# US 11,897,954 B2 · Anti-TSPAN8/anti-CD3 bispecific antibody and anti-TSPAN8 antibody > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,897,954 B2 - Document ID: US11897954B2 - Status: Granted - Kind: B2 - Application number: 17/639,783 - Filed: 2021-11-15 - Published: 2023-11-02 - Granted: 2024-02-13 - Earliest claimed: 2020-11-16 - Assignees: Astellas Pharma Inc.; National Cancer Center - Applicants: Astellas Pharma Inc.; National Cancer Center - Inventors: Yoshiyuki Tenda; Masatoshi Yuri; Daisuke Yamajuku; Takeshi Tsutsumi; Yuko Kusuzaki; Hiroki Sasaki; Fumiko Chiwaki; Masayuki Komatsu - Web page: https://patentgrep.com/patent/US11897954B2 ## CPC - C07K 16/28: against receptors, cell surface antigens or cell surface determinants - A61P 35/00: Antineoplastic agents - C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex} - C12N 5/12: Fused cells, e.g. hybridomas - A61K 2039/505: {comprising antibodies} - C07K 2317/21: from primates, e.g. man - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/31: multispecific - C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity - C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues - C07K 2317/40: characterized by post-translational modification - C07K 2317/52: Constant or Fc region; Isotype - C07K 2317/55: Fab or Fab' - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - C07K 2317/565: Complementarity determining region [CDR] - C07K 2317/622: Single chain antibody (scFv) - C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation - C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC] - C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding - C12N 2740/16043: viral genome or elements thereof as genetic vector - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract An objective of the present invention is to provide an anti-TSPAN8/anti-CD3 bispecific antibody and an anti-TSPAN8 antibody usable in treatment or prevention in human. A human monoclonal antibody producing mouse was immunized with a peritoneal disseminated cancer cell isolated from a patient, to obtain an antibody 16B11 and an antibody 16B12 that selectively bind to a peritoneal disseminated cancer cell. These antibodies were anti-TSPAN8 antibodies that bind to the region from amino acid positions 126 to 155 of TSPAN8 and exhibited strong binding activity to TSPAN8 expressed in the peritoneal disseminated cancer cell. Further, an anti-TSPAN8(16B11)-anti-CD3 bispecific antibody produced based on the sequence of 16B11 exhibited a cytotoxic activity against the TSPAN8-expressing cancer cell in vitro, exerted an anti-tumor action on TSPAN8-expressing cancer cell-bearing mice in vivo, and extended the lifetime of peritoneal dissemination model mice. ## Claims ### Claim 1 A bispecific antibody that binds to TSPAN8 and CD3, comprising: (a) a Fab region of an anti-TSPAN8 antibody consisting of: a heavy chain fragment comprising a heavy chain variable region of the anti-TSPAN8 antibody; and a light chain comprising a light chain variable region of the anti-TSPAN8 antibody; (b) an anti-CD3-scFv region comprising a heavy chain variable region and a light chain variable region of an anti-CD3 antibody; and (c) a Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region (a) and a second Fc polypeptide linked to the anti-CD3-scFv region (b), wherein the heavy chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting amino acids 31 to 35 of SEQ ID NO: 6, a CDR2 consisting of amino acids 50 to 66 of SEQ ID NO: 6, and a CDR3 consisting of amino acids 99 to 110 of SEQ ID NO: 6, and the light chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of amino acids 24 to 34 of SEQ ID NO: 8, a CDR2 consisting of amino acids 50 to 56 of SEQ ID NO: 8, and a CDR3 consisting of amino acids 89 to 96 of SEQ ID NO: 8; or the heavy chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of amino acids 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of amino acids 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of amino acids 99 to 110 of SEQ ID NO: 10, and the light chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of amino acids 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of amino acids 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of amino acids 89 to 96 of SEQ ID NO: 12. ### Claim 2 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting amino acids 31 to 35 of SEQ ID NO: 6, a CDR2 consisting of amino acids 50 to 66 of SEQ ID NO: 6, and a CDR3 consisting of amino acids 99 to 110 of SEQ ID NO: 6, and the light chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of amino acids 24 to 34 of SEQ ID NO: 8, a CDR2 consisting of amino acids 50 to 56 of SEQ ID NO: 8, and a CDR3 consisting of amino acids 89 to 96 of SEQ ID NO: 8. ### Claim 3 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of amino acids 31 to 35 of SEQ ID NO: 10, a CDR2 consisting of amino acids 50 to 66 of SEQ ID NO: 10, and a CDR3 consisting of amino acids 99 to 110 of SEQ ID NO: 10, and the light chain variable region of the anti-TSPAN8 antibody comprises a CDR1 consisting of amino acids 24 to 34 of SEQ ID NO: 12, a CDR2 consisting of amino acids 50 to 56 of SEQ ID NO: 12, and a CDR3 consisting of amino acids 89 to 96 of SEQ ID NO: 12. ### Claim 4 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain variable region of the anti-TSPAN8 antibody consists of amino acids 1 to 121 of SEQ ID NO: 6, and the light chain variable region of the anti-TSPAN8 antibody consists of amino acids 1 to 107 of SEQ ID NO: 8. ### Claim 5 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain variable region of the anti-TSPAN8 antibody consists of amino acids 1 to 121 of SEQ ID NO: 10, and the light chain variable region of the anti-TSPAN8 antibody consists of amino acids 1 to 107 of SEQ ID NO: 12. ### Claim 6 (depends on 1) The bispecific antibody according to claim 1, wherein the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of amino acids 1 to 219 of SEQ ID NO: 6 and a light chain consisting of an amino acid sequence of SEQ ID NO: 8. ### Claim 7 (depends on 1) The bispecific antibody according to claim 1, wherein the Fab region of the anti-TSPAN8 antibody consists of a heavy chain fragment consisting of an amino acid sequence from amino acid positions 1 to 219 of SEQ ID NO: 10 and a light chain consisting of an amino acid sequence of SEQ ID NO: 12. ### Claim 8 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of amino acids 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of amino acids 50 to 68 of SEQ ID NO: 14, and a CDR3 consisting of amino acids 101 to 114 of SEQ ID NO: 14, and the light chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of amino acids 168 to 181 of SEQ ID NO: 14, a CDR2 consisting of amino acids 197 to 203 of SEQ ID NO: 14, and a CDR3 consisting of amino acids 236 to 244 of SEQ ID NO: 14. ### Claim 9 (depends on 2) The bispecific antibody according to claim 2, wherein the heavy chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of amino acids 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of amino acids 50 to 68 of SEQ ID NO: 14, and a CDR3 consisting of amino acids 101 to 114 of SEQ ID NO: 14, and the light chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of amino acids 168 to 181 of SEQ ID NO: 14, a CDR2 consisting of amino acids 197 to 203 of SEQ ID NO: 14, and a CDR3 consisting of amino acids 236 to 244 of SEQ ID NO: 14. ### Claim 10 (depends on 3) The bispecific antibody according to claim 3, wherein the heavy chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of amino acids 31 to 35 of SEQ ID NO: 14, a CDR2 consisting of amino acids 50 to 68 of SEQ ID NO: 14, and a CDR3 consisting of amino acids 101 to 114 of SEQ ID NO: 14, and the light chain variable region of the anti-CD3 antibody comprises a CDR1 consisting of amino acids 168 to 181 of SEQ ID NO: 14, a CDR2 consisting of amino acids 197 to 203 of SEQ ID NO: 14, and a CDR3 consisting of amino acids 236 to 244 of SEQ ID NO: 14. ### Claim 11 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain variable region of the anti-CD3 antibody consists of amino acids 1 to 125 of SEQ ID NO: 14, and the light chain variable region of the anti-CD3 antibody consists of amino acids 146 to 254 of SEQ ID NO: 14. ### Claim 12 (depends on 4) The bispecific antibody according to claim 4, wherein the heavy chain variable region of the anti-CD3 antibody consists of amino acids 1 to 125 of SEQ ID NO: 14, and the light chain variable region of the anti-CD3 antibody consists of amino acids 146 to 254 of SEQ ID NO: 14. ### Claim 13 (depends on 5) The bispecific antibody according to claim 5, wherein the heavy chain variable region of the anti-CD3 antibody consists of amino acids 1 to 125 of SEQ ID NO: 14, and the light chain variable region of the anti-CD3 antibody consists of amino acids 146 to 254 of SEQ ID NO: 14. ### Claim 14 (depends on 1) The bispecific antibody according to claim 1, wherein the anti-CD3-scFv region consists of amino acids 1 to 254 of SEQ ID NO: 14. ### Claim 15 (depends on 1) The bispecific antibody according to claim 1, wherein the Fc region comprises a LALA mutation, a N297G mutation, and a knobs-into-holes mutation, wherein the knobs-into-holes mutation is T366W mutation in one Fc polypeptide included in the Fc region, and T366S, L368A and Y407V mutation in another Fc polypeptide included in the Fc region, wherein positions of mutation are amino acid positions in human Igyl constant region according to EU index. ### Claim 16 (depends on 1) The bispecific antibody according to claim 1, wherein the first Fc polypeptide consists of amino acids 235 to 451 of SEQ ID NO: 6 and the second Fc polypeptide consists of amino acids 270 to 486 of SEQ ID NO: 14. ### Claim 17 (depends on 1) The bispecific antibody according to claim 1, wherein the heavy chain fragment of the anti-TSPAN8 antibody and the first Fc polypeptide are linked through a hinge region, and the anti-CD3-scFv region and the second Fc polypeptide are linked through a hinge region. ### Claim 18 (depends on 1) The bispecific antibody according to claim 1 further comprising a post-translational modification selected from pyroglutamylation at a N-terminal of the heavy chain variable region of the anti-TSPAN8 antibody or the anti-CD3-scFv, a lysine deletion at a C-terminal of a heavy chain variable region of the anti-TSPAN8 antibody or the anti-CD3-scFv, or a combination thereof. ### Claim 19 A bispecific antibody that binds to TSPAN8 and CD3, comprising: (a) a Fab region of an anti-TSPAN8 antibody consisting of: a heavy chain fragment comprising a heavy chain variable region of the anti-TSPAN8 antibody; and a light chain comprising a light chain variable region of the anti-TSPAN8 antibody; (b) an anti-CD3-scFv region comprising a heavy chain variable region and a light chain variable region of an anti-CD3 antibody; and (c) a Fc region consisting of a first Fc polypeptide linked to the heavy chain fragment of the Fab region (a) and a second Fc polypeptide linked to the anti-CD3-scFv region (b), wherein the bispecific antibody comprises: a heavy chain of the anti-TSPAN8 antibody consisting of an amino acid sequence of SEQ ID NO: 6 in which the heavy chain fragment of the anti-TSPAN8 antibody is linked to the first Fc polypeptide; a light chain of the anti-TSPAN8 antibody consisting of an amino acid sequence of SEQ ID NO: 8; and a polypeptide consisting of an amino acid sequence of SEQ ID NO: 14 in which the anti-CD3-scFv region is linked to the second Fc polypeptide. ### Claim 20 (depends on 1) A method of treating cancer, comprising administering to a subject with cancer a bispecific antibody according to claim 1.