A combination of an antibody specific for a tumor antigen and a CD47 inhibitor

Abstract

The present invention relates to a CD47 inhibitor for use in the treatment or prevention of cancer by simultaneous or sequential administration with an antibody specific for a tumor antigen. The present invention also relates to a pharmaceutical composition comprising a CD47 inhibitor and an antibody specific for a tumor antigen.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition comprising a CD47 inhibitor. The present invention also relates to a CD47 inhibitor for use in the treatment or prevention of cancer, and also to a treatment method comprising the step of administering a CD47 inhibitor.

Background Art

[0002] CD47 (also known as integrin-associated protein: IAP) is a 50 kDa transmembrane protein receptor having an extracellular N-terminal IgV domain, a five-pass transmembrane domain, and a short C-terminal intracellular tail. CD47 is expressed as a marker for "self" on erythrocytes and is also highly expressed on tumor cells. High CD47 expression on tumor cells has been observed to act as a negative prognostic factor for survival in acute myeloid leukemia and some solid tumor cancers (Patent Document 1).

[0003] SIRPα (SHPS-1) is a single-pass transmembrane molecule belonging to the Ig superfamily that is present in myeloid cells such as macrophages, dendritic cells, and neutrophils, as well as glial cells (Non-Patent Document 1). Its extracellular region consists of a single IgV domain and two IgC domains. The IgV domain (also referred to as the "D1 domain" (Non-Patent Document 2)), which is the binding position to CD47, has been reported to have 10 variants in humans (Non-Patent Document 3). On the other hand, its intracellular region contains an immunoreceptor tyrosine-based inhibition motif (ITIM). When the extracellular region of SIRPα binds to CD47, the binding of SHP-1 and SEPHP-2, which are tyrosine dephosphorylating enzymes, is induced, and an inhibitory signal is transmitted.

[0004] The interaction between SIRPα and CD47 causes physiological phenomena, that is, it has been reported that CD47 on red blood cells binds to SIRPα on macrophages and transmits a "phagocytosis rejection" signal, as a result of which unwanted phagocytosis by red blood cells can be avoided (Non-Patent Document 4). Also under the tumor microenvironment, as mentioned above, CD47 highly expressed on tumor cells is suggested to bind to SIRPα on macrophages and dendritic cells, suppressing the phagocytic activity of phagocytosing tumor cells. When the phagocytic activity is suppressed, subsequent presentation of tumor antigens to T cells is also suppressed, and further subsequent tumor immune responses are also suppressed. Therefore, the immune phenomenon, that is, phagocytosis against tumor cells, is considered a checkpoint against the invasion of tumor antigens.

[0005] Patent Document 1 discloses a construct that binds to a CD47 protein for use in cancer treatment. The construct is a polypeptide comprising the D1 domain of SIRPα and an Fc domain monomer linked to its N-terminus or C-terminus.

[0006] An antibody-drug conjugate (ADC) contains a drug with cytotoxic effects conjugated to an antibody whose antigen binds to an antigen expressed on the surface of cancer cells and capable of internalization into cells. Therefore, ADC causes the accumulation of the drug in cancer cells and selectively delivers the drug to cancer cells to kill the cancer cells.

[0007] As one such antibody-drug conjugate, an antibody-drug conjugate containing an antibody and a derivative of exatecan, which is a topoisomerase I inhibitor, as its components is known (Patent Documents 2 to 8, Non-Patent Documents 5 to 8).

[0008] Patent Documents 2 to 8 disclose that the above-mentioned antibody-drug conjugate can be administered in combination with any one of various cancer therapeutics.

[0009] However, there are no test results showing a combinatorial effect superior to that of the aforementioned antibody-drug conjugate when used in combination with a CD47 inhibitor, nor is there any disclosure of scientific basis to suggest such results.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0012] Antibodies specific for tumor-associated antigens are known to have a therapeutic effect in cancer patients. However, when such antibodies alone do not bring about a complete remission in patients, or in situations where it is desired to reduce the dosage of such antibodies required for treatment, an improvement in treatment for treating cancer is needed. Therefore, it is necessary to obtain an excellent antitumor effect from such antibodies.

[0013] An object of the present invention is to provide a pharmaceutical composition in which an inhibitor of CD47 protein and an antibody specific for a tumor antigen are combined, and / or a treatment method including the step of administering an inhibitor of CD47 protein and an antibody specific for a tumor antigen to a subject in combination. [Means for Solving the Problems]

[0014] As a result of painstaking research to solve the above problems, the present inventors have found that the combined administration of a CD47 inhibitor and an antibody specific for a tumor antigen exhibits an excellent combined effect and complements the present invention. Specifically, the present invention includes the following aspects of the present invention. (1) i. A CD47 inhibitor; and ii. An antibody specific for a tumor antigen A pharmaceutical composition comprising the same. (2) The pharmaceutical composition according to (1), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative. (3) The pharmaceutical composition according to (2), wherein the SIRPα derivative comprises a polypeptide having a sequence identity of at least 80%, 90%, 95%, 99%, or 100% to residues 1 to 149 of SEQ ID NO: 15 or 16. (4) The pharmaceutical composition according to (1) or (2), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region. (5) The pharmaceutical composition according to (4), wherein the CD47 inhibitor comprises a polypeptide having a sequence identity of at least 80%, 90%, 95%, 99%, or 100% to SEQ ID NO: 15 or 16. (6) The pharmaceutical composition according to any one of (1) to (5), wherein the CD47 inhibitor is evolocumab. (7) The pharmaceutical composition according to (2), wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107, and SL-172154. (8) The pharmaceutical composition according to (1), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, and the antibody is specific for CD47. (9) The pharmaceutical composition according to (8), wherein the antibody specific for CD47 is selected from the group consisting of magrolimab, remsoipalimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (ligufalimab), and IMM-0306. (10) The pharmaceutical composition according to (1), wherein the CD47 inhibitor comprises a small molecule drug capable of binding to CD47. (11) The pharmaceutical composition according to (10), wherein the low molecular weight agent is selected from the group consisting of RRx-001 and IMM-01. (12) The pharmaceutical composition according to any one of (1) to (11), wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO: 13. (13) Further comprising an antibody-drug conjugate, wherein the antibody specific for a tumor antigen is part of the antibody-drug conjugate, the antibody-drug conjugate further comprises a linker and a drug, the antibody specific for a tumor antigen is connected to the drug via the linker, and the linker and the drug form a drug-linker. The pharmaceutical composition according to any one of (1) to (12). (13a) Further comprising an antibody-drug conjugate, the antibody-drug conjugate comprising an antibody specific for a tumor antigen connected to the drug via a linker, and the linker and the drug form a drug-linker. The pharmaceutical composition according to any one of (1) to (12). (14) The drug-linker is represented by the following formula:

[0015]

Chemical formula

[0016] [Chemical formula] [wherein A represents the connection position to the antibody] A CD47 inhibitor for the use according to (52), wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond. (54) A CD47 inhibitor for the use according to any one of (40) to (53), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-GPR20 antibody, or an anti-CDH6 antibody. (55) A CD47 inhibitor for the use according to (54), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody. (56) A CD47 inhibitor for the use according to (55), wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2. (57) A CD47 inhibitor for the use according to (55), wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2. A CD47 inhibitor for use according to any one of (55) to (57) as a dependent claim of (53), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. A CD47 inhibitor for use according to (54), wherein the antibody specific for the tumor antigen is an anti-HER3 antibody. A CD47 inhibitor for use according to (59), wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4. A CD47 inhibitor for use according to (60), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. A CD47 inhibitor for use according to any one of (59) to (61) as a dependent claim of (53), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. A CD47 inhibitor for use according to (54), wherein the antibody specific for the tumor antigen is an anti-TROP2 antibody. A CD47 inhibitor for use according to (63), wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 6. A CD47 inhibitor for use according to (64), wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. A CD47 inhibitor for use according to any one of (63) to (65) as a dependent claim of (53), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5. A CD47 inhibitor for use according to (54), wherein the antibody specific for the tumor antigen is an anti-B7-H3 antibody. (68) The CD47 inhibitor for use according to (67), wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 8. (69) The CD47 inhibitor for use according to (68), wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (70) The CD47 inhibitor for use according to any one of (67) to (69) as a dependent claim of (53), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5. (71) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-GPR20 antibody. (72) The CD47 inhibitor for use according to (71), wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 10. (73) The CD47 inhibitor for use according to (72), wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (74) The CD47 inhibitor for use according to any one of (71) to (73) as a dependent claim of (53), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. (75) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody. (76) The CD47 inhibitor for use according to (75), wherein the anti-CDH6 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 12. (77) The CD47 inhibitor for use according to (76), wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. A CD47 inhibitor for use as a dependent claim of (53), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. (79) A CD47 inhibitor for use as described in any one of (40) to (78), wherein the CD47 inhibitor and an antibody specific for a tumor antigen are active ingredients individually contained in different formulations. (80) A CD47 inhibitor for use as described in any one of (40) to (79), wherein the cancer is at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophagogastric junction adenocarcinoma, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine cancer, sarcoma, head and neck cancer, hepatocellular cancer, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia, and myelodysplastic syndrome. (81) A treatment method comprising the step of administering in combination a CD47 inhibitor and an antibody specific for a tumor antigen to a subject in need of treatment. (82) The treatment method according to (81), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative. (83) The treatment method according to (82), wherein the SIRPα derivative comprises a polypeptide having a sequence identity of at least 80%, 90%, 95%, 99%, or 100% to residues 1 to 149 of SEQ ID NO: 15 or 16. (84) The treatment method according to (81) or (82), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region. (85) The treatment method according to (84), wherein the CD47 inhibitor comprises a polypeptide having a sequence identity of at least 80%, 90%, 95%, 99%, or 100% to SEQ ID NO: 15 or 16. (86) The treatment method according to any one of (81) to (85), wherein the CD47 inhibitor is evolocumab. (87) The treatment method according to (82), wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107, and SL-172154. (88) The treatment method according to (81), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, and the antibody is specific for CD47. (89) The treatment method according to (88), wherein the antibody specific for CD47 is selected from the group consisting of magrolimab, remso-palrimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (ligufalimab), and IMM-0306. (90) The treatment method according to (81), wherein the CD47 inhibitor comprises a small molecule drug capable of binding to CD47. (91) The treatment method according to (90), wherein the small molecule drug is selected from the group consisting of RRx-001 and IMM-01. (92) The treatment method according to any one of (81) to (91), wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO: 13. (93) The treatment method according to any one of (81) to (92), further comprising the step of administering an antibody-drug conjugate, wherein the antibody specific for a tumor antigen is part of the antibody-drug conjugate, the antibody-drug conjugate further comprises a linker and a drug, the antibody specific for the tumor antigen is connected to the drug via the linker, and the linker and the drug form a drug-linker. (93a) The treatment method according to any one of (81) to (92), further comprising the step of administering an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody specific for a tumor antigen connected to the drug via a linker, and the linker and the drug form a drug-linker. (94) The drug-linker has the following formula:

[0017] [In the formula, A represents the connection position to the antibody] [wherein, A represents the connection position to the antibody] A drug-linker represented by (93) or (93a), wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond. (95) The therapeutic method according to any one of (81) to (94), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-GPR20 antibody, or an anti-CDH6 antibody. (96) The therapeutic method according to (95), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody. (97) The therapeutic method according to (96), wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence of amino acid residues 1 to 214 of SEQ ID NO: 2. (98) The therapeutic method according to (96), wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2. (99) The therapeutic method according to any one of (96) to (98) as a dependent claim of (94), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. (100) The therapeutic method according to (95), wherein the antibody specific for a tumor antigen is an anti-HER3 antibody. (101) The therapeutic method according to (100), wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4. (102) The therapeutic method according to (101), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (103) The therapeutic method according to any one of (100) to (102) as a dependent claim of (94), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. (104) The therapeutic method according to (95), wherein the antibody specific for a tumor antigen is an anti-TROP2 antibody. (105) The therapeutic method according to (104), wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 6. (106) The therapeutic method according to (105), wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (107) The therapeutic method according to any one of (104) to (106) as a dependent claim of (94), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5. (108) The therapeutic method according to (95), wherein the antibody specific for the tumor antigen is an anti-B7-H3 antibody. (109) The therapeutic method according to (108), wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 8. (110) The therapeutic method according to (109), wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (111) The therapeutic method according to any one of (108) to (110) as a dependent claim of (94), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5. (112) The therapeutic method according to (95), wherein the antibody specific for the tumor antigen is an anti-GPR20 antibody. (113) The therapeutic method according to (112), wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 10. (114) The therapeutic method according to (113), wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (115) The method of treatment according to any one of (112) to (114) as a dependent claim of (94), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. (116) The method of treatment according to (95), wherein the antibody specific for the tumor antigen is an anti-CDH6 antibody. (117) The method of treatment according to (116), wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 11 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 12. (118) The method of treatment according to (117), wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (119) The method of treatment according to any one of (116) to (118) as a dependent claim of (94), wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8. (120) The method of treatment according to any one of (81) to (119), wherein the CD47 inhibitor and the antibody specific for the tumor antigen are active ingredients individually contained in different formulations. (121) The method of treatment according to any one of (81) to (120), which is a method for treating at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophagogastric junction adenocarcinoma, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine carcinosarcoma, head and neck cancer, hepatocellular cancer, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia, and myelodysplastic syndrome. (122) An antibody specific for a tumor antigen for use in the treatment or prevention of cancer by simultaneous or sequential administration with a CD47 inhibitor. (123) The antibody specific for a tumor antigen for use according to (122), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative. An antibody specific for a tumor antigen for use according to (123), wherein the SIRPα derivative comprises a polypeptide having a sequence identity of at least 80%, 90%, 95%, 99%, or 100% to residues 1 to 149 of SEQ ID NO: 15 or 16. An antibody specific for a tumor antigen for use according to (122) or (123), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region. An antibody specific for a tumor antigen for use according to (125), wherein the CD47 inhibitor comprises a polypeptide having a sequence identity of at least 80%, 90%, 95%, 99%, or 100% to SEQ ID NO: 15 or 16. An antibody specific for a tumor antigen for use according to any one of (122) to (126), wherein the CD47 inhibitor is evolocumab. An antibody specific for a tumor antigen for use according to (123), wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107, and SL-172154. An antibody specific for a tumor antigen for use according to (122), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, and the antibody is specific for CD47. An antibody specific for a tumor antigen for use according to (129), wherein the antibody specific for CD47 is selected from the group consisting of magrolimab, remsoipalimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (ligufalimab), and IMM-0306. An antibody specific for a tumor antigen for use according to (122), wherein the CD47 inhibitor comprises a small molecule agent capable of binding to CD47. An antibody specific for a tumor antigen for use according to (131), wherein the small molecule agent is selected from the group consisting of RRx-001 and IMM-01. (133) An antibody specific for a tumor antigen for use in any one of (122) to (132), wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO: 13. (134) An antibody specific for a tumor antigen, which is part of an antibody-drug conjugate, the antibody-drug conjugate comprising an antibody specific for a tumor antigen connected to a drug via a linker, wherein the linker and the drug form a drug-linker, for use in any one of (122) to (133), an antibody specific for a tumor antigen. (135) The drug-linker has the following formula:

[0018]

Chemical formula

Advantages of the Invention

[0019] The present invention can provide a pharmaceutical composition in which a CD47 inhibitor and an antibody specific for a tumor antigen are administered in combination, and / or a treatment method including the step of administering a CD47 inhibitor and an antibody specific for a tumor antigen in combination to a subject.

[0020] Definition As used herein, the term "tumor antigen" refers to an antigenic substance produced within tumor cells. "Tumor antigen" includes both "tumor-specific antigen" (i.e., an antigen that exists only on tumor cells and not on other cell types) and "tumor-associated antigen" (i.e., an antigen that exists on some tumor cells and also on some normal cells, but is expressed at a high level on tumor cells). "Tumor antigen" includes HER2, HER3, TROP2, B7-H3, GPR20, and CDH6.

[0021] As used herein, the term "SIRPα" means signal regulatory protein α. In some embodiments, the amino acid sequence of SIRPα is the sequence of human SIRPα protein disclosed in GenBank accession number: NP_001035111 and set forth in SEQ ID NO: 14.

[0022] As used herein, the term "CD47" means CD (cluster of differentiation) 47 protein. In some embodiments, the amino acid sequence of CD47 is the sequence set forth in SEQ ID NO: 13.

[0023] As used herein, the term "small molecule" means an organic molecule having a small molecular weight, such as a molecular weight of less than 1000 Da.

[0024] As used herein, the term "antigen-binding fragment" of an antibody means a partial fragment of an antibody having antigen-binding activity, including Fab, F(ab’)2, scFv, etc. The term "antigen-binding fragment" also includes Fab’, which is a monovalent fragment within the variable region of an antibody obtained by treating F(ab’)2 under reducing conditions. However, the term "antigen-binding fragment" is not limited to these molecules as long as the fragment has binding affinity for the antigen. Furthermore, these antigen-binding fragments include not only fragments obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using a genetically modified antibody gene.

[0025] As used herein, "identity" between two amino acid sequences has the following meaning. The identity between two amino acid sequences having completely identical amino acid sequences is 100%. Assuming that one of the amino acid sequences has substitutions, deletions, or additions compared to the other amino acid sequence of one or two or more amino acids or amino acid residues, the identity between these two amino acid sequences is less than 100%. Examples of algorithms or programs for determining identity between two sequences considering gaps include those known to those skilled in the art, such as BLAST (Altschul et al., Nucleic Acids Res., Vol. 25, pp. 3389-3402, 1997), BLAST2 (Altschul et al., J. Mol. Biol., Vol. 215, pp. 403-410, 1990), and Smith-Waterman (Smith et al., J. Mol. Biol., Vol. 147, pp. 195-197, 1981).

[0026] As used herein, the term "antibody specific for a target antigen" means that the antibody preferentially binds to the target antigen as compared to non-related target antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0027]

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Mode for Carrying Out the Invention

[0028] In the following, preferred ways for carrying out the present invention will be described. The embodiments described below are given only for exemplifying an example of typical embodiments of the present invention and are not intended to limit the scope of the present invention.

[0029] 1. CD47 Inhibitor A CD47 inhibitor is an agent that blocks the binding between CD47 and SIRPα. Tumor cells highly express CD47. When SIRPα expressed on phagocytic cells having phagocytic activity binds to and interacts with CD47, a "phagocytosis rejection" signal is transmitted to the phagocytic cells. In this way, tumor cells escape from phagocytosis by phagocytic cells. A CD47 inhibitor inhibits the binding between CD47 and SIRPα, thereby inhibiting the transmission of the "phagocytosis rejection" signal from tumor cells to phagocytic cells, thereby enhancing phagocytosis of tumor cells by phagocytic cells. As a result, an antitumor effect can be exerted. Examples of phagocytic cells having phagocytic activity include macrophages such as M1 macrophages and M2 macrophages, and dendritic cells such as immature dendritic cells (imDC).

[0030] In some embodiments of the present invention, the CD47 inhibitor is a polypeptide comprising the SIRPα protein. For example, in some embodiments, the CD47 inhibitor is a polypeptide comprising the sequence of SEQ ID NO: 14. In other embodiments, the CD47 inhibitor comprises a derivative of the SIRPα protein, i.e., a fragment of the SIRPα protein and / or a protein having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14. The derivative of the SIRPα protein retains the binding specificity for the CD47 protein. For example, in some embodiments, the derivative of the SIRPα protein comprises the D1 domain of the SIRPα protein. In particular, in one example, the CD47 inhibitor comprises at least 80%, 90%, 95%, 99%, or 100% sequence identity to a polypeptide having residues 1 to 149 of SEQ ID NO: 15 or 16.

[0031] In some alternative embodiments, the CD47 inhibitor is a fusion protein comprising the SIRPα protein or a derivative thereof, linked to an additional polypeptide at the N-terminus or C-terminus of the SIRPα protein or a derivative thereof. In a preferred embodiment, the additional polypeptide comprises a region of the Fc domain. In some variations of this embodiment, the Fc domain is mutated compared to its wild-type version to abolish or reduce binding to the Fcγ receptor. In some embodiments comprising the SIRPα protein and the Fc domain, the SIRPα protein is directly conjugated to the Fc domain, whereas in other embodiments, the SIRPα protein is connected to the Fc domain via a linker (e.g., a spacer) between them. In some further embodiments, the CD47 inhibitor is further linked to one or more additional components, such as a polymer (e.g., a PEG polymer), to improve its pharmacokinetic properties.

[0032] In a particularly preferred embodiment, the CD47 inhibitor comprises a polypeptide comprising the sequence of SEQ ID NO: 15. It should be understood that amino acid residues 1 to 149 of SEQ ID NO: 15 correspond to the SIRPα protein, and the region of residues 150 to 376 of SEQ ID NO: 15 corresponds to the human IgG1 Fc protein. Thus, in some variants of this embodiment, the CD47 inhibitor has a first region having at least 80%, 90%, 95%, or 99% sequence identity to residues 1 to 149 of SEQ ID NO: 15, linked to a second region having at least 80%, 90%, 95%, or 99% sequence identity to residues 150 to 376 of SEQ ID NO: 15, and comprises a polypeptide that retains binding to the CD47 protein.

[0033] In another preferred embodiment, the CD47 inhibitor comprises a polypeptide comprising the sequence of SEQ ID NO: 16. It should be understood that amino acid residues 1 to 149 of SEQ ID NO: 16 correspond to the SIRPα protein, and the region of residues 150 to 371 of SEQ ID NO: 16 corresponds to the mouse IgG1 Fc protein. Thus, in some variants of this embodiment, the CD47 inhibitor has a first region having at least 80%, 90%, 95%, or 99% sequence identity to residues 1 to 149 of SEQ ID NO: 16, linked to a second region having at least 80%, 90%, 95%, or 99% sequence identity to residues 150 to 371 of SEQ ID NO: 16, and comprises a polypeptide that retains binding to the CD47 protein.

[0034] In a particularly preferred embodiment, the CD47 inhibitor is evolocumab, also known as ALX148.

[0035] In yet a further embodiment, the CD47 inhibitor is one of TTI-621 (WO2014 / 094122), TTI-622 (WO2014 / 094122), DSP-107 (WO2018 / 127919), and SL-172154.

[0036] In other embodiments of the present invention, the CD47 inhibitor is a small molecule drug. In certain embodiments, the small molecule drug is RRx-001 (J. Med. Chem., 2021, 64, 11, 7261-7271) or IMM-01.

[0037] In some embodiments, the CD47 inhibitor is an anti-CD47 antibody. In such embodiments, the antibody can be obtained in the same form as described in "2. Antibodies Specific for Tumor Antigens".

[0038] In particular, the anti-CD47 antibody, which is a monoclonal antibody, can be obtained by using CD47 or a fragment thereof as an immunogen to immunize mammals such as mice, rats, rabbits, hamsters, guinea pigs, horses, monkeys, dogs, pigs, cows, goats, and sheep, fusing spleen cells with myeloma cells to obtain hybridomas, and causing the hybridomas to produce and secrete the antibody. The hybridomas can be prepared by methods known in the art.

[0039] The CD47 used as an immunogen may be chemically synthesized based on sequence information, or may be obtained as a recombinant protein prepared according to methods known in the art based on the DNA sequence encoding the protein.

[0040] The antibody can preferably be screened by any method by cell ELISA using animal cells transfected with the DNA encoding CD47.

[0041] The anti-CD47 antibodies used in the embodiments of the present invention also include modified variants of the antibodies. The modified variants refer to variants obtained by subjecting the anti-CD47 antibodies used in the present invention to chemical modification or biological modification. Examples of chemically modified variants include variants having a linkage of a chemical moiety to the amino acid backbone, and variants having a linkage of a chemical moiety to an N-linked carbohydrate chain or an O-linked carbohydrate chain. Examples of biologically modified variants include variants obtained by post-translational modification (such as N-linked glycosylation or O-linked glycosylation, N-terminal processing or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants in which a methionine residue is added to the N-terminus by expression using a prokaryotic host cell. Furthermore, antibodies labeled so as to enable deletion or isolation of the anti-CD47 antibodies used in such embodiments of the present invention, for example, enzyme-labeled antibodies, fluorescent-labeled antibodies, and affinity-labeled antibodies are also included within the scope of the meaning of modified variants. Such modified variants of the anti-CD47 antibodies used in the present invention are useful, for example, for improving the stability of the antibody and its retention in the blood, reducing its antigenicity, or detecting or isolating the antibody.

[0042] Note that it is known that lysine residues at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells are deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells are deleted and the proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (e.g., complement activation, antibody-dependent cell cytotoxicity) of the antibody. Therefore, the anti-CD47 antibodies used in the embodiments of the present invention also include antibodies subjected to such modifications and functional fragments thereof, and deletion mutants in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain and which have an amidated residue (e.g., a heavy chain in which the carboxyl-terminal proline residue is amidated). Note that the types of deletion mutants having deletions at the carboxyl terminus of the heavy chain of the anti-CD47 antibodies used in the embodiments of the present invention are not limited to the above mutants as long as the antigen-binding affinity and effector functions are conserved. The two heavy chains constituting the anti-CD47 antibody used in the present invention may be a heavy chain selected from the group consisting of a full-length heavy chain and a heavy chain having the deletions described above, or may be two types of heavy chains selected in combination therefrom. The ratio of the amounts of the respective deletion mutants can be affected by the type of cultured mammalian cells producing the anti-CD47 antibody used in the present invention and the culture conditions, but an antibody in which one amino acid residue at the carboxyl terminus is deleted in either of the two heavy chains of the anti-CD47 antibody used in the present invention can preferably be exemplified.

[0043] The anti-CD47 antibodies used in the present invention include chimeric antibodies modified to reduce the heterologous antigenicity against human antibodies and humanized antibodies. Humanized antibodies are also referred to as CDR-grafted antibodies.

[0044] A chimeric antibody refers to an antibody composed of the light-chain variable region and heavy-chain variable region of an antibody from a non-human animal, and the light-chain constant region and heavy-chain constant region of a human antibody. A chimeric antibody is prepared by collecting cDNA encoding the light-chain variable region and cDNA encoding the heavy-chain variable region from a hybridoma that produces an anti-CD47 antibody, inserting the cDNA into an expression vector having cDNA encoding the light-chain constant region and heavy-chain constant region of a human antibody to construct an expression vector for the chimeric antibody, and introducing the expression vector for the chimeric antibody into a host cell to enable the expression of the antibody.

[0045] It is known that in antibodies produced in cultured mammalian cells, lysine residues at the carboxyl terminus of the heavy chain are deleted (Tsubaki et al., Int. J. Biol. Macromol, pages 139 - 147, 2013). However, the deletion of the heavy-chain sequence does not affect the antigen-binding affinity and effector functions of the antibody (e.g., complement activation and antibody-dependent cell-mediated cytotoxicity). Therefore, the present invention also includes antibodies lacking lysine residues at the carboxyl terminus of the heavy chain.

[0046] Specific examples of anti-CD47 antibodies include magrolimab (INN RN: 2169232 - 81 - 7), remsoipalrimab (INN RN: 2377483 - 71 - 9), AO - 176 (WO20198370), SRF - 231 (WO18236904), IBI - 188, IBI - 322, IMC - 002, MIL - 95, TG - 1801, ZL - 1201, AK - 117 (rigulimab), and IMM - 0306.

[0047] 2. Antibodies specific to tumor antigens The antibody specific for a tumor antigen used in the present invention can be derived from any species, preferably an antibody derived from human, rat, mouse, or rabbit. When the antibody is derived from a species other than the human species, the antibody is preferably chimerized or humanized using well-known techniques. The antibody specific for a tumor antigen may be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0048] The antibody specific for a tumor antigen is an antibody having a characteristic capable of targeting cancer cells, and preferably, for example, an antibody having the property of recognizing cancer cells, the property of binding to cancer cells, the property of internalizing into cancer cells, and / or the property of having cytotoxic activity against cancer cells.

[0049] The binding activity of the antibody to cancer cells can be confirmed using flow cytometry. The internalization of the antibody into tumor cells can be confirmed using (1) an assay that visualizes the antibody incorporated into cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008), 15, pp. 751 - 761), (2) an assay that measures the fluorescence intensity incorporated into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell, Vol. 15, pp. 5268 - 5282, December 2004), or (3) the Mab-ZAP assay that uses an antitoxin that binds to the therapeutic antibody and, when incorporated into cells, releases the toxin to inhibit cell proliferation (Bio Techniques, 28:162 - 165, January 2000). As the antitoxin, a recombinant complex protein of the diphtheria toxin catalytic domain and protein G can be used.

[0050] The antitumor activity of an antibody can be confirmed in vitro by determining its inhibitory activity against cell proliferation. For example, a cancer cell line that overexpresses the target protein of the antibody is cultured, and the antibody is added to the culture system while varying the concentration to determine its inhibitory activity against lesion formation, colony formation, and spheroid growth. The antitumor activity can be confirmed in vivo, for example, by administering the antibody to nude mice transplanted with a cancer cell line that highly expresses the target protein and determining the changes in the cancer cells.

[0051] Although it is preferable, it is not essential that an antibody specific for a tumor antigen itself exerts an antitumor effect. In embodiments where an antibody specific for a tumor antigen is part of an antibody-drug conjugate, for the purpose of specifically and selectively exerting the cytotoxic activity of the antitumor compound against cancer cells, it is also important, and furthermore preferably considered, that the antibody has internalization properties that allow it to migrate to cancer cells.

[0052] Antibodies specific for tumor antigens used in the present invention can be obtained by procedures known in the art. For example, the antibody can be obtained using methods commonly practiced in the art, which involve immunizing an animal with an antigenic polypeptide (i.e., a tumor antigen) and recovering and purifying the antibody produced in vivo. The origin of the antigen is not limited to humans, and the animal can be immunized with an antigen derived from a non-human animal such as a mouse or a rat. In this case, the cross-reactivity of the obtained antibody that binds to the heterologous antigen with human antigens can be examined to screen for the applicability of the antibody to human diseases.

[0053] Alternatively, antibody-producing cells that produce antibodies against an antigen are fused with myeloma cells according to methods known in the art (e.g., Kohler and Milstein, Nature (1975), 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)) to establish hybridomas from which monoclonal antibodies can be obtained.

[0054] An antigen can be obtained by genetically engineering a host cell to create a gene encoding the antigen protein. Specifically, a vector enabling the expression of the antigen gene is prepared and introduced into the host cell so that the gene is expressed. The antigen thus expressed can be purified. Antibodies can also be obtained by immunizing an animal by the method described above with the genetically engineered antigen-expressing cells or cell lines expressing the antigen.

[0055] Antibodies specific for tumor antigens are preferably recombinant antibodies obtained by artificial modification, such as chimeric antibodies or humanized antibodies, for the purpose of reducing their heterologous antigenicity to humans, or preferably antibodies having only the gene sequence of antibodies derived from humans, i.e., human antibodies. These antibodies can be produced using known methods.

[0056] Examples of chimeric antibodies include antibodies derived from chimeric antibodies in which the variable and constant regions of the antibody are connected to the constant region of an antibody derived from a human, such as the variable region of a mouse-derived antibody or a rat-derived antibody (Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855 (1984)).

[0057] Examples of humanized antibodies include antibodies obtained by incorporating only the complementarity-determining regions (CDRs) of a heterologous antibody into a human-derived antibody (Nature (1986), 321, pp. 522-525), antibodies obtained by grafting the CDR sequences of a heterologous antibody, along with some of the amino acid residues of the framework of the heterologous antibody, into a human antibody via the CDR grafting method (WO90 / 07861), and antibodies humanized using gene conversion mutagenesis strategies (U.S. Patent No. 5,821,337).

[0058] Examples of human antibodies include antibodies produced by using a human antibody-producing mouse having a human chromosome fragment containing the genes for the heavy and light chains of a human antibody (see Tomizuka, K. et al., Nature Genetics (1997), 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998), 26, pp. 3447-3448; Yoshida, H. et al., "Animal Cell Technology: Basic and Applied Aspects", Vol. 10, pp. 69-73 (edited by Kitagawa, Y., Matsuda, T., and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, pp. 722-727, etc.). As an alternative, antibodies obtained by phage display and selected from a human antibody library (see Wormstone, I.M. et al., Investigative Ophthalmology & Visual Science (2002), 43(7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1(2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002), 109(3), pp. 427-431, etc.) can be exemplified.

[0059] Antibodies specific for tumor antigens also include modified variants of the antibodies. The modified variant refers to a variant obtained by subjecting an antibody specific for a tumor antigen to chemical modification or biological modification. Examples of chemically modified variants include variants containing the linkage of a chemical moiety to the amino acid backbone, variants containing the linkage of a chemical moiety to an N-linked carbohydrate chain or an O-linked carbohydrate chain, and the like. Examples of biologically modified variants include variants obtained by post-translational modification (such as N-linked glycosylation or O-linked glycosylation, N-terminal processing or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants in which a methionine residue is added to the N-terminus by being expressed in a prokaryotic host cell. Furthermore, antibodies labeled so as to enable deletion or isolation of the antibody or antigen according to the present invention, for example, enzyme-labeled antibodies, fluorescent-labeled antibodies, and affinity-labeled antibodies are also included within the scope of the meaning of modified variants. Such modified variants of antibodies specific for tumor antigens improve the stability of the antibody and its retention in the blood, reduce its antigenicity, and are useful for detecting or isolating the antibody or antigen.

[0060] Furthermore, it is possible to enhance antibody-dependent cell-mediated cytotoxic activity by regulating the modification (such as glycosylation, defucosylation, etc.) of the glycan linked to an antibody specific for a tumor antigen. Techniques for regulating the modification of the glycan of an antibody are known, such as International Publication No. WO99 / 54342, International Publication No. WO00 / 61739, International Publication No. WO02 / 31140, International Publication No. WO2007 / 133855, and International Publication No. WO2013 / 120066. However, the techniques are not limited thereto. Antibodies specific for tumor antigens also include antibodies in which the modification of the glycan is regulated.

[0061] It is known that lysine residues at the carboxyl terminus of the heavy chain of antibodies produced in cultured mammalian cells are deleted (Journal of Chromatography A, 705: 129-134 (1995)), and two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of antibodies produced in cultured mammalian cells are deleted, and it is also known that a proline residue newly arranged at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (such as complement activation, antibody-dependent cell cytotoxicity, etc.) of the antibody. Therefore, antibodies specific for tumor antigens include such modified antibodies and functional fragments of the antibody, and deletion mutants in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain and which have an amidated residue (for example, a heavy chain in which the carboxyl-terminal proline residue is amidated), etc. are also included. The types of deletion mutants having deletions at the carboxyl terminus of the heavy chain of the antibody according to the present invention are not limited to the above mutants as long as the antigen-binding affinity and effector functions are conserved. The two heavy chains constituting the antibody specific for the tumor antigen may be of one type selected from the group consisting of the full-length heavy chain and the deletion mutants described above, or may be of two types selected in combination therefrom. The ratio of the amount of each deletion mutant can be affected by the type of cultured mammalian cells producing the antibody according to the present invention and the culture conditions, but an antibody in which one amino acid residue at the carboxyl terminus is deleted in both of the two heavy chains of the antibody specific for the tumor antigen can preferably be exemplified.

[0062] Examples of the isotype of the antibody specific for the tumor antigen may include, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably, IgG1, IgG2, or IgG4 may be exemplified.

[0063] Examples of antibodies specific for tumor antigens can include, but are not particularly limited to, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 antibody, anti-FOLR1 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD70 antibody, anti-PSMA antibody, anti-CEA antibody, anti-mesothelin antibody, anti-A33 antibody, anti-CanAg antibody, anti-Cripto antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-integrin antibody, anti-tenascin C antibody, anti-SLC44A4 antibody, anti-GPR20 antibody, and anti-CDH6 antibody, with anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody, and anti-CDH6 antibody being preferably exemplified.

[0064] In some embodiments, an antibody specific for a tumor-associated antigen comprises a plurality of antibodies, where each antibody is specific for a different tumor antigen. For example, in some embodiments, two antibodies are provided, each specific for a different tumor-associated antigen. More generally, in some embodiments, a plurality of antibodies are provided, where each antibody is specific for a different tumor antigen and each antibody is independently selected from the group consisting of: anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 antibody, anti-FOLR1 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD70 antibody, anti-PSMA antibody, anti-CEA antibody, anti-mesothelin antibody, anti-A33 antibody, anti-CanAg antibody, anti-Cripto antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-integrin antibody, anti-tenascin C antibody, anti-SLC44A4 antibody, anti-GPR20 antibody, and anti-CDH6 antibody, and are selected from anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody, and anti-CDH6 antibody.

[0065] As used herein, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (human epidermal growth factor receptor type 2; ErbB-2) and preferably has internalization activity into HER2-expressing cells by binding to HER2.

[0066] Examples of anti-HER2 antibodies include trastuzumab (U.S. Patent No. 5,821,337) and pertuzumab (International Publication No. WO01 / 00245), and preferably, trastuzumab can be exemplified.

[0067] The anti-HER2 antibody is preferably an antibody comprising a heavy chain containing CDRH1 consisting of the amino acid sequence consisting of amino acid residues 26 to 33 of SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence consisting of amino acid residues 51 to 58 of SEQ ID NO: 1, and CDRH3 consisting of the amino acid sequence consisting of amino acid residues 97 to 109 of SEQ ID NO: 1, and a light chain containing CDRL1 consisting of the amino acid sequence consisting of amino acid residues 27 to 32 of SEQ ID NO: 2, CDRL2 consisting of the amino acid sequence consisting of amino acid residues 50 to 52 of SEQ ID NO: 2, and CDRL3 consisting of the amino acid sequence consisting of amino acid residues 89 to 97 of SEQ ID NO: 2; more preferably, an antibody comprising a heavy chain containing a heavy chain variable region consisting of the amino acid sequence consisting of amino acid residues 1 to 120 of SEQ ID NO: 1 and a light chain containing a light chain variable region consisting of the amino acid sequence consisting of amino acid residues 1 to 107 of SEQ ID NO: 2; even more preferably, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2; or an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.

[0068] As used herein, the term "anti-HER3 antibody" refers to an antibody that specifically binds to HER3 (human epidermal growth factor receptor type 3; ErbB-3) and preferably has internalization activity into HER3-expressing cells by binding to HER3.

[0069] Examples of anti-HER3 antibodies include patritumab (U3-1287), U1-59 (International Publication No. WO2007 / 077028), the anti-ERBB3 antibody MM-121 (seribantumab), RG-7116 (lumretuzumab), and LJM-716 (ergatumumab) described in International Publication No. WO2008 / 100624, with patritumab and U1-59 being preferably exemplified.

[0070] The anti-HER3 antibody is preferably an antibody comprising a heavy chain consisting of a CDRH1 consisting of the amino acid sequence of amino acid residues 26 to 35 of SEQ ID NO: 3, a CDRH2 consisting of the amino acid sequence of amino acid residues 50 to 65 of SEQ ID NO: 3, and a CDRH3 consisting of the amino acid sequence of amino acid residues 98 to 106 of SEQ ID NO: 3, and a light chain consisting of a CDRL1 consisting of the amino acid sequence of amino acid residues 24 to 39 of SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence of amino acid residues 56 to 62 of SEQ ID NO: 4, and a CDRL3 consisting of the amino acid sequence of amino acid residues 95 to 103 of SEQ ID NO: 4; More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of amino acid residues 1 to 117 of SEQ ID NO: 3 and a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acid residues 1 to 113 of SEQ ID NO: 4; Even more preferably, it is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4, or a mutant of the antibody in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0071] As used herein, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: tumor-associated calcium signal transducer 2; EGP-1) and preferably has internalization activity into TROP2-expressing cells by binding to TROP2.

[0072] Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. WO2015 / 098099).

[0073] The anti-TROP2 antibody is preferably an antibody comprising a heavy chain consisting of a CDRH1 consisting of the amino acid sequence consisting of amino acid residues 50 to 54 of SEQ ID NO: 5, a CDRH2 consisting of the amino acid sequence consisting of amino acid residues 69 to 85 of SEQ ID NO: 5, and a CDRH3 consisting of the amino acid sequence consisting of amino acid residues 118 to 129 of SEQ ID NO: 5, and a light chain consisting of a CDRL1 consisting of the amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO: 6, a CDRL2 consisting of the amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO: 6, and a CDRL3 consisting of the amino acid sequence consisting of amino acid residues 109 to 117 of SEQ ID NO: 6; More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 5 and a light chain comprising a light chain variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 6; Even more preferably, it is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 6, or a mutant of the antibody in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0074] As used herein, the term "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B cell antigen 7 homolog 3; PD-L3; CD276) and preferably has internalization activity into B7-H3-expressing cells by binding to B7-H3.

[0075] Examples of anti-B7-H3 antibodies include M30-H1-L4 (International Publication No. WO2014 / 057687).

[0076] The anti-B7-H3 antibody preferably comprises a heavy chain consisting of CDRH1 comprising the amino acid sequence consisting of amino acid residues 50 to 54 of SEQ ID NO: 7, CDRH2 comprising the amino acid sequence consisting of amino acid residues 69 to 85 of SEQ ID NO: 7, and CDRH3 comprising the amino acid sequence consisting of amino acid residues 118 to 130 of SEQ ID NO: 7, and a light chain consisting of CDRL1 comprising the amino acid sequence consisting of amino acid residues 44 to 53 of SEQ ID NO: 8, CDRL2 comprising the amino acid sequence consisting of amino acid residues 69 to 75 of SEQ ID NO: 8, and CDRL3 comprising the amino acid sequence consisting of amino acid residues 108 to 116 of SEQ ID NO: 8; More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 7 and a light chain comprising a light chain variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 128 of SEQ ID NO: 8; Even more preferably, it is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 8, or a mutant of the antibody in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0077] As used herein, the term "anti-GPR20 antibody" refers to an antibody that specifically binds to GPR20 (G protein-coupled receptor 20) and preferably has internalization activity into GPR20-expressing cells by binding to GPR20.

[0078] Examples of anti-GPR20 antibodies include h046-H4e / L7 (International Publication No. WO2018 / 135501).

[0079] The anti-GPR20 antibody preferably comprises a heavy chain consisting of CDRH1 comprising the amino acid sequence consisting of amino acid residues 45 to 54 of SEQ ID NO: 9, CDRH2 comprising the amino acid sequence consisting of amino acid residues 69 to 78 of SEQ ID NO: 9, and CDRH3 comprising the amino acid sequence consisting of amino acid residues 118 to 131 of SEQ ID NO: 9, and a light chain consisting of CDRL1 comprising the amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO: 10, CDRL2 comprising the amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO: 10, and CDRL3 comprising the amino acid sequence consisting of amino acid residues 109 to 117 of SEQ ID NO: 10; More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 142 of SEQ ID NO: 9, and a light chain comprising a light chain variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 10; Even more preferably, it is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 10, or a mutant of the antibody in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0080] As used herein, the term "anti-CDH6 antibody" refers to an antibody that specifically binds to CDH6 (cadherin 6) and preferably has internalization activity into CDH6-expressing cells by binding to CDH6.

[0081] Examples of anti-CDH6 antibodies include H01L02 (International Publication No. WO2018 / 212136).

[0082] The anti-CDH6 antibody is preferably an antibody comprising a heavy chain consisting of a CDRH1 consisting of the amino acid sequence consisting of amino acid residues 45 to 54 of SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence consisting of amino acid residues 69 to 78 of SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence consisting of amino acid residues 118 to 130 of SEQ ID NO: 11, and a light chain consisting of a CDRL1 consisting of the amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO: 12, and a CDRL3 consisting of the amino acid sequence consisting of amino acid residues 109 to 116 of SEQ ID NO: 12; More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 11, and a light chain comprising a light chain variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 128 of SEQ ID NO: 12; Even more preferably, it is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 12, or a mutant of the antibody in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0083] 3. Antibody-drug conjugate In some embodiments of the present invention, the antibody specific for a tumor antigen is part of an antibody-drug conjugate comprising an antibody specific for a tumor antigen connected to a drug via a linker. The partial structure consisting of the linker and the drug within the antibody-drug conjugate is referred to as a "drug-linker".

[0084] The antibody-drug conjugate has the following formula:

[0085] [In the formula, A represents the connection position to the antibody] [wherein, A represents the connection position to the antibody] It is particularly preferred that the drug-linker represented by is conjugated to an antibody specific for a tumor antigen via a thioether bond, and is an antibody-drug conjugate.

[0086] The drug-linker is connected to thiol groups (in other words, the sulfur atoms of cysteine residues) formed at the interchain disulfide bond sites (two sites between heavy chains and two sites between heavy and light chains) within the antibody.

[0087] The drug-linker of a plurality of embodiments contains, as a component, exatecan which is a topoisomerase I inhibitor (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo [d] pyrano [3’,4’:6,7] indolizino [1,2-b] quinoline-10,13-dione (also represented as the chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo [d] pyrano [3’,4’:6,7] indolizino [1,2-b] quinoline-10,13(9H,15H)-dione)). Exatecan has an antitumor effect and is represented by the following formula:

[0088]

Chem.

[0089] The antibody-drug conjugate used in the embodiments of the present invention is also represented by the following formula:

[0090]

Chem.

[0091] After migrating to cancer cells, the antibody-drug conjugate used in the embodiments of the present invention is cleaved at the linker moiety to give the following formula (hereinafter referred to as compound (A) in this specification):

[0092]

Chemical formula

[0093] The aforementioned compound is presumed to be the original source for at least part of the antitumor activity of the antibody-drug conjugate used in such embodiments of the present invention, and has been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, October 15, 2016, 22(20):5097 - 5108, Epub, March 29, 2016).

[0094] Topoisomerase I is an enzyme that cleaves a single strand of DNA, reconnects the cleaved fragment, and is involved in the conversion of DNA higher-order structure and DNA synthesis. Therefore, a drug having a topoisomerase I inhibitory effect inhibits DNA synthesis, terminates cell division during the S phase (DNA synthesis phase) in the cell cycle, induces apoptosis (cell death), and suppresses the growth of cancer cells.

[0095] It is also known that the antibody-drug conjugate used in the embodiments of the present invention has a bystander effect (Ogitani Y. et al., Cancer Science (2016), 107, 1039 - 1046).

[0096] The bystander effect is exerted through a process such that the antibody-drug conjugate used in such embodiments of the present invention is internalized into cancer cells expressing the target, the aforementioned compound is released, and then exerts an antitumor effect on cancer cells that are present in the vicinity thereof and do not express the target.

[0097] The bystander effect is also exerted as an excellent antitumor effect when the antibody-drug conjugate according to such embodiments of the present invention is used in combination with a CD47 inhibitor.

[0098] 4. Preparation of Antibody-Drug Conjugate The drug-linker intermediate for use in the preparation of the antibody-drug conjugate according to the present invention has the following formula:

[0099]

Chemical formula

[0100] The drug-linker intermediate is represented by the chemical name: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide, and can be prepared by referring to the descriptions of International Publication No. WO2014 / 057687, International Publication No. WO2015 / 098099, International Publication No. WO2015 / 115091, International Publication No. WO2015 / 155998, and International Publication No. WO2019 / 044947.

[0101] The antibody-drug conjugate used in the embodiments of the present invention can be prepared by reacting the drug-linker intermediate described above with an antibody having a thiol group (alternatively referred to as a sulfhydryl group) that is specific for a tumor antigen.

[0102] Antibodies having sulfhydryl groups can be obtained by methods well known in the art (Hermanson, G.T., Bioconjugate Techniques, pages 56-136, 456-493, Academic Press (1996)). For example, by using a reducing agent such as 0.3-3 molar equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) per interchain disulfide in the antibody and reacting it in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an antibody having sulfhydryl groups in which the interchain disulfides in the antibody are partially or completely reduced can be obtained.

[0103] Furthermore, by using 2-20 molar equivalents of the drug-linker intermediate per antibody having a sulfhydryl group, an antibody-drug conjugate in which 2-8 drug molecules are conjugated per antibody molecule can be prepared.

[0104] The average number of conjugated drug molecules per antibody molecule of the prepared antibody-drug conjugate can be determined, for example, by a calculation method (UV method) based on the measurement of UV absorbance at two wavelengths of 280 nm and 370 nm for the antibody-drug conjugate and its conjugation precursor, or by a calculation method (HPLC method) based on the quantification via HPLC measurement for the fragments obtained by treating the antibody-drug conjugate with a reducing agent.

[0105] The conjugation of an antibody with a drug-linker intermediate and the calculation of the average number of conjugated drug molecules per antibody molecule of the antibody-drug conjugate can be carried out by referring to the descriptions in International Publication No. WO2014 / 057687, International Publication No. WO2015 / 098099, International Publication No. WO2015 / 115091, International Publication No. WO2015 / 155998, International Publication No. WO2018 / 135501, and International Publication No. WO2018 / 212136, etc.

[0106] In the present invention, the "anti-HER2 antibody-drug conjugate" represents an antibody-drug conjugate in which the antibody in the antibody-drug conjugate according to the present invention is an anti-HER2 antibody.

[0107] The average number of conjugated drug-linker units per antibody molecule in the anti-HER2 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, still more preferably 7.5 to 8, and still more preferably about 8.

[0108] The anti-HER2 antibody-drug conjugate can be prepared by referring to the descriptions in International Publication No. WO2015 / 115091, etc.

[0109] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate according to the present invention is an anti-HER3 antibody.

[0110] The average number of conjugated drug-linker units per antibody molecule in the anti-HER3 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, still more preferably 7.5 to 8, and still more preferably about 8.

[0111] An anti-HER3 antibody-drug conjugate can be prepared by referring to the description in International Publication No. WO2015 / 155998 etc.

[0112] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody within the antibody-drug conjugate according to the present invention is an anti-TROP2 antibody.

[0113] The average number of conjugated drug-linker units per antibody molecule in the anti-TROP2 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 5, still more preferably 3.5 to 4.5, and even more preferably about 4.

[0114] An anti-TROP2 antibody-drug conjugate can be prepared by referring to the description in International Publication No. WO2015 / 098099 etc.

[0115] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody within the antibody-drug conjugate according to the present invention is an anti-B7-H3 antibody.

[0116] The average number of conjugated drug-linker units per antibody molecule in the anti-B7-H3 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 5, still more preferably 3.5 to 4.5, and even more preferably about 4.

[0117] The anti-B7-H3 antibody-drug conjugate used in the present invention can be prepared by referring to the description in International Publication No. WO2014 / 057687 etc.

[0118] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody within the antibody-drug conjugate according to the present invention is an anti-GPR20 antibody.

[0119] The average number of conjugated drug-linker units per antibody molecule in the anti-GPR20 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, still more preferably 7.5 to 8, and still more preferably about 8.

[0120] The anti-GPR20 antibody-drug conjugate can be prepared by referring to the description in International Publication No. WO2018 / 135501 and the like.

[0121] In the present invention, the term "anti-CDH6 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate according to the present invention is an anti-CDH6 antibody.

[0122] The average number of conjugated drug-linker units per antibody molecule in the anti-CDH6 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, still more preferably 7 to 8, still more preferably 7.5 to 8, and still more preferably about 8.

[0123] The anti-CDH6 antibody-drug conjugate can be prepared by referring to the description in International Publication No. WO2018 / 212136 and the like.

[0124] 5. Relationship between immunogenic cell death and CD47 inhibitor Immunogenic cell death (ICD) is a type of cell death characterized by the massive release of intracellular molecules such as ATP and HMGB1 (high-mobility group box 1 protein), and the exposure of calreticulin (CRT) on the cell surface. These are danger signals that activate immune cells. ATP contributes to the recruitment and activation of dendritic cells (DCs) and macrophages; HMGB1 contributes to type I IFN, such as enhancing the production of inflammatory cytokines; CRT is used as an "eat-me signal" and has been reported to enhance the uptake of antigens from dead cells (Nature Reviews Immunology., 2017, 17, pp. 97-111). Briefly, ICD of cancer cells can occur and immunity against cancer cells (anti-tumor immunity) can be induced. As anti-cancer agents that induce ICD, for example, oxaliplatin and cyclophosphamide, which are anthracycline drugs, are known, but for example, docetaxel and mitomycin C have not been confirmed to induce ICD. The presence or absence of the ICD effect can be evaluated not only in vitro based on whether a danger signal is detected or not by adding a drug, but also in vivo based on a vaccination assay. In the latter assay, cancer cells are treated with a drug and transplanted into immunocompetent mice. One week later, cancer cells not treated with the drug are transplanted to the opposite side. At this time, if immune memory has already been formed by the cancer cells that caused ICD, the engraftment and growth of the transplanted cancer cells are inhibited (Cancer Research, 2017, 77, pp. 2686-2698).

[0125] For the formation of immune memory by ICD, it is important that myeloid cells such as dendritic cells and macrophages take up cancer antigens. At this time, the "phagocytosis rejection signal" is presumably transmitted between myeloid cells and cancer cells by SIRPα-CD47. When a CD47 inhibitor is administered, signal transduction may be inhibited and phagocytosis may be enhanced. As a result, the uptake of cancer antigens can be enhanced. Cancer antigens taken up by dendritic cells and / or macrophages are processed intracellularly into 8-30mer peptide fragments, and these peptide fragments are presented on MHC. MHC has two classes, class I and class II. An antigen peptide of about 9mer presented to MHC class I activates CD8+ T cells, while an antigen peptide of about 15mer presented to MHC class II activates CD4+ T cells. Generally, foreign antigens are processed within myeloid cells and presented to MHC class II, but a part of the DC subset presents foreign antigens to MHC class I, activates CD8+ T, and exerts cytotoxic activity against cancer cells. Briefly, a part of the DC subset has cross-presentation ability.

[0126] Among them, in the case of an antibody-drug conjugate incorporating tubulysin, pyrrolobenzodiazepine (PBD), and MMAE, the relationship between the antibody-drug conjugate incorporating a drug having cytotoxic activity therein and the induction of ICD has already been reported (Cancer Research, 2017, 77, pp. 2686-2698; or ONCOIMMUNOLOGY, 2019, 8(4), e1565859). Regarding the antibody-drug conjugate used in the present invention containing the TpoI inhibitor compound (A) as a payload, it has been reported that HMGB1 is released from cells treated with compound (A), but the antitumor effect exerted by vaccinating the treated cells is limited (Clin. Invest., 2020:130(1):374-388). Now, the following have been found: 1) Compound (A) induces the release of not only HMGB1 but also other Danger signals from dying cancer cells; and 2) Compound (A) enhances the cancer antigen-specific T cell population so as to activate immune cells present in the cancer microenvironment and induce antitumor immunity.

[0127] Here, the mechanism of action of the present invention is explained. First, an antibody specific for a tumor antigen binds to cancer cells. As a result, the antibody induces ADCP via neighboring immune cells. Second, the CD47 inhibitor enhances ADCP activity by neutralizing the "phagocytosis rejection signal" presented by cancer cells. Third, in an embodiment where the antibody is part of the ADC and is specific for a tumor antigen, the antibody delivers the drug to cancer cells, and the drug payload thereby induces immunogenic cell death of the cancer cells (as long as phagocytosis of the cells is not also performed), and the immunogenic cell death of the cancer cells releases inflammatory molecules such as ATP and HMGB1. These molecules can activate immune cells. Thus, the immunogenic cell death caused by the drug payload and the ADCP enhanced by the antibody portion of the antibody-drug conjugate in the presence of the CD47 inhibitor contribute to antitumor immunity.

[0128] 6. Treatment, Dosage, and Combination In accordance with the treatment method of the present invention, a CD47 inhibitor and an antibody specific for a tumor antigen are administered in combination.

[0129] In some embodiments of the treatment method of the present invention, the CD47 inhibitor and the antibody specific for the tumor antigen are individually contained as active ingredients in different formulations. In some embodiments, the CD47 inhibitor and the antibody specific for the tumor antigen are administered simultaneously, while in other embodiments they are administered at different times (i.e., sequentially). In still further embodiments, the CD47 inhibitor and the antibody specific for the tumor antigen are contained as active ingredients in a single formulation and are administered together.

[0130] The pharmaceutical composition or treatment method of the present invention is preferably used to treat cancer, including breast cancer, gastric cancer (also called gastric adenocarcinoma), colorectal cancer (also called colon cancer and rectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, head and neck cancer (including salivary gland cancer and pharyngeal cancer), esophagogastric junction adenocarcinoma, cholangiocarcinoma (including bile duct cancer), gallbladder cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, renal cancer, vulvar cancer, thyroid cancer, thymic cancer, penile cancer, leukemia, lymphoma, malignant lymphoma, plasmacytoma, multiple myeloma, myelodysplastic syndrome, brain tumor, glioma, glioblastoma multiforme, osteosarcoma, and melanoma; more preferably, it may be used to treat at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophagogastric junction adenocarcinoma, cholangiocarcinoma, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, and uterine carcinosarcoma; even more preferably, it can be used to treat at least one cancer selected from the group consisting of breast cancer, gastric cancer, lung cancer, and ovarian cancer.

[0131] In a preferred embodiment, the specificity of the antibody specific for the tumor antigen is determined by considering the types of cancer markers and / or tumor markers present. For example, in cancer, if the expression of HER2 is found, it is preferable to use an anti-HER2 antibody; in cancer, if the expression of HER3 is found, it is preferable to use an anti-HER3 antibody; in cancer, if the expression of TROP2 is found, it is preferable to use an anti-TROP2 antibody; in cancer, if the expression of B7-H3 is found, it is preferable to use an anti-B7-H3 antibody; in cancer, if the expression of GPR20 is found, it is preferable to use an anti-GPR20 antibody; in cancer, if the expression of CDH6 is found, it is preferable to use an anti-CDH6 antibody.

[0132] The presence or absence of HER2, HER3, TROP2, B7-H3, GPR20, and CDH6, as well as other tumor markers, may be examined, for example, by subjecting tumor tissue collected from a cancer patient to scrutiny at the gene product (protein) level using immunohistochemistry (IHC), flow cytometry, Western blotting, etc., or at the gene translation level using in situ hybridization (ISH), quantitative PCR (q-PCR), microarray analysis, etc. Alternatively, it may also be examined by subjecting cell-free circulating tumor DNA (ctDNA) collected from a cancer patient to scrutiny using methods such as next-generation sequencing (NGS).

[0133] The pharmaceutical composition or treatment method of the present invention can preferably be used for mammals, and more preferably can be used for humans.

[0134] The antitumor effect of the pharmaceutical composition or treatment method of the present invention can be confirmed, for example, by creating a model in which cancer cells are transplanted into a test animal and measuring the reduction in tumor volume and the life-prolonging effect resulting from treatment with the pharmaceutical composition or treatment method of the present invention. Furthermore, comparison of the antitumor effect of each single administration of the CD47 inhibitor used in the present invention and the antibody specific to the tumor antigen can provide confirmation of the combined effect of the CD47 inhibitor used in the present invention and the antibody specific to the tumor antigen.

[0135] In addition, the antitumor effect of the pharmaceutical composition or treatment method of the present invention can be confirmed in clinical studies by assessment methods such as RECIST (Response Evaluation Criteria in Solid Tumors), assessment methods by WHO, assessment methods by Macdonald, body weight measurement, and other methods; and can be determined by indicators such as complete response (CR), partial response (PR), progression (PD), objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), and overall survival (OS).

[0136] The above methods can provide confirmation of the superiority of the pharmaceutical composition or treatment method of the present invention for antitumor effects over existing pharmaceutical compositions and treatment methods for cancer treatment.

[0137] The pharmaceutical composition or treatment method of the present invention can delay the growth of cancer cells, suppress their growth, and further destroy cancer cells. These effects enable the treatment effect to be achieved by enabling cancer patients to avoid the symptoms caused by cancer or achieve an improvement in the QOL of cancer patients and maintain the lives of cancer patients. Even when the pharmaceutical composition or treatment method of the present invention does not achieve the killing of cancer cells, it can still achieve a higher QOL for cancer patients while achieving longer-term survival by inhibiting, controlling, or preventing the growth of cancer cells.

[0138] The pharmaceutical composition of the present invention can exert a treatment effect by application as a systemic therapy to a patient, and in addition, can also exert a treatment effect by local application to cancer tissue.

[0139] The pharmaceutical composition of the present invention can be administered containing at least one pharmaceutically suitable component. The pharmaceutically suitable component can be appropriately selected and applied from formulation additives generally used in the art, considering the dosage, administration concentration, etc. of the CD47 inhibitor used in the present invention and the antibody specific to the tumor antigen. For example, the antibody specific to the tumor antigen used in the present invention can be administered as a pharmaceutical composition containing a buffer such as a histidine buffer, an excipient such as sucrose or trehalose, and a surfactant such as polysorbate 80 or polysorbate 20. The pharmaceutical composition containing the antibody specific to the tumor antigen used in the present invention is preferably capable of being used as an injection, more preferably capable of being used as an aqueous injection or a lyophilized injection, and still more preferably capable of being used as a lyophilized injection.

[0140] When the pharmaceutical composition containing the antibody specific to the tumor antigen used in the present invention is an aqueous injection, the pharmaceutical composition is preferably diluted with a suitable diluent and then administered as an intravenous infusion. Examples of the diluent include a dextrose solution, physiological saline, etc., preferably a dextrose solution may be exemplified, and more preferably a 5% dextrose solution may be exemplified.

[0141] When the pharmaceutical composition containing the antibody specific to the tumor antigen used in the present invention is a lyophilized injection, the pharmaceutical composition is preferably dissolved in water for injection, and then the required amount is diluted with a suitable diluent and then can be administered as an intravenous infusion. Examples of the diluent include a dextrose solution, physiological saline, etc., preferably a dextrose solution may be exemplified, and more preferably a 5% dextrose solution may be exemplified.

[0142] Examples of the administration routes that can be used to administer the pharmaceutical composition of the present invention include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes; preferably including the intravenous route.

[0143] The antibody-drug conjugate specific for a tumor antigen used in the present invention can be administered to humans once at intervals of 1 to 180 days, preferably once a week, once every two weeks, once every three weeks, or once every four weeks, and more preferably can be administered once every three weeks. The antibody-drug conjugate specific for a tumor antigen used in the present invention can also be administered at a dose of about 0.001 to 100 mg / kg, preferably can also be administered at a dose of 0.8 to 12.4 mg / kg. When the antibody-drug conjugate specific for a tumor antigen in the present invention is an anti-HER2 antibody-drug conjugate, the antibody-drug conjugate can preferably be administered once every three weeks at a dose of 0.8 mg / kg, 1.6 mg / kg, 3.2 mg / kg, 5.4 mg / kg, 6.4 mg / kg, 7.4 mg / kg, or 8 mg / kg. When the antibody-drug conjugate specific for a tumor antigen used in the present invention is an anti-HER3 antibody-drug conjugate, the antibody-drug conjugate can preferably be administered once every three weeks at a dose of 1.6 mg / kg, 3.2 mg / kg, 4.8 mg / kg, 5.6 mg / kg, 6.4 mg / kg, 8.0 mg / kg, 9.6 mg / kg, or 12.8 mg / kg. When the antibody-drug conjugate specific for a tumor antigen used in the present invention is an anti-TROP2 antibody-drug conjugate, the antibody-drug conjugate can preferably be administered once every three weeks at a dose of 0.27 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6.0 mg / kg, 8.0 mg / kg, or 10.0 mg / kg. The CD47 inhibitor according to the present invention can be administered to humans once at intervals of 1 to 180 days, preferably can be administered once a week, once every two weeks, once every three weeks, or once every four weeks. The CD47 inhibitor according to the present invention can also be administered at a dose of about 0.001 to 100 mg / kg per administration.

[0144] The pharmaceutical composition or treatment method of the present invention may further contain a CD47 inhibitor and a cancer therapeutic agent other than an antibody specific to a tumor antigen according to the present invention. The pharmaceutical composition or treatment method of the present invention may also be administered in combination with another cancer therapeutic agent, thereby enhancing the anti-tumor effect. Another cancer therapeutic agent used for such a purpose may be administered to a subject simultaneously with, individually, or sequentially with the pharmaceutical composition of the present invention, and may also be administered with a variable dosing interval. Such cancer therapeutic agents are not limited as long as they are agents having anti-tumor activity, and include irinotecan (CPT-11), cisplatin, carboplatin, oxaliplatin, fluorouracil (5-FU), gemcitabine, capecitabine, doxorubicin, epirubicin, cyclophosphamide, mitomycin C, tegafur / gimeracil / oteracil combination agent, panitumumab, bevacizumab, ramucirumab, regorafenib, trifluridine / tipiracil combination agent, gefitinib, erlotinib, afatinib, methotrexate, pemetrexed, tamoxifen, toremifene, fulvestrant, leuprorelin, goserelin, letrozole, anastrozole, progesterone preparation, and lapatinib, and may be exemplified by at least one cancer therapeutic agent selected from the group consisting of them.

[0145] The pharmaceutical composition or treatment method of the present invention may further contain, as a cancer therapeutic agent used in combination with an immune checkpoint inhibitor and an antibody-drug that specifically binds to a cancer antigen and has ADCC activity and / or ADCP activity, in addition to a CD47 inhibitor and an antibody specific to a tumor antigen according to the present invention. Examples of the immune checkpoint inhibitor include an inhibitor of the binding between PD-1 and its ligand PD-L1, or a CTLA4 inhibitor. Specific examples thereof include anti-PD-1 antibodies (nivolumab, pembrolizumab, semiprimumab, spartalizumab, PDR-001, or BI754091), anti-PD-L1 antibodies (atezolizumab, avelumab, or durvalumab), and anti-CTLA4 antibodies (ipilimumab or tremelimumab). Examples of the antibody-drug that specifically binds to a cancer antigen and has ADCC activity and / or ADCP activity include anti-CD20 antibody (rituximab), anti-HER2 antibodies (trastuzumab or pertuzumab), anti-EGFR antibody (cetuximab), and anti-CD52 antibody (alemtuzumab).

[0146] ADCC refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) expressing Fcγ receptors recognize an antibody bound to a target cell and then cause lysis of the target cell. In NK cells, which are the main cells contributing to ADCC, FcγRIIC and FcγRIIIA are expressed. In monocytes, FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA are expressed. On the other hand, ADCP refers to a cell-mediated reaction in which phagocytic cells (e.g., macrophages, neutrophils) expressing Fc receptors recognize an antibody bound to a target cell and then phagocytose the target cell within the cell. In monocytes, which are the main cells contributing to ADCP, FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA are expressed.

[0147] The pharmaceutical composition or treatment method of the present invention may also be used in combination with radiotherapy. For example, a cancer patient is subjected to radiotherapy before and / or after being treated with the pharmaceutical composition of the present invention, or simultaneously therewith.

[0148] The pharmaceutical composition or treatment method of the present invention can also be used as adjuvant chemotherapy combined with surgery. The pharmaceutical composition of the present invention may be administered before surgery for the purpose of reducing the size of a tumor (referred to as preoperative adjuvant chemotherapy or neoadjuvant therapy), or after surgery for the purpose of preventing tumor recurrence (referred to as postoperative adjuvant chemotherapy or adjuvant therapy).

[0149] In some embodiments, the CD47 inhibitor and / or the antibody specific for a tumor antigen are formulated in combination with a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, adjuvant, or other agent. The "pharmaceutically acceptable carrier" and other agents can be appropriately selected from a wide range according to the type of the target disease and the dosage form of the drug. The method for administering the CD47 inhibitor and / or the antibody specific for a tumor antigen according to the present invention allows for appropriate selection, and for example, administration by injection can be selected. Examples of injections that can be used include local injection, intraperitoneal injection, selective intravenous injection, intravenous injection, subcutaneous injection, and organ perfusion fluid injection. The injection solution can be formulated by using a carrier consisting of any one of saline, glucose solution, or a mixture of saline and glucose solution, and a buffer solution. Alternatively, the injection solution can be prepared by formulating a powder preparation and mixing the powder preparation with the aforementioned liquid carrier at the time of use.

[0150] Other administration methods can also be appropriately selected in conjunction with the development of the preparation. For example, for oral administration, oral solutions, powders, pills, capsules, and tablets can be applied. In the case of oral solutions, oral liquid preparations such as suspensions and syrups can be prepared by using water; sugars such as sucrose, sorbitol, and fructose; glycols such as polyethylene glycol; oils such as sesame oil and soybean oil; preservatives such as alkyl parahydroxybenzoic acid; and flavoring agents such as strawberry flavor and peppermint flavor. Powders, pills, capsules, and tablets can be formulated by using, for example, excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin. Tablets and capsules are easily administered. In this regard, tablets and capsules are preferred unit dosage forms of the composition of the present invention. In the preparation of tablets and capsules, solid carriers for preparation are used.

Example

[0151] The present invention is specifically described with the examples illustrated below in mind. However, the present invention is not limited to these examples. Furthermore, the present invention should in no way be construed in a limiting manner.

[0152] Preparation Example 1 Preparation of Antibody-Drug Conjugate (1) According to the production method described in International Publication No. WO2015 / 115091, a humanized anti-HER2 antibody (a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2, which will be referred to as "humanized anti-HER2(1)" in the following description of this specification) was used to prepare the following formula:

[0153]

Chemical formula

[0154] Production Example 2 Production of Antibody-Drug Conjugate (2) According to the production methods described in International Publication Nos. WO2015 / 098099 and WO2017 / 002776, a humanized anti-TROP2 antibody (a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 6, hereinafter referred to as "humanized anti-TROP2 antibody (1)" in the present specification) was used to produce the following formula:

[0155] [Chemical formula] [wherein, A represents the connection position to the antibody] A drug-linker represented by the following formula: was conjugated to an anti-TROP2 antibody via a thioether bond to produce an antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (2)" in the present specification). The DAR of the antibody-drug conjugate (2) can be controlled within the range of 0 to 8. In the present specification, an antibody-drug conjugate was produced such that the average number of conjugated drugs was 3.5 to 4.5.

[0156] Production Example 3 Production of Antibody-Drug Conjugate (3) According to the production methods described in International Publication Nos. WO2014 / 057867 and WO2017 / 002776, a humanized anti-B7-H3 antibody (a heavy chain consisting of an amino acid sequence composed of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence composed of amino acid residues 21 to 233 of SEQ ID NO: 8, which will be referred to as the humanized anti-B7-H3 antibody (3) later in this specification) was used to prepare the following formula:

[0157] [Chemical formula] [In the formula, A represents the connection position to the antibody] A drug-linker represented by the formula was conjugated to the anti-B7-H3 antibody via a thioether bond to prepare an antibody-drug conjugate (which will be referred to as the "antibody-drug conjugate (3)" later in this specification). The DAR of the antibody-drug conjugate (3) can be controlled within the range of 0 to 8. In this specification, an antibody-drug conjugate was prepared with an average number of conjugated drugs of 3.5 to 4.5.

Example

[0158] ADCP activity of the antibody-drug conjugate with compound (A) and the anti-CD47 blocker against HER2-positive cancer cell lines and TROP2-positive cancer cell lines 1-1 Preparation of target cells AGS (CD47 / TROP2 positive), a human gastric cancer cell line, and JIMT-1 (CD47 / HER2 positive), a human breast cancer cell line, were collected, washed twice with PBS, and resuspended in PBS. The number of viable cells was counted according to the trypan blue exclusion test. Cell Trace Violet (CTV, Thermo Fisher Scientific) solution was added at a volume of 1 μL per 1 × 10 6 cells / mL. The mixture was allowed to stand at room temperature in the dark for 10 minutes. After incubation, the cells were washed twice with 10 mL of 10% FBS containing RPMI1640 culture medium (R10). Finally, 1 × 10 6A cell suspension was prepared so that the concentration was 10 cells / mL and used as the target cells.

[0159] 1-2 Preparation of effector cells On day 0, Ficoll-Paque PLUS (GE Healthcare) was dispensed into SepMate-50 tubes (STEMCELL Technologies) at a volume of 15 mL per tube. Whole blood diluted 2-fold with PBS containing 2% FBS was layered at a volume of 33-34 mL per tube. Centrifugation was performed at 1200 g for 10 min at room temperature. The supernatant containing PBMCs was collected by decantation into a fresh 50 mL tube, 10 mL of PBS containing 2% FBS was added, and centrifuged at 300 g for 5-8 min at room temperature. After removing the supernatant, the PBMCs were washed twice with 10 mL of PBS containing 2% FBS. The number of viable PBMCs was counted, and the PBMCs were resuspended in RoboSep buffer (STEMCELL Technologies) at 5 × 10 cells. 7 The EasySep human monocyte enrichment cocktail in the Human Monocyte Enrichment Kit without CD16 Depletion (STEMCELL Technologies) was added to 5 × 10 PBMCs / mL. 7 After incubation at 4° C. for 10 minutes, EasySep Magnetic Particles (STEMCELL Technologies) were added to the PBMC suspension at a concentration of 5×10 PBMC. 7It was added in an amount of 50 μL per well. After a reaction for 5 minutes at 4°C, RoboSep buffer was added to the PBMC up to 2.5 mL. The PBMC was introduced into a 5 mL tube and placed in the magnetic field of an EasySep Magnet (STEMCELL Technologies). After 2 minutes and 30 seconds, the supernatant containing human peripheral blood monocytes was collected by decantation and centrifuged. To generate macrophages, the isolated monocytes were suspended in R10 (PEPROTEC) containing 20 ng / mL of M-CSF and seeded into a flask of 225 cm 2 and cultured at 37°C and 5% CO 2 for 7 days. On day 7, the culture supernatant was removed and R10 containing 20 ng / mL of M-CSF was added. On day 11, the culture supernatant was removed and R10 (PEPROTEC) containing 20 ng / mL of M-CSF and 20 ng / mL of IL-10 was added. On day 13, the macrophages were washed once with PBS, TrypLE Express (Thermo Fisher Scientific) was added, and held at 37°C for 10 - 30 minutes. After harvesting, the macrophages were suspended in PBS at a cell density of 1×10 6 cells / mL. CellTrace Far Red solution (Thermo Fisher Scientific) was added to the macrophages at 1 μL per 10 6 cells / mL. After incubation for 10 minutes at room temperature in the dark, the macrophages were washed twice with 10 mL of R10. The cells were suspended in R10 at a cell density of 1×10 6 cells / mL and used as effector cells.

[0160] Assay of 1 - 3 ADCP activity First, 100 μL of each of the indicated agents was added to the cells in a U-shaped 96-well microplate. Briefly, the antibody-drug conjugate (1) or (2) diluted with R10, or the control antibody-drug conjugate, was added at a final concentration of 5 nM. ALX148_hIgG1 or the isotype-matched control Ab diluted with R10 was added at a final concentration of 50 nM. ALX148_hIgG1 was prepared with reference to US Patent Application Publication No. 2019 / 0169266A1. The amino acid sequence of ALX148_hIgG1 is specified in SEQ ID NO: 15 in the Sequence Listing. Second, 50 μL of the effector cells (cells 1×10 6 cells / mL) prepared as in steps 1-2 above was added to the wells. Finally, 50 μL of the target cells (cells 1×10 6 cells / mL) prepared as in step 1-1 above was added to the wells, and the cells were mixed by gently pipetting. The cells were incubated at 37 °C and 5% CO 2 for 4 hours. After centrifugation at 510 g for 3 minutes, the supernatant was removed. The cells were washed once with 200 μL of Hanks’ Balanced Salt Solution containing 0.1% BSA. The cells were suspended in 50 μL of 1×BD Stabilizing Fixative (BD Biosciences) and kept at 4 °C in the dark until flow cytometry analysis. ADCP was evaluated using a BD LSRFortessa X-20 flow cytometer (BD Biosciences). The data obtained were analyzed using Flowjo (TreeStar). Cancer cells were determined as CTV-positive (CTV+) cells. Within the cancer cell population, cancer cells phagocytosed by macrophages were defined as CTV+ / Far Red-positive (Far Red+), and are referred to herein later as CTV+ Far Red+ cells. ADCP (%) was calculated as follows: ADCP (%) = 100 × [(CTV+ Far Red+ cell count) ÷ (CTV+ cell count)] as calculated above.

[0161] Data were analyzed using GraphPad Prism 9.1.0 (GraphPad Software), and statistical analysis was performed using SAS System Release 9.2 (SAS Institute).

[0162] As shown in Figure 17, antibody-drug conjugates (1) or (2) showed ADCP activity against JIMT-1 cells and AGS cells, respectively, and showed higher ADCP activity in the presence of ALX148_hIgG1 (Figures 17A and 17B).

Example

[0163] ADCP activity of antibody-drug conjugates with compound (A) and anti-CD47 blocker against B7-H3 positive cancer cell lines 2-1 Preparation of target cells NCI-H322 (CD47 / B7-H3 positive), a human lung cancer cell line, was collected, washed twice with PBS, and resuspended in PBS. The number of viable cells was counted according to the trypan blue exclusion test. CellTrace Violet (CTV, Thermo Fisher Scientific) solution was added at a volume of 1 μL per 1 × 10 6 cells / mL. The mixture was allowed to stand at room temperature in the dark for 10 minutes. After incubation, the cells were washed twice with 10 mL of 10% FBS containing RPMI1640 culture medium (R10). Finally, a cell suspension was prepared to a concentration of 1 × 10 6 cells / mL and used as target cells.

[0164] 2-2 Preparation of effector cells On day 0, Ficoll-Paque PLUS (GE healthcare) was dispensed into SepMate-50 tubes (STEMCELL Technologies) at a volume of 15 mL per tube. Whole blood diluted 2-fold with PBS containing 2% FBS was overlaid at a volume of 33 - 34 mL per tube. Centrifugation was performed at room temperature at 1200 g for 10 minutes. The supernatant containing PBMCs was collected into an unused 50 mL tube by decantation, 10 mL of PBS containing 2% FBS was added, and centrifuged at room temperature at 300 g for 5 minutes. After removing the supernatant, the PBMCs were washed twice with 10 mL of PBS containing 2% FBS. The number of viable PBMCs was counted, and the PBMCs were suspended in RoboSep buffer (STEMCELL Technologies) at a cell density of 5×10 7 cells / mL. The EasySep human Monocyte enrichment cocktail in the Human monocyte Enrichment Kit Without CD16 Depletion (STEMCELL Technologies) was added at a volume of 50 μL per 5×10 7 cells. After a 10-minute reaction at 4°C, EasySep Magnetic Particles (STEMCELL Technologies) were added to the PBMC suspension at a volume of 50 μL per 5×10 7 cells. After a 5-minute reaction at 4°C, RoboSep buffer was added to the PBMCs up to 2.5 mL. The PBMCs were transferred to a 5 mL tube and placed in the magnetic field of an EasySep Magnet (STEMCELL Technologies). After 2 minutes and 30 seconds, the supernatant containing human peripheral blood monocytes was collected by decantation and centrifuged. To generate macrophages, the isolated monocytes were suspended in R10 (PEPROTEC) containing 20 ng / mL of M-CSF, seeded into a 225 cm 2 flask, and incubated at 37°C and 5% CO 2Then, it was cultured for 7 days. On the 7th day, the culture supernatant was removed, and R10 containing 20 ng / mL of M-CSF was added. On the 11th day, the culture supernatant was removed, and R10 (PEPROTEC) containing 20 ng / mL of M-CSF and 20 ng / mL of IL-10 was added. On the 13th day, the macrophages were washed once with PBS, TrypLE Express (Thermo Fisher Scientific) was added, and they were held at 37 °C for 10 minutes. After collection, the macrophages were suspended in PBS at 1×10 6 cells / mL. CellTrace Far Red solution (Thermo Fisher Scientific) was added to the macrophages at 1 μL per 10 6 cells / mL. After incubation for 10 minutes at room temperature in the dark, the macrophages were washed twice with 10 mL of R10. The cells were suspended in R10 at 1×10 6 cells / mL and used as effector cells.

[0165] 2 - 3 Assay of ADCP activity First, 100 μL each of the indicated agents shown in Fig. 18 was added to a U-shaped 96-well microplate. Briefly, an antibody-drug conjugate (3) diluted with R10 or a control antibody-drug conjugate was added at a final concentration of 5 nM. ALX148_hIgG1 or an isotype-matched control Ab diluted with R10 was added at a final concentration of 50 nM. ALX148_hIgG1 was prepared with reference to US Patent Application Publication No. 2019 / 0169266A1. The amino acid sequence of ALX148_hIgG1 is specified in SEQ ID NO: 15 in the Sequence Listing. Second, 50 μL of the effector cells (1×10 6 cells / mL) prepared as in step 2-2 above was added to the wells. Finally, 50 μL of the target cells (1×10 6 cells / mL) prepared as in step 2-1 above was added to the wells, and the cells were mixed by gently pipetting. The cells were incubated at 37 °C and 5% CO 2Then, it was incubated for 4 hours. After centrifugation at 510 g for 3 minutes, the supernatant was removed. The cells were washed once with 200 μL of Hanks’ Balanced Salt Solution containing 0.1% BSA. The cells were suspended in 50 μL of 1×BD Stabilizing Fixative (BD Biosciences) and kept at 4°C in the dark until flow cytometry analysis. ADCP was evaluated using a BD LSRFortessa X-20 flow cytometer (BD Biosciences). The obtained data were analyzed using Flowjo (TreeStar). Cancer cells were determined as CTV-positive (CTV+) cells. Within the cancer cell population, cancer cells phagocytosed by macrophages were defined as CTV+ / Far Red-positive (Far Red+), and hereinafter in this specification, they are referred to as CTV+ Far Red+ cells. ADCP (%) was as follows: ADCP (%) = 100 × [(number of CTV+ Far Red+ cells) ÷ (number of CTV+ cells)] It was calculated as follows. The data were analyzed using GraphPad Prism 9.1.0 (GraphPad Software), and the statistical analysis was performed using SAS System Release 9.2 (SAS Institute).

[0166] As shown in Figure 18, the antibody-drug conjugate (3) showed ADCP activity against NCI-H322 cells and showed a higher level of ADCP activity in the presence of ALX148_hIgG1.

Example

[0167] Antitumor Research (1) Mice: Six-week-old female BALB / c mice (BALB / c AnNCrlCrlj) (Jackson Laboratory Japan, INC.) were used in the experiment.

[0168] Measurement and calculation formula: The major and minor diameters of the tumor were measured twice a week using an electronic digital caliper (Mitutoyo Corporation), and the tumor volume (mm 3 ) was calculated as follows: Tumor volume (mm 3 ) = 0.5 × major diameter (mm) × [minor diameter (mm)] 2 as calculated.

[0169] Antibody-drug conjugate (1) (drug-to-antibody ratio: 7.8) was diluted with buffer and administered intravenously at a dose of 10 mL / kg via the tail vein. ALX148_mIgG1 was prepared with reference to U.S. Patent Application Publication No. 2019 / 0169266A1. The amino acid sequence of ALX148_mIgG1 is set forth in SEQ ID NO: 16 in the Sequence Listing. ALX148_mIgG1 was diluted with saline and administered intraperitoneally at a dose of 10 mL / kg.

[0170] CT26.WT (CRL-2638), a mouse colon cancer cell line, was purchased from the American Type Culture Collection and transfected with the human HER2 gene using a retroviral vector. The established CT26.WT-hHER2 was used for the experiment. On the day of inoculation of CT26.WT-hHER2 cells into mice, CT26.WT-hHER2 cells were collected and resuspended in PBS(-) at a cell density of 3.0 × 10 cells per mouse 6They were suspended and subcutaneously transplanted into the right abdominal region of each animal (day 0). Six days later, the mice were randomly grouped based on tumor volume (day 6). The antibody-drug conjugate (1) was intravenously administered into the tail vein at a dose of 10 mg / kg on days 6 and 13 (a total of 2 times). ALX148_mIgG1 was intraperitoneally administered at a dose of 30 mg / kg on days 7, 10, and 14 (a total of 3 times). A combination administration group with the antibody-drug conjugate (1) and recombinant ALX148_mIgG1 was set; a vehicle administration group was set as a control group. The number of mice per group was 8. The measurement of tumor volume continued until day 27. The comparison between each of the single administration group and the combination administration group was performed according to Dunnett's multiple comparison. A p-value of less than 0.05 was defined as statistically significant.

[0171] The results are shown in Figure 19. Figure 19A shows the treatment schedule of the experiment. Figure 19B summarizes the number of animals that achieved complete remission on day 27. The combination of ADC(1) and ALX148_mIgG1 induced complete remission in a certain number of animals (2 out of 8 mice), while monotherapy with ADC(1) or ALX148_mIgG1 did not induce complete remission (0 out of 8 mice).

Example

[0172] Antitumor Research (2) Mice: Six-week-old female B6J mice (C57BL / 6J) (Jackson Laboratory Japan, INC.) were used in the experiment.

[0173] Measurement and calculation formula: The major and minor axes of the tumor were measured twice a week using an electronic digital caliper (Mitutoyo Corporation), and the tumor volume [mm 3 was as follows: Tumor volume [mm 3 =0.5 × major axis [mm] × minor axis [mm] 2 was calculated as above.

[0174] The antibody-drug conjugate (2) (drug-to-antibody ratio: 3.9) was diluted with ABS, and a dose of 10 mL / kg was intravenously administered via the tail vein. Recombinant ALX148_mIgG1 was diluted with PBS and intraperitoneally administered at a dose of 10 mL / kg.

[0175] The mouse colon cancer MC38 cell line was purchased from the National Cancer Institute and transfected with the human TROP2 gene using a retroviral vector. MC38-hTROP2 cells were suspended in saline, and 0.5×10 6 cells were subcutaneously transplanted into each right axilla of B6J mice (day 0). Four days later, the mice were randomly grouped based on tumor volume (day 4). The antibody-drug conjugate (2) was intravenously administered via the tail vein at a dose of 1 mg / kg on days 4 and 11 (a total of 2 times). ALX148_mIgG1 was intraperitoneally administered at a dose of 30 mg / kg on days 5, 8, and 12 (a total of 3 times). A combination administration group of the antibody-drug conjugate (2) and ALX148_mIgG1 was set up, and a vehicle administration group was set up. The number of mice per group was 12. Measurement of tumor volume continued until day 18. The comparison between each of the single-dose group and the combination administration group was performed according to Dunnett's multiple comparison. A p-value of less than 0.05 was defined as statistically significant.

[0176] The results are shown in Figure 20. Figure 20A shows the experimental treatment schedule. Figure 20B shows the tumor growth of each administration group. The vertical axis represents the tumor volume [mm 3 , and the horizontal axis represents the number of days after tumor inoculation. On day 18, the combination of ADC (2) with ALX148_mIgG1 showed an excellent antitumor effect compared to the single-agent therapies of ADC (2) and ALX148_mIgG1 respectively (Figure 20B).

[0177] From the above examples, it was found that the ADC according to the present invention showed an excellent antitumor effect when administered in combination with ALX148_mIgG1.

[0178] Since the mechanism of action of ALX148 is based on the blockade of the activity of the CD47-SIRPα interaction, other CD47 targeting agents with this blocking activity will also have the same combined effect.

Claims

1. i. A CD47 inhibitor; and ii. An antibody specific for a tumor antigen A pharmaceutical composition comprising the same.

2. The pharmaceutical composition according to claim 1, wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

3. The pharmaceutical composition according to claim 2, wherein the SIRPα derivative comprises a polypeptide having at least 80%, 90%, 95%, 99%, or 100% sequence identity to residues 1 to 149 of SEQ ID NO: 15 or 16.

4. The pharmaceutical composition according to claim 1 or 2, wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

5. The pharmaceutical composition according to claim 4, wherein the CD47 inhibitor comprises a polypeptide having at least 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 15 or 16.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the CD47 inhibitor is evolocumab.

7. The pharmaceutical composition according to claim 2, wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107, and SL-172154.

8. The pharmaceutical composition according to claim 1, wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, and the antibody is specific for CD47.

9. The pharmaceutical composition according to claim 8, wherein the antibody specific for CD47 is selected from the group consisting of magrolimab, remsoipalimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (ligufalimab), and IMM-0306.

10. The pharmaceutical composition according to claim 1, wherein the CD47 inhibitor comprises a small molecule drug capable of binding to CD47.

11. The pharmaceutical composition according to claim 10, wherein the small molecule drug is selected from the group consisting of RRx-001 and IMM-01.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO:

13.

13. Further comprising an antibody-drug conjugate, wherein the antibody specific for a tumor antigen is part of the antibody-drug conjugate, the antibody-drug conjugate further comprises a linker and a drug, the antibody specific for a tumor antigen is connected to the drug via the linker, and the linker and the drug form a drug-linker. The pharmaceutical composition according to any one of claims 1 to 12.

14. The drug-linker is the following formula: 【Chemical 1】 [In the formula, A represents the connection position to the antibody] The pharmaceutical composition according to claim 13, wherein the drug-linker is a drug-linker represented by the formula and is conjugated to an antibody specific for a tumor antigen via a thioether bond.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the antibody specific for a tumor antigen is an anti-HER2 antibody, an anti-HER3 antibody, an anti-Trop2 antibody, an anti-B7-H3 antibody, an anti-GPR20 antibody, or an anti-CDH6 antibody.

16. The pharmaceutical composition according to claim 15, wherein the antibody specific for a tumor antigen is an anti-HER2 antibody.

17. The pharmaceutical composition according to claim 16, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence of amino acid residues 1 to 214 of SEQ ID NO:

2.

18. The pharmaceutical composition according to claim 16, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

2.

19. The pharmaceutical composition according to any one of claims 16 to 18 as a dependent claim of claim 14, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

20. The pharmaceutical composition according to claim 15, wherein the antibody specific for a tumor antigen is an anti-HER3 antibody.

21. The pharmaceutical composition according to claim 20, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

22. The pharmaceutical composition according to claim 21, wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

23. The pharmaceutical composition according to any one of claims 20 to 22 as dependent claims of claim 14, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

24. The pharmaceutical composition according to claim 15, wherein the antibody specific for the tumor antigen is an anti-TROP2 antibody.

25. The pharmaceutical composition according to claim 24, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

6.

26. The pharmaceutical composition according to claim 25, wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

27. The pharmaceutical composition according to any one of claims 24 to 26 as dependent claims of claim 14, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

28. The pharmaceutical composition according to claim 15, wherein the antibody specific for the tumor antigen is an anti-B7-H3 antibody.

29. The pharmaceutical composition according to claim 28, wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

8.

30. The pharmaceutical composition according to claim 29, wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

31. The pharmaceutical composition according to any one of claims 28 to 30 as dependent claims of claim 14, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

32. The pharmaceutical composition according to claim 15, wherein the antibody specific for the tumor antigen is an anti-GPR20 antibody.

33. The pharmaceutical composition according to claim 32, wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

10.

34. The pharmaceutical composition according to claim 33, wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

35. The pharmaceutical composition according to any one of claims 32 to 34 as a dependent claim of claim 14, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

36. The pharmaceutical composition according to claim 15, wherein the antibody specific for a tumor antigen is an anti-CD16 antibody.

37. The pharmaceutical composition according to claim 36, wherein the anti-CD16 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

12.

38. The pharmaceutical composition according to claim 37, wherein the anti-CD16 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

39. The pharmaceutical composition according to any one of claims 36 to 38 as a dependent claim of claim 14, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

40. A CD47 inhibitor for use in the treatment or prevention of cancer by simultaneous or sequential administration with an antibody specific for a tumor antigen.

41. The CD47 inhibitor for use according to claim 40, comprising SIRPα or a SIRPα derivative.

42. The CD47 inhibitor for use according to claim 41, wherein the SIRPα derivative comprises a polypeptide having at least 80%, 90%, 95%, 99%, or 100% sequence identity to residues 1 to 149 of SEQ ID NO: 15 or 16.

43. The CD47 inhibitor for use according to claim 40 or 41, which is a fusion protein and further comprises an Fc region.

44. The CD47 inhibitor for use according to claim 43, comprising a polypeptide having at least 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 15 or 16.

45. The CD47 inhibitor for use according to any one of claims 40 to 44, which is evolocumab.

46. The CD47 inhibitor for use according to claim 41, selected from the group consisting of TTI-621, TTI-622, DSP-107, and SL-172154.

47. The CD47 inhibitor for use according to claim 40, wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, and the antibody is specific for CD47.

48. The CD47 inhibitor for use according to claim 47, wherein the antibody specific for CD47 is selected from the group consisting of magrolimab, remsoipalimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (ligufalimab), and IMM-0306.

49. The CD47 inhibitor for use according to claim 40, which comprises a small molecule drug capable of binding to CD47.

50. The CD47 inhibitor for use according to claim 49, wherein the small molecule drug is selected from the group consisting of RRx-001 and IMM-01.

51. The CD47 inhibitor for use according to any one of claims 40 to 50, wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO:

13.

52. The CD47 inhibitor for use according to any one of claims 40 to 51, wherein the antibody specific for a tumor antigen is part of an antibody-drug conjugate, the antibody-drug conjugate comprises an antibody specific for a tumor antigen connected to a drug via a linker, and the linker and the drug form a drug-linker.

53. The drug-linker is represented by the following formula: 【Chemical 2】 [wherein A represents the connection position to the antibody] The CD47 inhibitor for use according to claim 52, wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

54. The CD47 inhibitor for use according to any one of claims 40 to 53, wherein the antibody specific for a tumor antigen is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-GPR20 antibody, or an anti-CDH6 antibody.

55. The CD47 inhibitor for use according to claim 54, wherein the antibody specific for a tumor antigen is an anti-HER2 antibody.

56. The CD47 inhibitor for use according to claim 55, wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:

2.

57. The CD47 inhibitor for use according to claim 55, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

2.

58. The CD47 inhibitor for use according to any one of claims 55 to 57 as a dependent claim of claim 53, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

59. The CD47 inhibitor for use according to claim 54, wherein the antibody specific for the tumor antigen is an anti-HER3 antibody.

60. The CD47 inhibitor for use according to claim 59, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

61. The CD47 inhibitor for use according to claim 60, wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

62. The CD47 inhibitor for use according to any one of claims 59 to 61 as a dependent claim of claim 53, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

63. The CD47 inhibitor for use according to claim 54, wherein the antibody specific for the tumor antigen is an anti-TROP2 antibody.

64. The CD47 inhibitor for use according to claim 63, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

6.

65. The CD47 inhibitor for use according to claim 64, wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

66. The CD47 inhibitor for use according to any one of claims 63 to 65 as a dependent claim of claim 53, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

67. The CD47 inhibitor for use according to claim 54, wherein the antibody specific for the tumor antigen is an anti-B7-H3 antibody.

68. The CD47 inhibitor for use according to claim 67, wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

8.

69. The CD47 inhibitor for use according to claim 68, wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

70. The CD47 inhibitor for use according to any one of claims 67 to 69 as a dependent claim of claim 53, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

71. The CD47 inhibitor for use according to claim 54, wherein the antibody specific for a tumor antigen is an anti-GPR20 antibody.

72. The CD47 inhibitor for use according to claim 71, wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

10.

73. The CD47 inhibitor for use according to claim 72, wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

74. The CD47 inhibitor for use according to any one of claims 71 to 73 as a dependent claim of claim 53, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

75. The CD47 inhibitor for use according to claim 54, wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody.

76. The CD47 inhibitor for use according to claim 75, wherein the anti-CDH6 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

12.

77. The CD47 inhibitor for use according to claim 76, wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

78. A CD47 inhibitor for use according to any one of claims 75 to 77 as a dependent claim of claim 53, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

79. A CD47 inhibitor for use according to any one of claims 40 to 78, wherein the CD47 inhibitor and an antibody specific for a tumor antigen are active ingredients individually contained in different formulations.

80. A CD47 inhibitor for use according to any one of claims 40 to 79, wherein the cancer is at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophagogastric junction adenocarcinoma, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine cancer, sarcoma, head and neck cancer, hepatocellular cancer, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia, and myelodysplastic syndrome.

81. A treatment method comprising the step of administering in combination a CD47 inhibitor and an antibody specific for a tumor antigen to a subject in need of treatment.

82. The treatment method according to claim 81, wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

83. The treatment method according to claim 82, wherein the SIRPα derivative comprises a polypeptide having at least 80%, 90%, 95%, 99%, or 100% sequence identity to residues 1 to 149 of SEQ ID NO: 15 or 16.

84. The treatment method according to claim 81 or 82, wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

85. The treatment method according to claim 84, wherein the CD47 inhibitor comprises a polypeptide having at least 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 15 or 16.

86. The treatment method according to any one of claims 81 to 85, wherein the CD47 inhibitor is evolocumab.

87. The treatment method according to claim 82, wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107, and SL-172154.

88. The treatment method according to claim 81, wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, and the antibody is specific for CD47.

89. The treatment method according to claim 88, wherein the antibody specific for CD47 is selected from the group consisting of magrolimab, remsoipalimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (ligufalimab), and IMM-0306.

90. The treatment method according to claim 81, wherein the CD47 inhibitor comprises a small molecule drug capable of binding to CD47.

91. The treatment method according to claim 90, wherein the small molecule drug is selected from the group consisting of RRx-001 and IMM-01.

92. The treatment method according to any one of claims 81 to 91, wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO:

13.

93. The treatment method according to any one of claims 81 to 92, further comprising the step of administering an antibody-drug conjugate, wherein the antibody specific for a tumor antigen is part of the antibody-drug conjugate, the antibody-drug conjugate further comprises a linker and a drug, the antibody specific for the tumor antigen is connected to the drug via the linker, and the linker and the drug form a drug-linker.

94. The drug-linker is represented by the following formula: [Chemical Formula 3] [wherein A represents the connection position to the antibody] The treatment method according to claim 93, wherein the drug-linker is conjugated to the antibody specific for the tumor antigen via a thioether bond.

95. The treatment method according to any one of claims 81 to 94, wherein the antibody specific for the tumor antigen is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-GPR20 antibody, or an anti-CDH6 antibody.

96. The treatment method according to claim 95, wherein the antibody specific for the tumor antigen is an anti-HER2 antibody.

97. The treatment method according to claim 96, wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:

2.

98. The treatment method according to claim 96, wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

2.

99. The treatment method according to any one of claims 96 to 98 as dependent claims of claim 94, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

100. The treatment method according to claim 95, wherein the antibody specific for the tumor antigen is an anti-HER3 antibody.

101. The treatment method according to claim 100, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

102. The treatment method according to claim 101, wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

103. The treatment method according to any one of claims 100 to 102 as dependent claims of claim 94, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

104. The treatment method according to claim 95, wherein the antibody specific for the tumor antigen is an anti-TROP2 antibody.

105. The treatment method according to claim 104, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

6.

106. The treatment method according to claim 105, wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

107. The treatment method according to any one of claims 104 to 106 as dependent claims of claim 94, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

108. The treatment method according to claim 95, wherein the antibody specific for the tumor antigen is an anti-B7-H3 antibody.

109. The treatment method according to claim 108, wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

8.

110. The treatment method according to claim 109, wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

111. The therapy according to any one of claims 108 to 110 as dependent claims of claim 94, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

112. The therapy according to claim 95, wherein the antibody specific for the tumor antigen is an anti-GPR20 antibody.

113. The therapy according to claim 112, wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 9 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

10.

114. The therapy according to claim 113, wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

115. The therapy according to any one of claims 112 to 114 as dependent claims of claim 94, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

116. The therapy according to claim 95, wherein the antibody specific for the tumor antigen is an anti-CDH6 antibody.

117. The therapy according to claim 116, wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 11 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

12.

118. The therapy according to claim 117, wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

119. The therapy according to any one of claims 116 to 118 as dependent claims of claim 94, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

120. The therapy according to any one of claims 81 to 119, wherein the CD47 inhibitor and the antibody specific for the tumor antigen are active ingredients individually contained in different formulations.

121. The treatment method according to any one of claims 81 to 120, which is a method for treating at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, adenocarcinoma of the esophagogastric junction, bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine carcinosarcoma, head and neck cancer, hepatocellular cancer, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymus cancer, gallbladder cancer, lymphoma, leukemia, and myelodysplastic syndrome.

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