispecific antibody according to any one of [24] to [39], wherein the cancer is a primary, metastatic, or peritoneal disseminated solid cancer.
  • the cancer is esophageal cancer, colon cancer, pancreatic cancer, stomach cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer.
  • Specific antibodies are any one of [24] to [41], 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-binding fragment thereof. Bispecific antibodies as described.
  • the bispecific antibody according to [42], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody.
  • the bispecific antibody according to [43], wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spartalizumab, or cemiplimab.
  • the bispecific antibody according to [42], wherein the PD-1 signal inhibitor is an anti-PD-L1 antibody.
  • the bispecific antibody according to [45] wherein the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab.
  • a method for treating cancer comprising administering to a subject an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor,
  • the anti-TSPAN8-anti-CD3 bispecific antibody : (a) Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, 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); including methods.
  • the heavy chain variable region of the anti-TSPAN8 antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and SEQ ID NO:
  • the light chain variable region of the anti-TSPAN8 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6.
  • the method according to [47] comprising CDR2 consisting of the amino acid sequence from 50 to 56, and CDR3 consisting of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6.
  • the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence of amino acid number 1 to 107 of SEQ ID NO: 6.
  • the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6 [47] to [49] The method described in any of the above.
  • the heavy chain variable region of the anti-CD3 antibody has CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 68 of SEQ ID NO: 8, and SEQ ID NO:
  • the light chain variable region of the anti-CD3 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO.
  • the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 146 to 254 of SEQ ID NO: 8.
  • the anti-TSPAN8-anti-CD3 bispecific antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and A heavy chain of an anti-TSPAN8 antibody in which a first Fc polypeptide is linked to a heavy chain fragment of an anti-TSPAN8 antibody that includes a heavy chain variable region including CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, SEQ ID NO: CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO.
  • CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8
  • CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8
  • the heavy chain variable region of an anti-CD3 antibody including CDR3 consisting of the amino acid sequence from amino acid numbers 101 to 114 of SEQ ID NO: 8, and CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO: 8
  • the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and a first Fc polypeptide.
  • [56] The group consisting of LALA mutations (L234A and L235A), N297G mutation, and Knobs into holes mutations (wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region)
  • the method according to any one of [47] to [55] comprising an Fc region containing the LALA mutation, the N297G mutation, and the knobs into holes mutation.
  • Knobs into holes mutations are the T366W mutation in one Fc polypeptide forming the Fc region and the T366S, L368A and Y407V mutations in another Fc polypeptide forming the Fc region (wherein The method according to [56] or [57], wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region.
  • a method for treating cancer comprising administering to a subject an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor,
  • the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an amino acid sequence of SEQ ID NO: 8.
  • a method comprising: a polypeptide in which an anti-CD3 scFv region consisting of a second Fc polypeptide is linked; [60] The method according to any one of [47] to [59], wherein the anti-TSPAN8-anti-CD3 bispecific antibody is post-translationally modified. [61] The method according to any one of [47] to [60], wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are applied to the target simultaneously, sequentially, or sequentially. The method by which it is administered.
  • [62] The method according to any one of [47] to [60], wherein the anti-TSPAN8-anti-CD3 bispecific antibody and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition. (ii) in separate pharmaceutical compositions and administered to the subject simultaneously, sequentially or sequentially.
  • the cancer is a primary, metastatic, or peritoneal disseminated solid cancer.
  • the cancer is esophageal cancer, colon cancer, pancreatic cancer, stomach cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer.
  • the PD-1 signal inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof.
  • Method described [66] The method according to [65], wherein the PD-1 signal inhibitor is an anti-PD-1 antibody.
  • the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spartalizumab, or cemiplimab.
  • the PD-1 signal inhibitor is an anti-PD-L1 antibody.
  • an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition used in combination with a PD-1 signal inhibitor to treat cancer in a subject, comprising: The anti-TSPAN8-anti-CD3 bispecific antibody: (a) Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising an anti-CD3 antibody heavy chain variable region and an anti-CD3 antibody light chain variable region, 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-
  • the heavy chain variable region of the anti-TSPAN8 antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and SEQ ID NO:
  • the light chain variable region of the anti-TSPAN8 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6, and CDR3 consisting of the amino acid sequence from amino acid numbers 24 to 34 of SEQ ID NO: 6.
  • the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence of amino acid number 1 to 107 of SEQ ID NO: 6.
  • the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence from amino acid number 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6. Use as described in any of the above.
  • the heavy chain variable region of the anti-CD3 antibody has CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid numbers 50 to 68 of SEQ ID NO: 8, and SEQ ID NO:
  • the light chain variable region of the anti-CD3 antibody contains CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO.
  • the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid numbers 1 to 125 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid numbers 146 to 254 of SEQ ID NO: 8.
  • the use according to any one of [70] to [75], wherein the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO:8.
  • the anti-TSPAN8-anti-CD3 bispecific antibody has CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and A heavy chain of an anti-TSPAN8 antibody in which a first Fc polypeptide is linked to a heavy chain fragment of an anti-TSPAN8 antibody that includes a heavy chain variable region including CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, SEQ ID NO: CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO.
  • CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8
  • CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8
  • the heavy chain variable region of an anti-CD3 antibody including CDR3 consisting of the amino acid sequence from amino acid numbers 101 to 114 of SEQ ID NO: 8, and CDR1 consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO: 8
  • the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and a first Fc polypeptide.
  • An anti-CD3 scFv region and a second Fc polypeptide comprising a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence of The use according to any one of [70] to [77], comprising a linked polypeptide.
  • [79] The group consisting of LALA mutations (L234A and L235A), N297G mutation, and Knobs into holes mutation (wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region)
  • Knobs into holes mutations are the T366W mutation in one Fc polypeptide forming the Fc region and the T366S, L368A and Y407V mutations in another Fc polypeptide forming the Fc region (wherein The use according to [79] or [80], wherein the mutation position is an amino acid position according to the EU index in the human Ig ⁇ 1 constant region.
  • an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition used in combination with a PD-1 signal inhibitor to treat cancer in a subject, comprising:
  • the anti-TSPAN8-anti-CD3 bispecific antibody comprises a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an amino acid sequence of SEQ ID NO: 8.
  • a use comprising a polypeptide in which an anti-CD3 scFv region consisting of a second Fc polypeptide is linked.
  • the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, spartalizumab, or cemiplimab.
  • the PD-1 signal inhibitor is an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody is atezolizumab, durvalumab, or avelumab.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention binds to both the cancer antigen TSPAN8 and CD3, a T cell surface molecule, and reduces the physical distance between cancer cells and T cells. , enhances the cancer cell killing effect of T cells.
  • the combination of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signal inhibitor results in a significant antitumor effect compared to administration of the PD-1 signal inhibitor alone.
  • the invention thus provides the use of anti-TSPAN8-anti-CD3 bispecific antibodies in combination with PD-1 signal inhibitors in cancer therapy.
  • Figure 1 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-human in a co-culture system of RKO_hTSPAN8-GFP cells in which TSPAN8-GFP is stably expressed in human colon cancer cell line and Expanded PanT cells.
  • the effect of combined use with PD-1 antibody is shown in cancer cell proliferation inhibition.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the fluorescence intensity of GFP, which is an indicator of cell proliferation.
  • Each column shows the average value of fluorescence intensity. Error bars indicate standard error.
  • Figure 2 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-human in a co-culture system of RKO_hTSPAN8-GFP cells in which TSPAN8-GFP is stably expressed in human colon cancer cell line and Expanded PanT cells.
  • the effect of combination with PD-L1 antibody is shown in cancer cell proliferation inhibition.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the fluorescence intensity of GFP, which is an indicator of cell proliferation.
  • Each column shows the average value of fluorescence intensity. Error bars indicate standard error.
  • Figure 3-1 shows the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells in which TSPAN8-GFP was stably expressed in a human colon cancer cell line.
  • Activation of CD4 T cells by combined use with anti-human PD-1 antibody is shown.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the average fluorescence intensity of the fluorescently labeled antibody against CD25, which is an activation marker for CD4T cells.
  • Each column shows the average value of the average fluorescence intensity. Error bars indicate standard error.
  • Figure 3-2 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells in which TSPAN8-GFP was stably expressed in human colon cancer cell line.
  • Activation of CD8 T cells by combined use with anti-human PD-1 antibody is shown.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the average fluorescence intensity of the fluorescently labeled antibody against CD25, which is an activation marker for CD8 T cells.
  • Each column shows the average value of the average fluorescence intensity. Error bars indicate standard error.
  • Figure 4-1 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells in which TSPAN8-GFP is stably expressed in human colon cancer cell line.
  • Activation of CD4 T cells by combination with human PD-L1 antibody is shown.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the average fluorescence intensity of the fluorescently labeled antibody against CD25, which is an activation marker for CD4T cells.
  • Each column shows the average value of the average fluorescence intensity. Error bars indicate standard error.
  • Figure 4-2 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells in which TSPAN8-GFP is stably expressed in human colon cancer cell line.
  • Activation of CD8 T cells by combination with human PD-L1 antibody is shown.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the average fluorescence intensity of the fluorescently labeled antibody against CD25, which is an activation marker for CD8 T cells.
  • Each column shows the average value of the average fluorescence intensity. Error bars indicate standard error.
  • Figure 5 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-human PD in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells in which TSPAN8-GFP is stably expressed in a human colon cancer cell line.
  • the effect of combined use with -1 antibody is shown by the amount of interferon- ⁇ produced.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the average value of interferon- ⁇ production. Error bars indicate standard error.
  • Figure 6 shows anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-human in a co-culture system of human RKO_hTSPAN8-GFP cells in which TSPAN8-GFP is stably expressed in human colon cancer cell line and Expanded PanT cells.
  • the effect of combined use with PD-L1 antibody is shown by the amount of interferon- ⁇ produced.
  • the vertical axis of the figure shows the test antibody, and the horizontal axis shows the average value of interferon- ⁇ production. Error bars indicate standard error.
  • Figure 7-1 shows the combined effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody on tumor growth of human TSPAN8-expressing B16-F10 cells in the B-hCD3E mouse model. It is shown as the average value of tumor volume on each day after initiation. Error bars indicate standard error of tumor volume.
  • the vertical axis of the figure shows the tumor volume, and the horizontal axis shows the number of days from the start date of administration of the test antibody.
  • Figure 7-2 shows the effect of the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody on tumor growth of human TSPAN8-expressing B16-F10 cells in the B-hCD3E mouse model after administration.
  • the tumor volume of each individual after 13 days is shown. Error bars indicate standard error of tumor volume.
  • the vertical axis of the figure shows the tumor volume, and the horizontal axis shows the test antibody. Horizontal lines indicate the mean value and standard error.
  • the significance probability P value was calculated by comparing the tumor volume of the combination group with the tumor volume of the anti-TSPAN8 (16B11)-anti-CD3 single agent administration group or the anti-mouse PD-1 single agent administration group using an unpaired Student's t test. I asked for it. * and ** in the figure indicate groups whose P values are smaller than the significance level of 0.05 and 0.01, respectively.
  • Antibodies have a basic four-chain structure with a symmetrical Y-shaped structure consisting of two heavy chains with a single sequence and two light chains with a single sequence. Refers to glycoprotein. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE.
  • the basic structure of antibody molecules is common to each class; two heavy chains with a molecular weight of 50,000 to 70,000 and two light chains with a molecular weight of 20,000 to 30,000 are linked by disulfide bonds and non-covalent bonds, resulting in a molecular weight of 150,000 to 19.
  • An antibody molecule consisting of a four-stranded Y-shaped structure is formed.
  • Heavy chains usually consist of polypeptide chains containing about 440 amino acids, and each class has a characteristic structure, with Ig ⁇ , Ig ⁇ , Ig ⁇ , and Ig ⁇ corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. , is called Ig ⁇ .
  • IgG has subclasses of IgG1, IgG2, IgG3, and IgG4, and the corresponding heavy chains are called Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, and Ig ⁇ 4.
  • the light chain usually consists of a polypeptide chain containing about 220 amino acids, and two types, ⁇ type and ⁇ type, are known, and these are called Ig ⁇ and Ig ⁇ , respectively. The two types of light chains can be paired with either type of heavy chain.
  • variable region The domain located at the N-terminus of both heavy and light chains is called the variable region, and even antibodies produced from the same class (or subclass) of the same species have diverse amino acid sequences, and the interaction between the antibody and the antigen is Binding specificity is known to be involved in binding.
  • the amino acid sequence of the C-terminal domain downstream of the variable region is almost constant for each class or subclass, and is called a constant region.
  • the heavy chain has, from the N-terminus to the C-terminus, a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • CH is further divided into three domains from the N-terminal side: CH1 domain, CH2 domain, and CH3 domain.
  • the light chain has, from the N-terminus to the C-terminus, a light chain variable region (VL) and a light chain constant region (CL).
  • CDRs complementarity determining regions
  • VH and VL vary greatly and contribute to the variability of the variable regions.
  • CDRs are regions consisting of approximately 5 to 10 amino acid residues present in the order of CDR1, CDR2, and CDR3 at the N-terminus of heavy and light chains, respectively, and form an antigen-binding site.
  • the portion of the variable region other than the CDRs is called the framework region (FR), which consists of FR1 to FR4, and there are relatively few changes in the amino acid sequence.
  • FR framework region
  • Fab antigen-binding fragment, antigen binding regions.
  • the "Fab region” refers to a region consisting of the VH and CH1 domains of the heavy chain and the light chains (VL and CL), and binds to the antigen at the antigen-binding site at the distal end constituted by the Fab region.
  • the term "heavy chain fragment” refers to a fragment consisting of the VH and CH1 domains of the heavy chain that constitutes the Fab region. Further, the fragment on the C-terminal side is called an Fc (Fragment, crystallizable) region.
  • Fc polypeptide refers to a polypeptide consisting of the CH2 domain and CH3 domain of a heavy chain
  • Fc region refers to a complex consisting of two Fc polypeptides.
  • shinge region or “hinge” refers to a highly flexible peptide region that exists between the CH1 and CH2 domains of the heavy chain, and the heavy chain fragment and Fc polypeptide are referred to as the hinge region. connected in parts.
  • the two heavy chains of an antibody are disulfide bonded in the hinge region.
  • antigen is used in its commonly used meaning, and particularly refers to a molecule or 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.
  • an "antigen-binding fragment” is 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, F(ab') 2 fragments.
  • scFv single chain variable region fragments
  • Fab fragments are monovalent antigen-binding fragments composed of a light chain and a fragment containing the VH, CH1 domain of the heavy chain.
  • Fab' fragments are monovalent antigen-binding fragments that are composed of a light chain and a fragment that includes the VH and CH1 domains of the heavy chain and part of the hinge region, which includes the interheavy chain Contains cysteine residues that formed disulfide bonds.
  • F(ab') 2 fragments are divalent molecules in which Fab' fragments are joined by disulfide bonds. "Monovalent” means containing one antigen-binding site, and "bivalent” means containing two antigen-binding sites.
  • scFv region refers to a region containing a monovalent antigen-binding fragment comprising VH and VL connected by a linker.
  • 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.
  • antibody is used to include full-length antibodies, antigen-binding fragments, and bispecific antibodies of any structure, unless otherwise specifically limited by context.
  • human antibody refers to an antibody having a human immunoglobulin amino acid sequence.
  • humanized antibody refers to an antibody in which some, most, or all of the amino acid residues other than the CDRs are replaced with amino acid residues derived from human immunoglobulin molecules.
  • humanization method is not particularly limited, for example, humanized antibodies can be produced with reference to US Pat. No. 5,225,539, US Pat. No. 6,180,370, and the like.
  • Amino acid residue numbers of antibodies used herein are determined by Kabat numbering or EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed, 1991, NIH Publication). n No. 91-3242), They can be defined according to their numbering system.
  • linked refers to two or more components (e.g., Fab region and Fc polypeptide) that are linked together, either directly or through one or more intermediaries (e.g., a peptide linker). It means doing.
  • peptide linker or “linker” refers to one or more arbitrary amino acid residues that can be introduced by genetic engineering to connect variable regions.
  • the length of the peptide linker used in the present invention is not particularly limited, and can be appropriately selected by those skilled in the art depending on the purpose.
  • identity means an Identity value obtained using parameters provided by default using EMBOSS Needle (Nucleic Acids Res., 2015; Vol. 43: pW580-W584). .
  • analog refers to an antibody that has the same amino acid sequence in the Fab region as an antibody and can specifically bind to an antigen, but has a different amino acid sequence in the Fc region.
  • subject means a human or other animal in need of disease prevention or treatment. In one embodiment, the subject is a human in need of disease prevention or treatment. In one embodiment, the subject is a human with cancer.
  • treatment refers to treatment for the purpose of recovering, alleviating, improving, suppressing, or delaying the progression, onset, severity, or recurrence of disease symptoms, medical conditions, and biochemical signs associated with the disease.
  • treatment refers to any therapeutic intervention, treatment, or administration of an active ingredient to a subject.
  • the term "active ingredient” refers to a substance that exhibits some kind of physiological activity among substances contained in pharmaceutical compositions, drugs, etc. used for the prevention or treatment of diseases.
  • the active ingredient is an antibody, small molecule compound, nucleic acid, fusion protein, peptide.
  • the active ingredient is an antibody.
  • the active ingredient is a bispecific antibody.
  • the term "pharmaceutical composition” refers to an active ingredient and a pharmaceutically acceptable excipient (including, but not limited to, a pharmaceutical excipient, a pharmaceutical carrier, etc.) It is a drug prescribed for the treatment of.
  • “combination”, “combination”, or “use in combination” refers to the administration of multiple active ingredients simultaneously, continuously, or sequentially to the same subject for the prevention or treatment of diseases. It means to do.
  • the plurality of active ingredients may be contained in the same pharmaceutical composition or separately in different pharmaceutical compositions.
  • “simultaneously” means administering multiple active ingredients in parallel within one administration period, and “continuously” means administering a period after administration of one active ingredient is completed. It means that one active ingredient is administered immediately after the other, and “sequentially” means that multiple active ingredients are administered in sequence according to the administration schedule.
  • an "effective amount" of a drug refers to the amount of drug necessary to bring about a physiological change in the cells or tissues to which it is administered.
  • PD-1 signal inhibitor refers to a drug that releases suppression of immune cell activation by PD-1.
  • PD-1 signal inhibitors can inhibit immune checkpoint function caused by PD-1 by binding to PD-1 or its ligand PD-L1 or PD-L2 and inhibiting immunosuppressive signals.
  • the PD-1 signal inhibitor may be any substance as long as it has the effect of blocking PD-1 signals, such as antibodies, low molecular weight compounds, nucleic acids (DNA or RNA, or natural or artificial substances). nucleic acids), fusion proteins, peptides, etc.
  • anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies can inhibit PD-1 signals by inhibiting the binding between PD-1 and PD-L1 or PD-L2.
  • the present invention relates to the following (1) to (4): (1) A pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor to treat a target cancer (herein referred to as “the pharmaceutical composition of the present invention”) (also referred to as “composition”); (2) anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor to treat a target cancer (herein referred to as “duplex for the combination of the present invention”) (also referred to as “specific antibody”); (3) A method for treating cancer comprising administering to a subject an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor (herein also referred to as "the treatment method of the present invention”); or (4) the use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signal inhibitor to treat cancer in a subject. , also referred to as "the use of
  • anti-TSPAN8-anti-CD3 bispecific antibody of the present invention includes the following (a) to (c).
  • the structure consists of: (a) Fab region of an anti-TSPAN8 antibody consisting of a heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and a light chain containing the light chain variable region of the anti-TSPAN8 antibody, (b) an anti-CD3 scFv region comprising a heavy chain variable region and a light chain variable region of an anti-CD3 antibody, and (c) a first Fc polypeptide linked to the heavy chain fragment of the Fab region of (a); an Fc region consisting of a second Fc polypeptide linked to an anti-CD3 scFv region; Bispecific antibodies, including:
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention has a structure that includes one Fab region of the first antibody, an scFv region of the second antibody, and one 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 is a Fab of an anti-TSPAN8 antibody, which is 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. Contains areas.
  • the heavy chain variable region of the anti-TSPAN8 antibody is CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6.
  • CDR2 consists of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6
  • CDR3 consists of the amino acid sequence from amino acid number 89 to 96 of SEQ ID NO: 6.
  • the heavy chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO: 4, and the light chain variable region of the anti-TSPAN8 antibody consists of the amino acid sequence of amino acid number 1 to 107 of SEQ ID NO: 6. It consists of the amino acid sequence of
  • any constant region of Ig ⁇ , Ig ⁇ , Ig ⁇ , Ig ⁇ , or Ig ⁇ can be selected.
  • Ig ⁇ can be selected from, for example, Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, or Ig ⁇ 4.
  • the heavy chain fragment of the anti-TSPAN8 antibody comprises a CH1 domain derived from a human Ig ⁇ 1 constant region.
  • the CL of the light chain of the anti-TSPAN8 antibody either an Ig ⁇ or Ig ⁇ constant region can be selected.
  • the light chain of the anti-TSPAN8 antibody comprises a human Ig ⁇ constant region, CL.
  • the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of the amino acid sequence of amino acid numbers 1 to 219 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention contains an anti-CD3 scFv region that includes the heavy chain variable region and light chain variable region of an anti-CD3 antibody.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention was prepared based on the sequence information of the anti-CD3 scFv region known in the art or the heavy chain variable region and light chain variable region of the anti-CD3 antibody known in the art. Anti-CD3 scFv regions may also be used.
  • known anti-CD3 antibodies clones such as OKT3, UTCH1, L2K, and TR66 are known, and their sequences are used as bispecific antibodies (Pharmacol. Ther., 2018; Vol. 182: p.161-175).
  • the heavy chain variable region of the anti-CD3 antibody is CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 50 to 68 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and the light chain variable region of the anti-CD3 antibody comprises CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, CDR2 consists of the amino acid sequence from amino acid numbers 197 to 203 of SEQ ID NO: 8, and CDR3 consists of the amino acid sequence from amino acid numbers 236 to 244 of SEQ ID NO: 8.
  • the heavy chain variable region of the anti-CD3 antibody consists of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO:8, and the light chain variable region of the anti-CD3 antibody consists of the amino acid sequence of amino acid number 146 to 254 of SEQ ID NO:8. It consists of the amino acid sequence of
  • the type and length of the peptide linker that connects the heavy chain variable region and light chain variable region of the anti-CD3 antibody are not particularly limited, and can be appropriately selected by those skilled in the art.
  • 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 from 5 amino acids to 30 amino acids, preferably from 5 amino acids to 20 amino acids, particularly preferably from 15 amino acids to 20 amino acids.
  • a glycine-serine linker for example, a glycine-serine linker (GS linker) or a glycine-lysine-proline-glycine-serine linker (GKPGS linker) can be used.
  • GS linker glycine-serine linker
  • GKPGS linker glycine-lysine-proline-glycine-serine linkerine linker
  • 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 (GKPGS; SEQ ID NO: 17).
  • GKPGS Gly-Lys-Pro-Gly-Ser
  • the peptide linker connecting the heavy chain variable region and light chain variable region of the anti-CD3 antibody has an amino acid sequence of (Gly-Lys-Pro-Gly-Ser)n.
  • the peptide linker connecting the heavy chain variable region and light chain variable region of the anti-CD3 antibody has an amino acid sequence of (Gly-Lys-Pro-Gly-Ser) 4 .
  • the anti-CD3 scFv region consists of the amino acid sequence from amino acid number 1 to 254 of SEQ ID NO:8.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, and amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4.
  • the light chain of the anti-TSPAN8 antibody includes a light chain variable region including CDR3 consisting of the amino acid sequence, CDR1 consisting of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 8, and amino acid numbers 50 to 68 of SEQ ID NO: 8.
  • a heavy chain variable region of an anti-CD3 antibody including CDR2 consisting of the amino acid sequence and CDR3 consisting of the amino acid sequence from amino acid numbers 101 to 114 of SEQ ID NO: 8, and consisting of the amino acid sequence from amino acid numbers 168 to 181 of SEQ ID NO: 8.
  • An anti-CD3 scFv comprising the light chain variable region of an anti-CD3 antibody, including CDR1, CDR2 consisting of the amino acid sequence from amino acid numbers 197 to 203 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence from amino acid numbers 236 to 244 of SEQ ID NO: 8. It includes a polypeptide in which a region and a second Fc polypeptide are linked.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO:4.
  • the heavy chain constant region from which the first Fc polypeptide and second Fc polypeptide constituting the Fc region are derived include Ig ⁇ , Ig ⁇ , Ig ⁇ , Ig ⁇ , or Ig ⁇ . Any constant region can be selected. Ig ⁇ can be selected from, for example, Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, or Ig ⁇ 4. In one embodiment, the first Fc polypeptide and the second Fc polypeptide are Fc polypeptides derived from human Ig ⁇ 1 constant region.
  • the Fc region of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may contain mutations that reduce antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
  • L234A is a substitution of leucine with alanine at amino acid position 234 according to the EU index in the human Ig ⁇ 1 constant region.
  • L235A is a substitution of leucine with alanine at amino acid position 235 according to the EU index in the human Ig ⁇ 1 constant region.
  • Amino acid mutations in human Ig ⁇ 1 constant regions L234A and L235A are referred to as "LALA mutations.” This mutation is known to reduce the antibody-dependent cytotoxic activity and complement-dependent cytotoxic 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 techniques.
  • the Fc region can be modified by the N297G mutation according to the EU index in the human Ig ⁇ 1 constant region (Protein cell, 2018; Vol. 9: p. 63-73) or the mutation based on the knobs into holes technology (hereinafter referred to as (also referred to as the "knobs into holes mutation").
  • Knobs into holes technology replaces the amino acid side chain present in the CH3 region of one heavy chain with a larger side chain (knob), and replaces the amino acid side chain present in the CH3 region of the other heavy chain with a larger side chain (knob).
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the invention comprises an Fc region that includes a LALA mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the invention comprises an Fc region that includes the N297G mutation. In one embodiment, an anti-TSPAN8-anti-CD3 bispecific antibody of the invention comprises an Fc region that includes a knobs into holes mutation. In one embodiment, the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an Fc comprising one or more mutations selected from the group consisting of LALA mutation, N297G mutation, and knobs into holes mutation. Contains areas.
  • an anti-TSPAN8-anti-CD3 bispecific antibody of the invention comprises an Fc region that includes a LALA mutation, a N297G mutation, and a knobs into holes mutation.
  • the knobs-into-holes mutation contained in the anti-TSPAN8-anti-CD3 bispecific antibody of the invention comprises a T366W mutation in one Fc polypeptide that forms its Fc region, as well as a T366W mutation in one Fc polypeptide that forms its Fc region.
  • Another Fc polypeptide that forms is the T366S, L368A and Y407V mutations (see WO 1998/050431).
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a first Fc polypeptide consisting of the amino acid sequence from amino acid number 235 to 451 of SEQ ID NO: 4 and a first Fc polypeptide consisting of the amino acid sequence from amino acid number 270 to SEQ ID NO: 8. It comprises an Fc region consisting of a second Fc polypeptide consisting of a sequence of up to 486 amino acids.
  • amino acid mutations such as the LALA mutation, N297G mutation, and knobs-into-holes mutation are based on the amino acid positions according to the EU index in the human Ig ⁇ 1 constant region.
  • L234A is a substitution of leucine with alanine at amino acid position 234 according to the EU index in the human Ig ⁇ 1 constant region.
  • the "heavy chain of anti-TSPAN8 antibody” refers to the heavy chain fragment containing the heavy chain variable region of the anti-TSPAN8 antibody and the Fc polypeptide (first Fc polypeptide). It also includes a polypeptide in which a heavy chain fragment containing the heavy chain variable region of an anti-TSPAN8 antibody and a first Fc polypeptide are linked via the 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, and may be linked in particular via the hinge region.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is an anti-TSPAN8-anti-CD3 bispecific antibody in which a heavy chain fragment comprising the heavy chain variable region of an anti-TSPAN8 antibody and a first Fc polypeptide are linked via the hinge region. Contains the heavy chain of the TSPAN8 antibody.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a polypeptide in which an anti-CD3 scFv region and a second Fc polypeptide are linked via a hinge region.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is an anti-TSPAN8-anti-CD3 bispecific antibody in which a heavy chain fragment comprising the heavy chain variable region of an anti-TSPAN8 antibody and a first Fc polypeptide are linked via the hinge region. It includes a heavy chain of a TSPAN8 antibody, a light chain of an anti-TSPAN8 antibody, and a polypeptide in which an anti-CD3 scFv region and a second Fc polypeptide are linked via a hinge region.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 4, and amino acid sequence from amino acid number 50 to 66 of SEQ ID NO: 4.
  • a heavy chain fragment of an anti-TSPAN8 antibody including a heavy chain variable region including CDR2 consisting of the sequence and CDR3 consisting of the amino acid sequence from amino acid number 99 to 110 of SEQ ID NO: 4 and the first Fc polypeptide are linked via the hinge region.
  • CDR1 consisting of the amino acid sequence from amino acid number 24 to 34 of SEQ ID NO: 6
  • CDR2 consisting of the amino acid sequence from amino acid number 50 to 56 of SEQ ID NO: 6, and the amino acid number of SEQ ID NO: 6.
  • the light chain of an anti-TSPAN8 antibody comprising a light chain variable region comprising CDR3 consisting of the amino acid sequence from 89 to 96, and CDR1 consisting of the amino acid sequence from amino acid number 31 to 35 of SEQ ID NO: 8, and the amino acid number of SEQ ID NO: 8.
  • a heavy chain variable region of an anti-CD3 antibody comprising CDR2 consisting of the amino acid sequence from 50 to 68, and CDR3 consisting of the amino acid sequence from amino acid number 101 to 114 of SEQ ID NO: 8, and amino acid number 168 to 181 of SEQ ID NO: 8.
  • a light chain variable of an anti-CD3 antibody comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence of amino acids 197 to 203 of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of amino acids 236 to 244 of SEQ ID NO: 8.
  • the anti-CD3 scFv region and the second Fc polypeptide are linked via a hinge region.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises a heavy chain fragment of an anti-TSPAN8 antibody comprising a heavy chain variable region consisting of the amino acid sequence from amino acid number 1 to 121 of SEQ ID NO:4.
  • a heavy chain of an anti-TSPAN8 antibody to which a first Fc polypeptide is linked via a hinge region a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence from amino acid number 1 to 107 of SEQ ID NO: 6, and , a heavy chain variable region of an anti-CD3 antibody consisting of the amino acid sequence from amino acid number 1 to 125 of SEQ ID NO: 8, and a light chain variable region of an anti-CD3 antibody consisting of the amino acid sequence of amino acid number 146 to 254 of SEQ ID NO: 8.
  • the anti-CD3 scFv region and the second Fc polypeptide are linked via a hinge region.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises an anti-TSPAN8 antibody heavy chain consisting of the amino acid sequence of SEQ ID NO: 4, and an anti-TSPAN8 antibody light chain consisting of the amino acid sequence of SEQ ID NO: 6. , and a polypeptide in which an anti-CD3 scFv region consisting of the amino acid sequence of SEQ ID NO: 8 and a second Fc polypeptide are linked.
  • post-translational modification refers to modification of an antibody after translation when the antibody is expressed within a cell.
  • post-translational modifications include modifications such as pyroglutamylation, glycosylation, oxidation, deamidation, and saccharification of glutamine or glutamic acid at the N-terminus of the heavy chain, and deletion of lysine due to cleavage of the lysine at the C-terminus of the heavy chain by carboxypeptidase. can be mentioned. It is known that such post-translational modifications occur in various antibodies (J. Pharm. Sci., 2008; Vol. 97: p. 2426-2447).
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention may be post-translationally modified.
  • the post-translational modification is heavy chain variable region N-terminal pyroglutamylation and/or heavy chain C-terminal lysine deletion. It is known in the art that post-translational modification by N-terminal pyroglutamylation or C-terminal lysine deletion does not affect antibody activity (Analytical Biochemistry, 2006; Vol. 348: p. 24- 39).
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention comprises human TSPAN8 (gene number: NM_004616.2) and human CD3 ⁇ complex protein (CD3 ⁇ gene number: NM_000733.3, CD3 ⁇ gene number: NM_000732.4 or NM_001040651.1). ). Whether or not it binds to human TSPAN8 and human CD3 ⁇ complex protein can be confirmed using a known method for measuring binding activity. Examples of methods for measuring binding activity include methods such as Enzyme-Linked ImmunoSorbent Assay (ELISA) method and flow cytometry method.
  • ELISA Enzyme-Linked ImmunoSorbent Assay
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention can be prepared by those skilled in the art based on the sequence information of the heavy chain variable region and light chain variable region of the anti-TSPAN8 antibody and anti-CD3 scFv region disclosed herein. Based on the above, it can be produced by a method known in the art. Furthermore, a person skilled in the art will be able to determine the anti-CD3 scFv region of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention based on the sequence information of the heavy chain variable region and light chain variable region of known anti-CD3 antibodies. It can be produced by methods known in the art.
  • the anti-TSPAN8-anti-CD3 bispecific antibodies of the invention are humanized or human antibodies.
  • back mutations may be introduced as appropriate using methods well known to those skilled in the art (Bioinformatics, 2015; Vol. 31: p. 434-435).
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is not particularly limited, but can be produced, for example, according to the method described in WO 2022/102768. Method for producing anti-TSPAN8-anti-CD3 bispecific antibody described in International Publication No.
  • the present invention provides a pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody (pharmaceutical composition of the present invention) used in combination with a PD-1 signal inhibitor to treat cancer in a subject.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for treating target cancer, which contains the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention, and is used in combination with a PD-1 signal inhibitor. Ru.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for use in a method of treating cancer in a subject, and the method of treatment comprises combining the pharmaceutical composition with a PD-1 signal inhibitor. Including administering to a subject.
  • the pharmaceutical composition of the present invention is produced using the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention described above, and is prepared using the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a pharmaceutically acceptable Contains excipients.
  • the anti-TSPAN8-anti-CD3 bispecific antibody contained in the pharmaceutical composition of the present invention has the molecular structure, peptide linker, and mutation described in the above section ⁇ Anti-TSPAN8-anti-CD3 bispecific antibody of the present invention>. , post-translational modifications, etc.
  • the pharmaceutical composition of the present invention can be prepared by a commonly used method using excipients commonly used in the field, ie, pharmaceutical excipients, pharmaceutical carriers, and the like.
  • Examples of the dosage form of these pharmaceutical compositions include parenteral preparations such as injections and infusion preparations, and can be administered by intravenous administration, subcutaneous administration, intraperitoneal administration, etc.
  • parenteral preparations such as injections and infusion preparations
  • excipients, carriers, additives, etc. depending on the dosage form can be used within pharmaceutically acceptable ranges.
  • the pharmaceutical composition of the present invention may contain post-translational modifications of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention.
  • the pharmaceutical compositions of the present invention include antibodies that have undergone deletion of the C-terminal lysine and/or pyroglutamylation of the N-terminus.
  • the pharmaceutical composition of the invention is a pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody of the invention and/or a post-translational modification of said antibody as described below:
  • CDR1 consists of the amino acid sequence from amino acid numbers 31 to 35 of SEQ ID NO: 4
  • CDR2 consists of the amino acid sequence from amino acid numbers 50 to 66 of SEQ ID NO: 4
  • CDR2 consists of the amino acid sequence from amino acid numbers 99 to 110 of SEQ ID NO: 4.
  • the heavy chain variable region of an anti-CD3 antibody comprising CDR3, and CDR1 consisting of the amino acid sequence from amino acid number 168 to 181 of SEQ ID NO: 8, CDR2 consisting of the amino acid sequence from amino acid number 197 to 203 of SEQ ID NO: 8, and the sequence A bispecific antibody comprising a polypeptide in which a second Fc polypeptide is linked to an anti-CD3 scFv region comprising the light chain variable region of an anti-CD3 antibody comprising CDR3 consisting of the amino acid sequence of amino acid numbers 236 to 244 of No. 8. .
  • the pharmaceutical composition of the invention is a pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody of the invention and/or a post-translational modification of said antibody as described below:
  • a heavy chain of an anti-TSPAN8 antibody in which 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 are linked an amino acid of SEQ ID NO: 6
  • a light chain of an anti-TSPAN8 antibody comprising a light chain variable region consisting of the amino acid sequence No. 1 to 107, and a heavy chain variable region consisting of the amino acid sequence No.
  • a bispecific antibody comprising a polypeptide in which an anti-CD3 scFv region containing a light chain variable region consisting of an amino acid sequence from 146 to 254 is linked to a second Fc polypeptide.
  • the pharmaceutical composition of the present invention comprises a heavy chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 4, a light chain of an anti-TSPAN8 antibody consisting of the amino acid sequence of SEQ ID NO: 6, and an amino acid sequence of SEQ ID NO: 8.
  • This is a pharmaceutical composition containing an anti-TSPAN8-anti-CD3 bispecific antibody comprising a polypeptide in which an anti-CD3 scFv region consisting of the sequence and a second Fc polypeptide are linked, and/or a post-translational modification of the antibody.
  • the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention to be added in the formulation of the pharmaceutical composition of the present invention depends on the severity and age of the patient's symptoms, the dosage form of the preparation used, the binding strength of the antibody, etc. Although it varies depending on the patient, for example, the anti-TSPAN8-anti-CD3 bispecific antibody can be used in the preparation in an amount of about 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 in formulation is in the range of 0.0001 mg/kg to 1000 mg/kg in terms of human dosage.
  • the amount of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention added in formulation is in the range of 0.001 mg/kg to 100 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 in formulation is in the range of 0.01 mg/kg to 10 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 in formulation is preferably in the range of 0.01 mg/kg to 10 mg/kg in terms of human dosage. .
  • a PD-1 signal inhibitor is used in combination with an anti-TSPAN8-anti-CD3 bispecific antibody of the present invention or a pharmaceutical composition of the present invention for the treatment of cancer in a subject.
  • the mechanism of action and therapeutic modality of the PD-1 signal inhibitor are not particularly limited as long as they block PD-1 signals.
  • the mechanism of action is, for example, inhibition of binding between molecules involved in PD-1 signal, reduction of expression level of PD-1 signal molecule (for example, inhibition of protein production or induction of protein degradation, etc.).
  • the therapeutic modality may be, for example, an antibody, a small molecule compound, a nucleic acid (which may include DNA or RNA, natural or artificial nucleic acid), a fusion protein, a peptide, or other therapeutic modality.
  • PD-1 signal inhibitors are determined by a method that measures the binding inhibition effect of two proteins, PD-1 and PD-L1 or PD-L2, and the expression reduction effect using the expression level of PD-1 signal molecules as an indicator. can be obtained.
  • an inhibitor of binding between PD-1 and PD-L1 or PD-L2 can be obtained by obtaining an inhibitor that binds to PD-1 and either PD-L1 or PD-L2, and then using the obtained inhibitor. They can be selected based on their ability to inhibit the binding between PD-1 and PD-L1 or PD-L2.
  • inhibitors that reduce the expression level of PD-1 signal molecules such as PD-1, PD-L1, or PD-L2 may reduce the amount of proteins such as PD-1, PD-L1, or PD-L2 in cells. It can be obtained as an index.
  • the inhibitory effect on PD-1 signals can be confirmed by the effects of T cell proliferation, interferon- ⁇ release, reporter assay, and the like.
  • the effect of an inhibitor to reduce 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.
  • Examples of PD-1 signal inhibitors include antibodies that inhibit PD-1 signals, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. Such antibodies may be humanized, chimeric, murine, human antibodies, as well as antigen-binding fragments thereof.
  • Known anti-PD-1 antibodies include, but are not limited to, US Pat. No. 8,008,449, US Pat. No. 6,808,710, US Pat. No. 7,488,802, US Pat. There are also antibodies described in WO 2006/121168 and WO 2012/145493.
  • Known anti-PD-L1 antibodies include, but are not limited to, WO 2007/005874, WO 2010/077634, WO 2011/066389, and WO 2013/079174.
  • the PD-1 signal inhibitor used in the invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or an antigen-binding fragment thereof.
  • the PD-1 signal inhibitor used in the invention is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.
  • the anti-PD-1 antibody may be an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, spartalizumab, cemiplimab.
  • the anti-PD-L1 antibody may be an anti-PD-L1 antibody such as atezolizumab, durvalumab, avelumab.
  • PD-1 signal inhibitors for example, AMP-224 (International Publication No. 2010/027827 and International Publication No. 2011/066342), BMS-1166 (Oncotarget, 2017; Vol. 8: p. 72167-72181), etc. Also included are fusion proteins and low molecular weight compounds that inhibit the binding of PD-1 to PD-L1.
  • Various PD-1 signal inhibitors are known in the art (Non-Patent Document 8).
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the invention and the PD-1 signal inhibitor are contained in the same pharmaceutical composition and are administered simultaneously to the subject; or (ii) contained in separate pharmaceutical compositions and administered to the subject simultaneously, sequentially, or sequentially.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for treating cancer in a subject, comprising an anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signal inhibitor.
  • a pharmaceutical composition of the invention is used in combination with another pharmaceutical composition comprising a PD-1 signal inhibitor.
  • the pharmaceutical composition of the present invention is a combination of pharmaceutical compositions (herein also referred to as "combination medicine of the present invention") for treating cancer in a subject, the combination includes a pharmaceutical composition comprising an anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and a PD-1 signal inhibitor.
  • the pharmaceutical combination of the invention is a combination of pharmaceutical compositions for use in a method of treating cancer in a subject, wherein the method of treatment comprises an anti-TSPAN8-anti-CD3 bispecific of the invention.
  • the method includes administering to the subject a pharmaceutical composition comprising a sex antibody and a PD-1 signal inhibitor.
  • the present invention also provides an anti-TSPAN8-anti-CD3 bispecific antibody used in combination with a PD-1 signal inhibitor to treat cancer in a subject (bispecific antibody for combination of the present invention).
  • the bispecific antibody for the combination of the invention is an anti-TSPAN8-anti-CD3 bispecific antibody for use in a method of treating cancer in a subject, the method of treatment comprising: , comprising administering the bispecific antibody and a PD-1 signal inhibitor to the subject.
  • the present invention also provides the use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for use in combination with a PD-1 signal inhibitor to treat cancer in a subject. use of bispecific antibodies).
  • the use of the bispecific antibody of the present invention is the use of an anti-TSPAN8-anti-CD3 bispecific antibody for the manufacture of a pharmaceutical composition for treating cancer in a subject, the pharmaceutical composition comprising: Used in combination with PD-1 signal inhibitors to treat target cancers.
  • the use of the bispecific antibodies of the invention includes the use of anti-TSPAN8-anti-CD3 bispecific antibodies for the manufacture of a pharmaceutical composition for use in a method of treating cancer in a subject.
  • the therapeutic method comprises administering the pharmaceutical composition and a PD-1 signal inhibitor to a subject.
  • the anti-TSPAN8-anti-CD3 bispecific antibodies used in the bispecific antibodies for the combination of the present invention and the use of the bispecific antibodies of the present invention are as described above.
  • the antibody may have the molecular structure, peptide linker, mutation, post-translational modification, etc. described in the section ⁇ Bispecific Antibodies''.
  • the present invention provides a method for treating cancer (the treatment method of the present invention) comprising administering to a subject an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor.
  • the anti-TSPAN8-anti-CD3 bispecific antibody used in the therapeutic method of the present invention has the molecular structure, peptide linker, and mutations described in the above section ⁇ Anti-TSPAN8-anti-CD3 bispecific antibody of the present invention>. , post-translational modifications, etc.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is administered to a subject simultaneously, sequentially, or sequentially with a PD-1 signal inhibitor.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered to the subject; (ii) in separate pharmaceutical compositions and administered to the subject simultaneously, sequentially, or sequentially.
  • the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention and the PD-1 signal inhibitor are (i) contained in the same pharmaceutical composition and administered to the subject; or (ii) in separate pharmaceutical compositions and administered to the subject on the same day.
  • administering is started after completion of administration of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention to the subject, or (b) After completion of administration of the PD-1 signal inhibitor to the subject, administration of the anti-TSPAN8-anti-CD3 bispecific antibody of the present invention is started.
  • the treatment method of the invention comprises sequential administration of an anti-TSPAN8-anti-CD3 bispecific antibody of the invention and a PD-1 signal inhibitor to a subject according to an administration regimen that includes administration cycles. This is a typical use.
  • the therapeutic methods of the invention begin administering the anti-TSPAN8-anti-CD3 bispecific antibody to the subject in at least one or all administration cycles, followed by Begin administration of inhibitor.
  • the treatment methods of the invention begin administering the PD-1 signal inhibitor to the subject in at least one or all cycles of administration, followed by administering the anti-TSPAN8-anti-CD3 bispecific Administration of the antibody or pharmaceutical composition is initiated.
  • the cancer treated by the pharmaceutical composition or treatment method of the present invention may be either solid cancer or blood cancer.
  • Cancers treated according to the present invention may be primary, metastatic, or peritoneally disseminated.
  • Cancers treated by the present invention are not particularly limited, but include, for example, gastric cancer, lung cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, Multiple myeloma, blood cancers such as T-cell lymphoma, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small Cellular lung cancer, mesothelioma, skin cancer, cutaneous T-cell lymphoma, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer,
  • the cancer to be treated according to the present invention is esophageal cancer, colon cancer, pancreatic cancer, stomach cancer, gastroesophageal junction cancer, liver cancer, biliary tract cancer, or prostate cancer.
  • the cancer is preferably esophageal cancer, colon cancer, pancreatic cancer, gastric cancer, or gastroesophageal junction cancer.
  • the dose of the anti-TSPAN8-anti-CD3 bispecific antibody or PD-1 signal inhibitor of the present invention to be administered to a subject depends on the degree of symptoms and age of the subject, the antibody used, the pharmaceutical composition, the dosage form of the inhibitor, etc. Alternatively, although it varies depending on the activity strength of the active ingredient, for example, about 0.0001 mg/kg to 1000 mg/kg can be used. In one embodiment, the amount of anti-TSPAN8-anti-CD3 bispecific antibodies of the invention administered to a subject is 0.0001 mg/kg to 1000 mg/kg. In one embodiment, the amount of anti-TSPAN8-anti-CD3 bispecific antibodies of the invention administered to a subject is from 0.001 mg/kg to 100 mg/kg. In one embodiment, the amount of anti-TSPAN8-anti-CD3 bispecific antibodies of the invention administered to a subject is from 0.01 mg/kg to 10 mg/kg.
  • Example 1 Production of anti-TSPAN8-anti-CD3 bispecific antibody
  • Example 1-1 Production of bispecific antibody vector for anti-TSPAN8 antibody
  • 16B11.1_HC_H shown in SEQ ID NO: 4 was created by introducing a mutation into the heavy chain (16B11.1_HC) of 16B11.1 shown in SEQ ID NO: 2. Specifically, the following three mutations were introduced into SEQ ID NO:2.
  • LALA mutations L234A and L235A in which leucine (L) at amino acid numbers 238 and 239 (EU index: 234 and 235) is replaced with alanine (A), (2) amino acid numbers 370, 372 and 411 (EU index: : 366, 368, and 407), respectively. 3) A mutation in which asparagine (N) is replaced with glycine (G) at amino acid number 301 (EU index: 297).
  • the designed heavy chain amino acid sequence of 16B11.1_HC_H is shown in SEQ ID NO: 4.
  • a polynucleotide encoding 16B11.1_HC_H of SEQ ID NO: 4 was introduced into a pcDNA3.4-TOPO vector (Thermo Fisher scientific).
  • the constructed vector is called pcDNA3.4-16B11.1_HC_H.
  • a polypeptide was designed in which the human ⁇ chain constant region amino acid sequence (sequence from amino acid number 108 to 213 of SEQ ID NO: 6) was linked to the C-terminal side of the light chain variable region of 16B11.1 (SEQ ID NO: 6). ).
  • a polynucleotide encoding the designed polypeptide was introduced into a pcDNA3.4 TOPO vector.
  • the constructed light chain vector is called pcDNA3.4-16B11_LC.
  • Example 1-2 Production of bispecific antibody vector for anti-human CD3 antibody
  • the sequence of the humanized anti-CD3 antibody was determined from the literature (Front Biosci., 2008; Vol. 13: It was designed according to the method described in p. 1619-1633). MOLSIS Inc.
  • the three-dimensional structure information (PDB Code: 5FCS) was analyzed using MOE, an integrated computational chemistry system provided by the company, and back mutations were introduced into the framework region.
  • Anti-CD3 scFv-Fc was prepared by combining a heavy chain variable region (SEQ ID NO: 8, amino acid numbers 1 to 125), a linker (SEQ ID NO: 8, amino acids 126 to 145), a light chain variable region (SEQ ID NO: 8, amino acids 146 to 254), a hinge ( It was designed to be arranged in the following order: SEQ ID NO: 8, amino acid numbers 255 to 269), CH2 domain (SEQ ID NO: 8, amino acids 270 to 379), and CH3 domain (SEQ ID NO: 8, amino acids 380 to 486).
  • SEQ ID NO: 8 has (1) a mutation in which the amino acids corresponding to amino acid numbers 44 and 247 are replaced with cysteine (C), and (2) the amino acid at amino acid number 259 (EU index: 220) is changed from C to S. (3) LALA mutation that substitutes amino acid number 273 and 274 (EU index: 234 and 235) from L to A; (4) substitution mutation that substitutes amino acid number 405 (EU index: 366) from T to tryptophan. A knobs-into-holes mutation in which (W) is substituted, and (5) a mutation in which amino acid number 336 (EU index: 297) is substituted from N to G have been introduced.
  • polynucleotides encoding amino acid sequences containing the respective mutation points were synthesized and inserted into the pcDNA3.1(+) vector (Thermo Fisher scientific, V79020).
  • the constructed vector is called pcDNA3.1-m7_scFV_K.
  • the nucleotide sequence and amino acid sequence of the produced anti-CD3scFv-Fc are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • Example 1-3 Production of anti-TSPAN8-anti-CD3 bispecific antibody
  • pcDNA3.4-16B11.1_HC_H a bispecific antibody composed of the Fab region, anti-CD3scFv region, and Fc region of anti-TSPAN8 antibody
  • pcDNA3.4-16B11.1_HC_H a bispecific antibody composed of the Fab region, anti-CD3scFv region, and Fc region of anti-TSPAN8 antibody
  • pcDNA3.4-16B11.1_HC_H To prepare a bispecific antibody composed of the Fab region, anti-CD3scFv region, and Fc region of anti-TSPAN8 antibody, pcDNA3.4-16B11.1_HC_H, pcDNA3.4-16B11_LC, and pcDNA3.1-m7_scFV_K were prepared using a conventional method.
  • ExpiCHO-S cells Thermo Fischer Scientific, A29127
  • the culture supernatant was purified using MabSelect SuRe (Cytiva, 17-5438-02), and further gel filtration column HiLoad (registered trademark) 26/600 Superdex (registered trademark) 200 pg (Cytiva, 28-9893-36)
  • HiLoad registered trademark
  • Superdex registered trademark
  • a purified antibody with a purity of 95% or more was obtained.
  • the resulting antibody is referred to as anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody.
  • Example 2 Effect of in vitro 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] RPMI 1640 (Thermo Fisher Scientific, 11875-119) with a final concentration of 10% FBS (Cytiva, SH30084.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070).
  • Anti-CD3 antibody BioLegend, 317315
  • PBS phosphate buffered saline
  • PanT cells Human peripheral blood mononuclear cells (LON) were isolated using PanT Cell Isolation Kit, human (Miltenyi Biotec, 130-096-535) according to the manufacturer's protocol. ZA, CC-2702) to PanT cells ( PanT cells, including both CD4 and CD8 T cells (hereinafter referred to as "PanT cells") were isolated. The isolated PanT cells were centrifuged, the supernatant was removed, and then suspended in effector culture medium. Suspended PanT cells were seeded at 1.5x10 6 cells/well in a 24-well plate immobilized with the aforementioned anti-CD3 antibody.
  • 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 ⁇ g/mL were added, and the total volume was adjusted to 1 mL/well with effector culture medium. .
  • Culture was performed at 37° C. in a 5% CO 2 incubator. Two days later, the PanT cells were collected, suspended in effector culture medium, and seeded in a 6-well tissue culture microplate (IWAKI, 3810-006) (hereinafter referred to as "6-well plate"). Further, human IL-2 at a final concentration of 10 ng/mL was added, and culture was performed at 37° C. in a 5% CO 2 incubator.
  • Example 2-2 Production of RKO cells stably expressing human TSPAN8-GFP
  • RKO cells a human colon cancer cell line, were obtained from the American Type Culture Collection (ATCC, CRL-2577).
  • RKO cells were grown in RPMI-1640 medium (Merck, R8758) containing FBS at a final concentration of 10% and penicillin-streptomycin at a final concentration of 1% (hereinafter referred to as "culture medium”) at 37°C and 5% CO2 . cultivated under.
  • TSPAN8 In order to produce cells expressing a fusion protein of TSPAN8 and GFP, a polypeptide containing human TSPAN8 was cut from TSPAN8 (Myc-DDK-tagged)-Human tetraspanin 8 (TSPAN8) (ORIGENE, RC202694) using a restriction enzyme. The polynucleotide encoding the peptide was subcloned into pCMV6-AC-GFP vector (ORIGENE, PS100010) (hereinafter referred to as "human TSPAN8-GFP expression vector").
  • the prepared human TSPAN8-GFP expression vector was lipofectioned into RKO cells using Lipofectamine 3000 (Thermo Fisher Scientific, L3000008) according to the manufacturer's recommended protocol.
  • Selective culture was performed in a culture medium supplemented with Geneticin Selective Antibiotic (Thermo Fisher Scientific, 10131-027) at a final concentration of 600 ⁇ g/mL.
  • Cells were separated using MACS Cell Separation (Miltenyi Biotec) to obtain RKO cell clones stably expressing human TSPAN8-GFP (hereinafter referred to as "RKO_hTSPAN8-GFP cells").
  • Example 2-3 Effect of combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody on in vitro cancer cell growth inhibition effect.
  • 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 inhibition was evaluated in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells. did.
  • RKO_hTSPAN8-GFP cells prepared in culture medium were seeded in a half-area 96-well microplate (Greiner Bio-One, 675090) (hereinafter referred to as "half-area plate") at 10 cells/well, and incubated at 37°C for 50 minutes. % CO2 incubator. After 3 hours, Expanded PanT cells prepared in the culture medium were seeded at 3 ⁇ 10 4 cells/well on the half area plate during culture.
  • the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody obtained in Example 1-3, and the anti-human PD-1 antibody nivolumab, pembrolizumab, or the anti-human PD-L1 antibody atezolizumab analog Dur Valumab analog was used as the test antibody.
  • Nivolumab was designed based on the full-length heavy chain and full-length light chain amino acid sequences (SEQ ID NOs: 17 and 18) of nivolumab described in International Publication No. 2014/055648.
  • Pembrolizumab was designed based on the full-length heavy chain and full-length light chain amino acid sequences (SEQ ID NOs: 31 and 36) of pembrolizumab described in WO 2008/156712.
  • Nivolumab and pembrolizumab were obtained from the designed sequences according to the method described in Example 11 of International Publication No. 2021/241616.
  • RecombiMAb human IgG4 (S228P) isotype control, anti-hen egg lysozyme (Bio X Cell, BE0349) was used as an isotype control for nivolumab and pembrolizumab.
  • V-bottom microplate After collecting floating cells in PBS containing 10% FBS on a PP plate 96V (Sumitomo Bakelite Co., Ltd., MS-3396P) (hereinafter referred to as "V-bottom microplate"), the adherent cells remaining on the half area plate were transferred to BD Phosflow Fix. After fixation with Buffer I (Becton, Dickinson and Company, 557870), the fluorescence intensity of GFP was measured using Infinite M200PRO (TECAN), and the value after subtracting the background was used as an index of cell proliferation. The results of four independent trials are shown in FIGS. 1 and 2. As shown in FIGS.
  • the anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody inhibited the proliferation of RKO_hTSPAN8-GFP cells in a co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells.
  • the RKO_hTSPAN8-GFP cell growth inhibition effect of the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab was stronger than that of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody, nivolumab or pembrolizumab alone. ( Figure 1).
  • Example 2-4 Effect of combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody on in vitro T cell activation
  • the floating cells collected in the V-bottom microplate in Example 2-3 were centrifuged at 540 ⁇ g for 5 minutes, the supernatant was removed, and 1/40 was added to autoMACS Running Buffer (Miltenyi Biotec, 130-091-221).
  • a liquid containing a certain amount of Human BD Fc Block (Becton, Dickinson and Company, 564220) was added at 20 ⁇ L/well.
  • PE anti-human CD4 Antibody BioLegend, 317410
  • autoMACS Running Buffer APC-H7 Mouse anti-Human CD8 (Becton, Dickinson) were added to each well. and Company, 560179
  • BV421 Mouse Anti-Human CD45 Becton, Dickinson and Company, 563879
  • APC anti-human CD25 BioLegend, 302610 were added at 10 ⁇ L/well and allowed to stand at 4° C. for 1 hour.
  • CD25 which is an activation marker for CD4- or CD8-positive cells
  • APC anti-human CD25 which is a fluorescently labeled antibody against CD25.
  • the results of three independent trials are shown in Figures 3-1, 3-2, 4-1, and 4-2.
  • Anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody was used to inhibit CD4T cells ( Figure 3-1, Figure 4-1) and CD8T cells ( Figure 3-2, Figure 4-2) induced CD25 expression.
  • CD25 in CD4T cells ( Figure 3-1) and CD8T cells ( Figure 3-2) by the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab was It was higher than the CD3 bispecific antibody, nivolumab, or pembrolizumab alone.
  • expression of CD25 in CD4T cells ( Figure 4-1) and CD8T cells ( Figure 4-2) by the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and atezolizumab analog or durvalumab analog was higher than anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody, atezolizumab analog, or durvalumab analog alone.
  • Example 2-5 Effect of combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-PD-1 antibody or anti-PD-L1 antibody on in vitro interferon- ⁇ production
  • the amount of interferon- ⁇ produced in the supernatant collected in Example 2-3 was measured using AlphaLISA Interferon- ⁇ Measurement Kit (Perkin Elmer, AL217F) according to the manufacturer's recommended protocol.
  • Figure 5 shows the combined effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab
  • Figure 6 shows the combination effect of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and atezolizumab analog or durval.
  • FIGS. 5 and 6 Shows combined action with mab analogues.
  • interferon- ⁇ production was detected in the co-culture system of RKO_hTSPAN8-GFP cells and Expanded PanT cells by adding anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody.
  • the amount of interferon- ⁇ produced by the combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and nivolumab or pembrolizumab was higher than that of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody, nivolumab or pembrolizumab alone. (Figure 5).
  • Example 3 Effect of in vivo combined use of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody.
  • B-hCD3E mice human CD3 ⁇ extracellular domain knock-in mice transplanted with human TSPAN8-expressing B16-F10 cells.
  • anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody was investigated.
  • Example 3-1 Construction of human TSPAN8-expressing B16-F10 cells
  • Mouse melanoma cell line B16-F10 cells were obtained from American Type Culture Collection (ATCC, CRL-6475). Dulbecco's modified Eagle's medium (SIGMA, D6429) supplemented with 10% FBS and 1% final concentration of penicillin-streptomycin (the prepared medium is hereinafter referred to as "Eagle culture medium”) at 37°C, 5% CO The cells were cultured under the following conditions.
  • SIGMA Dulbecco's modified Eagle's medium
  • Eagle culture medium penicillin-streptomycin
  • This vector was introduced into B16-F10 cells by electroporation.
  • a B16-F10 cell clone stably expressing human TSPAN8 (human TSPAN8-expressing B16-F10 cells) was obtained.
  • Example 3-2 Effect of combination of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody on in vivo antitumor action
  • B-hCD3E female mice C57BL/6-CD3e tm2 (CD3e) /Bcgen; Biocytogen, 110008) were obtained.
  • Human TSPAN8-expressing B16-F10 cells were suspended in Matrigel® Basement Membrane Matrix (Corning Inc., 356237) to prepare a cell suspension of 1 ⁇ 10 7 cells/mL.
  • the cell suspension was inoculated subcutaneously on the side of a 7- to 8-week-old mouse at 1 ⁇ 10 6 cells/100 ⁇ L.
  • Group 2 Anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody administration group Anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody adjusted to 0.1 mg/mL on day 0 and day 7 was administered intravenously at 1 mg/kg.
  • Group 3 Anti-mouse PD-1 antibody administration group On days 0, 3, 7, and 10, 10 mg/kg of anti-mouse PD-1 antibody (Bio Administered intravenously.
  • the tumor volumes of Group 2 and Group 3 on day 13 were compared with that of Group 4 by unpaired Student's t-test (FIG. 7-2).
  • the tumor volume in the combination group of anti-TSPAN8 (16B11)-anti-CD3 bispecific antibody and anti-mouse PD-1 antibody was It was significantly smaller than that of the single agent group and the anti-mouse PD-1 antibody single agent group.
  • the method for treating cancer using a combination of an anti-TSPAN8-anti-CD3 bispecific antibody and a PD-1 signal inhibitor of the present invention is expected to be useful for cancer treatment.
  • 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 in which the heavy chain fragment containing the heavy chain variable region of 16B11.1 and the first Fc polypeptide are linked, and the base sequence shown in SEQ ID NO: 3 is the amino acid sequence of the polypeptide shown in SEQ ID NO: 4. This is a base sequence encoding the amino acid sequence of a polypeptide in which a heavy chain fragment containing the heavy chain variable region of 16B11.1 and the first Fc polypeptide are linked.
  • sequence SEQ ID NO: 6 is the amino acid sequence of the light chain of 16B11.1, and the base sequence shown as SEQ ID NO: 5 is the base sequence encoding the light chain amino acid sequence of 16B11.1 shown in SEQ ID NO: 6.
  • SEQ ID NO: 8 is the amino acid sequence of a polypeptide in which the anti-CD3 scFv region and the second Fc polypeptide are linked, and the base sequence shown in SEQ ID NO: 7 is the amino acid sequence of the polypeptide in which the anti-CD3 scFv region and the second Fc polypeptide are linked together, as shown in SEQ ID NO: 8.
  • This is a base sequence that encodes the amino acid sequence of a polypeptide in which SEQ ID NOS: 9 to 17 are the amino acid sequences of various linkers described in the detailed description of the invention.

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Abstract

La présente invention aborde le problème consistant à fournir : un anticorps bispécifique anti-TSPAN8-anti-CD3 utilisé en association avec un inhibiteur de signal PD-1 pour le traitement d'un cancer cible ou une composition pharmaceutique contenant ledit anticorps bispécifique ; et une méthode de traitement du cancer comprenant l'administration, à une cible, de l'anticorps bispécifique anti-TSPAN8-anti-CD3 et de l'inhibiteur de signal PD-1.　L'association de l'anticorps bispécifique anti-TSPAN8 (16B11)-anti-CD3 et de l'inhibiteur de signal PD-1 est efficace pour le traitement du cancer exprimant TSPAN8.
PCT/JP2023/014792 2022-04-13 2023-04-12 Utilisation d'un anticorps bispécifique anti-tspan8-anti-cd3 associé à un inhibiteur de signal pd-1 pour le traitement du cancer Ceased WO2023199927A1 (fr)

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JP2016006026A (ja) * 2013-03-15 2016-01-14 ザ・トランスレーショナル・ジェノミクス・リサーチ・インスティチュート テトラスパニン８に対するハイブリドーマクローンおよびモノクローナル抗体
WO2016194992A1 (fr) * 2015-06-05 2016-12-08 中外製薬株式会社 Utilisation combinée d'activateurs immunitaires
JP2016540042A (ja) * 2013-11-05 2016-12-22 コグネート バイオサービシズ， インコーポレイテッド がんを処置するためのチェックポイント阻害剤および治療薬の組合せ
JP2019513701A (ja) * 2016-03-15 2019-05-30 ジェネロン（シャンハイ）コーポレイション リミテッド 多重特異性Ｆａｂ融合タンパクおよびその使用
JP2021508441A (ja) * 2017-12-05 2021-03-11 中外製薬株式会社 Ｃｄ３およびｃｄ１３７に結合する改変された抗体可変領域を含む抗原結合分子
WO2022102768A1 (fr) * 2020-11-16 2022-05-19 アステラス製薬株式会社 Anticorps bispécifique anti-tspan8-anti-cd3 et anticorps anti-tspan8

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Publication number Priority date Publication date Assignee Title
JP2011504933A (ja) * 2007-11-28 2011-02-17 メディミューン，エルエルシー タンパク質製剤
JP2016006026A (ja) * 2013-03-15 2016-01-14 ザ・トランスレーショナル・ジェノミクス・リサーチ・インスティチュート テトラスパニン８に対するハイブリドーマクローンおよびモノクローナル抗体
JP2016540042A (ja) * 2013-11-05 2016-12-22 コグネート バイオサービシズ， インコーポレイテッド がんを処置するためのチェックポイント阻害剤および治療薬の組合せ
WO2016194992A1 (fr) * 2015-06-05 2016-12-08 中外製薬株式会社 Utilisation combinée d'activateurs immunitaires
JP2019513701A (ja) * 2016-03-15 2019-05-30 ジェネロン（シャンハイ）コーポレイション リミテッド 多重特異性Ｆａｂ融合タンパクおよびその使用
JP2021508441A (ja) * 2017-12-05 2021-03-11 中外製薬株式会社 Ｃｄ３およびｃｄ１３７に結合する改変された抗体可変領域を含む抗原結合分子
WO2022102768A1 (fr) * 2020-11-16 2022-05-19 アステラス製薬株式会社 Anticorps bispécifique anti-tspan8-anti-cd3 et anticorps anti-tspan8

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