Combination of antibodies specific for tumor antigens and CD47 inhibitors

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 tumor antigens. The invention also relates to a pharmaceutical composition comprising: a CD47 inhibitor; and antibodies specific for tumor antigens.

Description

[Technical field]

[0001] The present invention relates to pharmaceutical compositions comprising CD47 inhibitors. The present invention also relates to CD47 inhibitors for use in treating or preventing cancer and methods of treatment comprising administering CD47 inhibitors. [Background Technology]

[0002] CD47 (also known as integrin-associated protein-IAP) is a 50 kDa transmembrane protein receptor with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. CD47 is expressed on erythrocytes as a marker of "self" and is also highly expressed on tumor cells. It has been observed that in acute myeloid leukemia and several solid tumor cancers, high CD47 expression on tumor cells can serve as a negative prognostic factor for survival (Patent Document 1).

[0003] SIRPα (SHPS-1) is a single transmembrane molecule belonging to the Ig superfamily, which is present in myeloid cells such as macrophages, dendritic cells and neutrophils, and glial cells (non-patent literature 1). Its extracellular region consists of a single IgV domain and two IgC domains. It is an IgV domain (also referred to as "D1 domain" (non-patent literature 2)) at the connection position with CD47, and it is reported that there are 10 variants in humans (non-patent literature 3). On the other hand, its intracellular region contains an inhibitory motif (ITIM) based on immunoreceptor tyrosine. After the extracellular region of SIRPα binds to CD47, it induces the combination with tyrosine dephosphorylase SHP-1 and SEPHP-2, and transmits a suppression signal.

[0004] It is reported that the SIRPα-CD47 interaction causes a physiological phenomenon, that is, CD47 on erythrocytes binds to SIRPα on macrophages to transmit a "don't eat me" signal, as a result of which unwanted phagocytosis by erythrocytes can be avoided (non-patent document 4). In addition, in the tumor microenvironment, it is suggested that when CD47, which is highly expressed on tumor cells as noted above, binds to SIRPα on macrophages and dendritic cells, the phagocytic activity of engulfing tumor cells is suppressed. When phagocytic activity is suppressed, subsequent tumor antigen presentation to T cells and further subsequent tumor immune responses are suppressed. Therefore, the immune phenomenon (i.e., phagocytosis of tumor cells) is regarded as a checkpoint for the entry of tumor antigens.

[0005] Patent Document 1 discloses a construct that binds to a CD47 protein for cancer treatment. The construct is a polypeptide that includes a SIRPαD1 domain and an Fc domain monomer linked to its N-terminus or C-terminus.

[0006] Antibody-drug conjugates (ADCs) contain cytotoxic drugs conjugated to antibodies whose antigens are expressed on the surface of cancer cells and also bind to antigens that can be internalized by cells. Therefore, ADCs can selectively deliver drugs to cancer cells to promote the accumulation of drugs in cancer cells and kill cancer cells.

[0007] As one such antibody-drug conjugate, an antibody-drug conjugate comprising an antibody and a derivative of exitecan, 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 antibody-drug conjugate as mentioned above can be administered in combination with any of various cancer therapeutic agents.

[0009] However, there are no test results showing excellent combined effects when the aforementioned antibody-drug conjugates are used in combination with a CD47 inhibitor, nor is there any disclosure regarding a scientific basis suggesting such results.

[0010] [Citation list]

[0011] [Patent Document]

[0012] [Patent Document 1] International Publication No. WO 2017 / 027422

[0013] [Patent Document 2] International Publication No. WO 2014 / 057687

[0014] [Patent Document 3] International Publication No. WO 2014 / 061277

[0015] [Patent Document 4] International Publication No. WO 2015 / 098099

[0016] [Patent Document 5] International Publication No. WO 2015 / 115091

[0017] [Patent Document 6] International Publication No. WO 2015 / 146132

[0018] [Patent Document 7] International Publication No. WO 2015 / 155976

[0019] [Patent Document 8] International Publication No. WO 2015 / 155998

[0020] [Non-patent literature]

[0021] [Non-patent document 1] Matozaki et al. Trends in cell biol. 2009 (19) 2, 72-80

[0022] [Non-patent document 2] Lee WY, J Immunol, 2007, 7741-7750

[0023] [Non-patent document 3] Takenaka et al. Nat Immunol. 2007 (8) 12, 1313-1323

[0024] [Non-patent document 4] Matozaki et al. J. Biochem. 2014 (155) 6, 335-344

[0025] [Non-patent document 5] Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108.

[0026] [Non-patent document 6] Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046.

[0027] [Non-patent document 7] Doi T et al., Lancet Oncol 2017;18:1512-22.

[0028] [Non-patent document 8] Takegawa N et al., Int. J. Cancer: 141, 1682-1689 (2017) [Summary of the Invention]

[0030] [Technical issues]

[0031] Antibodies specific for tumor-associated antigens are known to have therapeutic effects in cancer patients. However, in situations where such antibodies alone do not produce complete patient remissions, or where it is desirable to reduce the necessary dose of such antibodies in order to be therapeutic, improved therapies for treating cancer are needed. Therefore, there is a need to obtain superior anti-tumor effects from such antibodies.

[0032] One 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 provided in combination, and / or a therapeutic method comprising administering an inhibitor of CD47 protein and an antibody specific for a tumor antigen in combination to a subject.

[0033] [Solution to the problem]

[0034] As a result of intensive studies to solve the above problems, the present inventors have found that combined administration of a CD47 inhibitor and an antibody specific for a tumor antigen shows an excellent combined effect, and have completed the present invention. Specifically, the present invention includes the following aspects of the present invention.

[0035] (1) A pharmaceutical composition comprising:

[0036] i. CD47 inhibitors; and

[0037] ii. Antibodies specific for tumor antigens.

[0038] (2) The pharmaceutical composition according to (1), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

[0039] (3) The pharmaceutical composition according to (2), wherein the SIRPα derivative comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with residues 1 to 149 of SEQ ID NO: 15 or 16.

[0040] (4) The pharmaceutical composition according to (1) or (2), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

[0041] (5) The pharmaceutical composition according to (4), wherein the CD47 inhibitor comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 15 or 16.

[0042] (6) The pharmaceutical composition according to any one of (1) to (5), wherein the CD47 inhibitor is Evorpacept.

[0043] (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.

[0044] (8) The pharmaceutical composition according to (1), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, wherein the antibody is specific for CD47.

[0045] (9) The pharmaceutical composition according to (8), wherein the antibody specific for CD47 is selected from: mololimab, leszolimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (lefalimumab) and IMM-0306.

[0046] (10) The pharmaceutical composition according to (1), wherein the CD47 inhibitor comprises a small molecule agent capable of binding to CD47.

[0047] (11) The pharmaceutical composition according to (10), wherein the small molecule agent is selected from RRx-001 and IMM-01.

[0048] (12) The pharmaceutical composition according to any one of (1) to (11), wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO:13.

[0049] (13) The pharmaceutical composition according to any one of (1) to (12), further comprising an antibody-drug conjugate, wherein the antibody specific for the tumor antigen is part of the antibody-drug conjugate, and wherein the antibody-drug conjugate further comprises a linker and a drug, the antibody specific for the tumor antigen is connected to the drug via a linker, and the linker and the drug form a drug-linker.

[0050] (13a) The pharmaceutical composition according to any one of (1) to (12), further comprising an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody specific for a tumor antigen linked to a drug via a linker, and the linker and the drug form a drug-linker.

[0051] (14) The pharmaceutical composition according to (13) or (13a), wherein the drug-linker is represented by the following formula:

[0052] [Formula 1]

[0053]

[0054] Where A represents the connection position with the antibody,

[0055] And wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

[0056] (15) The pharmaceutical composition according to any one of (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.

[0057] (16) The pharmaceutical composition according to (15), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody.

[0058] (17) The pharmaceutical composition according to (16), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:2.

[0059] (18) The pharmaceutical composition according to (16), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.

[0060] (19) A pharmaceutical composition according to any one of (16) to (18) as dependent on (14), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0061] (20) The pharmaceutical composition according to (15), wherein the antibody specific for a tumor antigen is an anti-HER3 antibody.

[0062] (21) The pharmaceutical composition according to (20), wherein the anti-HER3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 3, and the light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.

[0063] (22) The pharmaceutical composition according to (21), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0064] (23) A pharmaceutical composition according to any one of (20) to (22) as dependent on (14), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0065] (24) The pharmaceutical composition according to (15), wherein the antibody specific for a tumor antigen is an anti-TROP2 antibody.

[0066] (25) A pharmaceutical composition according to (24), wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:6.

[0067] (26) The pharmaceutical composition according to (25), wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0068] (27) A pharmaceutical composition according to any one of (24) to (26) as dependent on (14), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0069] (28) The pharmaceutical composition according to (15), wherein the antibody specific for a tumor antigen is an anti-B7-H3 antibody.

[0070] (29) The pharmaceutical composition according to (28), wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:8.

[0071] (30) The pharmaceutical composition according to (29), wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0072] (31) A pharmaceutical composition according to any one of (28) to (30) as dependent on (14), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0073] (32) The pharmaceutical composition according to (15), wherein the antibody specific for a tumor antigen is an anti-GPR20 antibody.

[0074] (33) The pharmaceutical composition according to (32), wherein the anti-GPR20 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO:9, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:10.

[0075] (34) The pharmaceutical composition according to (33), wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0076] (35) A pharmaceutical composition according to any one of (32) to (34) as dependent on (14), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0077] (36) The pharmaceutical composition according to (15), wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody.

[0078] (37) A pharmaceutical composition according to (36), wherein the anti-CDH6 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:12.

[0079] (38) The pharmaceutical composition according to (37), wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0080] (39) A pharmaceutical composition according to any one of (36) to (38) as dependent on (14), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0081] (40) A CD47 inhibitor for use in treating or preventing cancer by simultaneous or sequential administration with an antibody specific for a tumor antigen.

[0082] (41) The CD47 inhibitor for use according to (40), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

[0083] (42) A CD47 inhibitor for use according to (41), wherein the SIRPα derivative comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with residues 1 to 149 of SEQ ID NO: 15 or 16.

[0084] (43) The CD47 inhibitor for use according to claim (40) or (41), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

[0085] (44) The CD47 inhibitor for use according to (43), wherein the CD47 inhibitor comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 15 or 16.

[0086] (45) The CD47 inhibitor for use according to any one of (40) to (44), wherein the CD47 inhibitor is Evorpacept.

[0087] (46) The CD47 inhibitor for use according to (41), wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107 and SL-172154.

[0088] (47) The CD47 inhibitor for use according to (40), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, wherein the antibody is specific for CD47.

[0089] (48) The CD47 inhibitor for use according to (47), wherein the antibody specific for CD47 is selected from: Magrolimab, Lemsoparlimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (Ligufalimab) and IMM-0306.

[0090] (49) The CD47 inhibitor for use according to (40), wherein the CD47 inhibitor comprises a small molecule agent capable of binding to CD47.

[0091] (50) The CD47 inhibitor for use according to (49), wherein the small molecule agent is selected from RRx-001 and IMM-01.

[0092] (51) The CD47 inhibitor for use according to any one of (40) to (50), wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO:13.

[0093] (52) A CD47 inhibitor for use according to any one of (40) to (51), wherein the antibody specific for a tumor antigen is part of an antibody-drug conjugate, and wherein the antibody-drug conjugate comprises the antibody specific for a tumor antigen linked to a drug via a linker, the linker and the drug forming a drug-linker.

[0094] (53) The CD47 inhibitor for use according to (52), wherein the drug-linker is represented by the following formula:

[0095] [Formula 1]

[0096]

[0097] Where A represents the connection position with the antibody,

[0098] And wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

[0099] (54) A CD47 inhibitor for 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.

[0100] (55) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody.

[0101] (56) The CD47 inhibitor for use according to (55), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:2.

[0102] (57) The CD47 inhibitor for use according to (55), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO: 2.

[0103] (58) A CD47 inhibitor for use according to any one of (55) to (57) as dependent on (53), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0104] (59) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-HER3 antibody.

[0105] (60) The CD47 inhibitor for use according to (59), wherein the anti-HER3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:3, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:4.

[0106] (61) The CD47 inhibitor for use according to (60), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0107] (62) A CD47 inhibitor for use according to any one of (59) to (61) as dependent upon (53), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0108] (63) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-TROP2 antibody.

[0109] (64) A CD47 inhibitor for use according to (63), wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:6.

[0110] (65) The CD47 inhibitor for use according to (64), wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0111] (66) A CD47 inhibitor for use according to any one of (63) to (65) as dependent on (53), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0112] (67) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-B7-H3 antibody.

[0113] (68) The CD47 inhibitor for use according to (67), wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:8.

[0114] (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.

[0115] (70) A CD47 inhibitor for use according to any one of (67) to (69) as dependent upon (53), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0116] (71) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-GPR20 antibody.

[0117] (72) The CD47 inhibitor for use according to (71), wherein the anti-GPR20 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO:9, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:10.

[0118] (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.

[0119] (74) A CD47 inhibitor for use according to any one of (71) to (73) as dependent upon (53), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0120] (75) The CD47 inhibitor for use according to (54), wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody.

[0121] (76) The CD47 inhibitor for use according to (75), wherein the anti-CDH6 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:12.

[0122] (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.

[0123] (78) A CD47 inhibitor for use according to any one of (75) to (77) as dependent on (53), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0124] (79) The CD47 inhibitor for use according to any one of (40) to (78), wherein the CD47 inhibitor and the antibody specific for the tumor antigen are active ingredients contained in different preparations.

[0125] (80) A CD47 inhibitor for use according to any one of (40) to (79), wherein the cancer is at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophagogastric junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine cancer, sarcoma, head and neck cancer, hepatocellular carcinoma, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia and myelodysplastic syndrome.

[0126] (81) A method of treatment comprising administering to a subject in need of treatment a combination of a CD47 inhibitor and an antibody specific for a tumor antigen.

[0127] (82) The method of treatment according to (81), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

[0128] (83) The method of treatment according to (82), wherein the SIRPα derivative comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with residues 1 to 149 of SEQ ID NO:15 or 16.

[0129] (84) The treatment method according to (81) or (82), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

[0130] (85) The method of treatment according to (84), wherein the CD47 inhibitor comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 15 or 16.

[0131] (86) The method of any one of (81) to (85), wherein the CD47 inhibitor is Evorpacept.

[0132] (87) The method of treatment according to (82), wherein the CD47 inhibitor is selected from the group consisting of TTI-621, TTI-622, DSP-107 and SL-172154.

[0133] (88) The method of treatment according to (81), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, wherein the antibody is specific for CD47.

[0134] (89) The method of treatment according to (88), wherein the antibody specific for CD47 is selected from: mololimab, leszolimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (lefalimumab) and IMM-0306.

[0135] (90) The treatment method according to (81), wherein the CD47 inhibitor comprises a small molecule agent capable of binding to CD47.

[0136] (91) The method of treatment according to (90), wherein the small molecule agent is selected from RRx-001 and IMM-01.

[0137] (92) The method according to any one of (81) to (91), wherein CD47 comprises the polypeptide sequence represented by SEQ ID NO:13.

[0138] (93) The method of any one of (81) to (92), further comprising administering an antibody-drug conjugate, wherein the antibody specific for the tumor antigen is part of the antibody-drug conjugate, and wherein the antibody-drug conjugate further comprises a linker and a drug, the antibody specific for the tumor antigen is connected to the drug via a linker, and the linker and the drug form a drug-linker.

[0139] (93a) The method of any one of (81) to (92), further comprising administering an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody specific for a tumor antigen linked to a drug via a linker, the linker and the drug forming a drug-linker.

[0140] (94) The method of treatment according to (93) or (93a), wherein the drug-linker is represented by the following formula:

[0141] [Formula 1]

[0142]

[0143] Where A represents the connection position with the antibody,

[0144] And wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

[0145] (95) A treatment 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.

[0146] (96) The treatment method according to (95), wherein the antibody specific for the tumor antigen is an anti-HER2 antibody.

[0147] (97) The method of treatment according to (96), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:2.

[0148] (98) The method of (96), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:2.

[0149] (99) According to any one of the treatment methods of (96) to (98) as dependent on (94), the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0150] (100) The treatment method according to (95), wherein the antibody specific for the tumor antigen is an anti-HER3 antibody.

[0151] (101) The method according to (100), wherein the anti-HER3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consists of the amino acid sequence represented by SEQ ID NO:3, and the light chain consists of the amino acid sequence represented by SEQ ID NO:4.

[0152] (102) The treatment method according to (101), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0153] (103) A method of treating according to any one of (100) to (102) as dependent upon (94), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0154] (104) The treatment method according to (95), wherein the antibody specific for the tumor antigen is an anti-TROP2 antibody.

[0155] (105) A method of treating according to (104), wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:6.

[0156] (106) The treatment method according to (105), wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0157] (107) A method of treating according to any one of (104) to (106) as dependent upon (94), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0158] (108) The treatment method according to (95), wherein the antibody specific for the tumor antigen is an anti-B7-H3 antibody.

[0159] (109) The method of treatment according to (108), wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain is composed of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7, and the light chain is composed of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:8.

[0160] (110) The treatment method according to (109), wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0161] (111) A method of treating according to any one of (108) to (110) as dependent upon (94), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0162] (112) The treatment method according to (95), wherein the antibody specific for the tumor antigen is an anti-GPR20 antibody.

[0163] (113) The method of treatment according to (112), wherein the anti-GPR20 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO:9, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:10.

[0164] (114) The treatment method according to (113), wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0165] (115) A method of treating according to any one of (112) to (114) as dependent upon (94), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0166] (116) The treatment method according to (95), wherein the antibody specific for the tumor antigen is an anti-CDH6 antibody.

[0167] (117) The method of treatment according to (116), wherein the anti-CDH6 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:12.

[0168] (118) The treatment method according to (117), wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0169] (119) A method of treating according to any one of (116) to (118) as dependent upon (94), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0170] (120) The method according to any one of (81) to (119), wherein the CD47 inhibitor and the antibody specific for the tumor antigen are active ingredients contained in different preparations.

[0171] (121) A method of treating a cancer according to any one of (81) to (120), wherein the method is used to treat at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine carcinosarcoma, head and neck cancer, hepatocellular carcinoma, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia and myelodysplastic syndrome.

[0172] (122) An antibody specific for a tumor antigen for use in treating or preventing cancer by simultaneous or sequential administration with a CD47 inhibitor.

[0173] (123) The antibody specific for a tumor antigen for the use according to (122), wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

[0174] (124) An antibody specific for a tumor antigen for the use according to (123), wherein the SIRPα derivative comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with residues 1 to 149 of SEQ ID NO: 15 or 16.

[0175] (125) The antibody specific for a tumor antigen for use according to claim (122) or (123), wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

[0176] (126) An antibody specific for a tumor antigen for the use according to (125), wherein the CD47 inhibitor comprises a polypeptide having a sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 15 or 16.

[0177] (127) The antibody specific for a tumor antigen for use according to any one of (122) to (126), wherein the CD47 inhibitor is Evorpacept.

[0178] (128) The antibody specific for a tumor antigen for the use according to (123), wherein the CD47 inhibitor is selected from the following: TTI-621, TTI-622, DSP-107 and SL-172154.

[0179] (129) The antibody specific for a tumor antigen for the use according to (122), wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, wherein the antibody is specific for CD47.

[0180] (130) An antibody specific for a tumor antigen for the use described in (129), wherein the antibody specific for CD47 is selected from: mololimab, leszolimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (lefalimumab) and IMM-0306.

[0181] (131) An antibody specific for a tumor antigen for the use according to (122), wherein the CD47 inhibitor comprises a small molecule agent capable of binding to CD47.

[0182] (132) An antibody specific for a tumor antigen for the use according to (131), wherein the small molecule agent is selected from RRx-001 and IMM-01.

[0183] (133) The antibody specific for a tumor antigen for use according to any one of (122) to (132), wherein CD47 comprises a polypeptide sequence represented by SEQ ID NO:13.

[0184] (134) An antibody specific for a tumor antigen for use according to any one of (122) to (133), wherein the antibody specific for a tumor antigen is part of an antibody-drug conjugate, and wherein 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.

[0185] (135) The antibody specific for a tumor antigen for use according to (134), wherein the drug-linker is represented by the following formula:

[0186] [Formula 1]

[0187]

[0188] Where A represents the connection position with the antibody,

[0189] And wherein the drug linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

[0190] (136) An antibody specific for a tumor antigen for the use according to any one of (133) to (135), 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.

[0191] (137) An antibody specific for a tumor antigen for the use according to (136), wherein the antibody specific for a tumor antigen is an anti-HER2 antibody.

[0192] (138) An antibody specific for a tumor antigen for the use described in (137), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:2.

[0193] (139) An antibody specific for a tumor antigen for the use according to (137), wherein the anti-HER2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:2.

[0194] (140) An antibody specific for a tumor antigen for use according to any one of (137) to (139) as dependent on (135), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0195] (141) The antibody specific for a tumor antigen for the use according to (136), wherein the antibody specific for a tumor antigen is an anti-HER3 antibody.

[0196] (142) An antibody specific for a tumor antigen for the use according to (141), wherein the anti-HER3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:3, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:4.

[0197] (143) An antibody specific for a tumor antigen for the use according to (142), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0198] (144) An antibody specific for a tumor antigen for use according to any one of (141) to (143) as dependent on (135), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0199] (145) The antibody specific for a tumor antigen for the use according to (136), wherein the antibody specific for a tumor antigen is an anti-TROP2 antibody.

[0200] (146) An antibody specific for a tumor antigen for the use described in (145), wherein the anti-TROP2 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:6.

[0201] (147) An antibody specific for a tumor antigen for the use according to (146), wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0202] (148) An antibody specific for a tumor antigen for use according to any one of (145) to (147) as dependent on (135), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0203] (149) The antibody specific for a tumor antigen for the use according to (136), wherein the antibody specific for a tumor antigen is an anti-B7-H3 antibody.

[0204] (150) An antibody specific for a tumor antigen for the use described in (149), wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:8.

[0205] (151) An antibody specific for a tumor antigen for use according to (150), wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0206] (152) An antibody specific for a tumor antigen for use according to any one of (149) to (151) as dependent on (135), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0207] (153) The antibody specific for a tumor antigen for the use according to (136), wherein the antibody specific for a tumor antigen is an anti-GPR20 antibody.

[0208] (154) An antibody specific for a tumor antigen for the use according to (153), wherein the anti-GPR20 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO:9, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:10.

[0209] (155) The antibody specific for a tumor antigen for use according to (154), wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0210] (156) An antibody specific for a tumor antigen for use according to any one of (153) to (155) as dependent on (135), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0211] (157) The antibody specific for a tumor antigen for the use according to (136), wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody.

[0212] (158) An antibody specific for a tumor antigen for the use described in (157), wherein the anti-CDH6 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:12.

[0213] (159) An antibody specific for a tumor antigen for the use according to (158), wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0214] (160) An antibody specific for a tumor antigen for use according to any one of (157) to (159) as dependent on (135), wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

[0215] (161) The antibody specific for a tumor antigen for use according to any one of (122) to (160), wherein the CD47 inhibitor and the antibody specific for a tumor antigen are active ingredients contained in different preparations.

[0216] (162) An antibody specific for a tumor antigen for use according to any one of (122) to (161), wherein the cancer is at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine cancer, sarcoma, head and neck cancer, hepatocellular carcinoma, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia and myelodysplastic syndrome.

[0217] [Advantageous Effects of the Invention]

[0218] The present invention may provide a pharmaceutical composition in which a CD47 inhibitor and an antibody specific for a tumor antigen are administered in combination, and / or a therapeutic method comprising administering a CD47 inhibitor and an antibody specific for a tumor antigen in combination to a subject.

[0219] [definition]

[0220] In this specification, "tumor antigen" is an antigenic substance produced in tumor cells. It includes both "tumor-specific antigens" (i.e., antigens that exist only on tumor cells and not on any other cell types) and "tumor-associated antigens" (i.e., antigens that exist on some tumor cells and some normal cells but are expressed at elevated levels on tumor cells). It includes HER2, HER3, TROP2, B7-H3, GPR20 and CDH6.

[0221] In the present specification, 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 No.: NP_001035111 and shown in SEQ ID NO:14.

[0222] In the present specification, the term "CD47" means cluster of differentiation 47 protein. In some embodiments, the amino acid sequence of CD47 is the sequence shown in SEQ ID NO:13.

[0223] In this specification, the term "small molecule" means an organic molecule with a low molecular weight, for example, less than 1000 Da.

[0224] In this specification, the term "antigen-binding fragment" of an antibody means a partial fragment of an antibody having antigen-binding activity, and includes Fab, F(ab')2, scFv, etc. The term also encompasses Fab', which is a monovalent fragment in an antibody variable region obtained by treating F(ab')2 under reducing conditions. However, the term is not limited to these molecules, as long as the fragment has binding affinity for the antigen. Further, these antigen-binding fragments include not only fragments obtained by treating the full-length molecules of an antibody protein with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0225] In this specification, " identity " between two amino acid sequences has the following meaning. The identity between two amino acid sequences with identical amino acid sequences is 100%. If one of the amino acid sequences has a replacement, deletion or addition of one or two or more amino acids or amino acid residues compared with another amino acid sequence, the identity between the two amino acid sequences is less than 100%. Considering that the example of the algorithm or program for determining the identity between the two sequences in the room can include those algorithms or programs 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).

[0226] In the present specification, the term "antibody specific for a target antigen" means that the antibody preferentially binds to the target antigen compared to an irrelevant target antigen. [Brief Description of the Figures]

[0228] [ Figure 1 ] Figure 1 is a diagram showing the amino acid sequence (SEQ ID NO: 1) of the heavy chain of the anti-HER2 antibody.

[0229] [ Figure 2 ] Figure 2is a diagram showing the amino acid sequence (SEQ ID NO: 2) of the light chain of the anti-HER2 antibody.

[0230] [ Figure 3 ] Figure 3 is a diagram showing the amino acid sequence (SEQ ID NO: 3) of the heavy chain of the anti-HER3 antibody.

[0231] [ Figure 4 ] Figure 4 is a diagram showing the amino acid sequence (SEQ ID NO: 4) of the light chain of the anti-HER3 antibody.

[0232] [ Figure 5 ] Figure 5 is a diagram showing the amino acid sequence (SEQ ID NO: 5) of the heavy chain of the anti-TROP2 antibody.

[0233] [ Figure 6 ] Figure 6 is a diagram showing the amino acid sequence (SEQ ID NO: 6) of the light chain of the anti-TROP2 antibody.

[0234] [ Figure 7 ] Figure 7 is a diagram showing the amino acid sequence (SEQ ID NO: 7) of the heavy chain of the anti-B7-H3 antibody.

[0235] [ Figure 8 ] Figure 8 is a diagram showing the amino acid sequence (SEQ ID NO: 8) of the light chain of the anti-B7-H3 antibody.

[0236] [ Fig. 9 ] Fig. 9 is a diagram showing the amino acid sequence (SEQ ID NO: 9) of the heavy chain of the anti-GPR20 antibody.

[0237] [ Fig.10 ] Fig.10 is a diagram showing the amino acid sequence (SEQ ID NO: 10) of the light chain of the anti-GPR20 antibody.

[0238] [ Fig.11 ] Fig.11 is a diagram showing the amino acid sequence (SEQ ID NO: 11) of the heavy chain of the anti-CDH6 antibody.

[0239] [ Fig.12 ] Fig.12 is a diagram showing the amino acid sequence (SEQ ID NO: 12) of the light chain of the anti-CDH6 antibody.

[0240] [ Fig.13 ] Fig.13is a diagram showing the amino acid sequence of the CD47 protein (SEQ ID NO: 13).

[0241] [ Fig.14 ] Fig.14 It is a diagram showing the amino acid sequence (SEQ ID NO: 14) of the SIRPα protein with GenBank Accession No.: NP_001035111.

[0242] [ Fig.15 ] Fig.15 is a diagram showing the amino acid sequence of the ALX148_hIgG1 construct.

[0243] [ Fig.16 ] Fig.16 is a diagram showing the amino acid sequence of the ALX148_mIgG1 construct.

[0244] [ Fig.17 ] Fig.17 It is a graph showing the ADCP (%) activity of (A) antibody-drug conjugate (1) in JIMT-1 cells and (B) antibody-drug conjugate (2) in AGS cells.

[0245] [ Fig.18 ] Fig.18 It is a graph showing the ADCP (%) activity of the antibody-drug conjugate (3) in NCI-H322 cells.

[0246] [ Fig.19 ] Fig.19 A is a diagram showing the treatment schedule for antibody-drug conjugate (1) and / or ALX148_mIgG1. Fig.19 B is a summary of the number of animals with complete remission at day 27 in the indicated treatment groups.

[0247] [ Fig. 20 ] Fig. 20 A is a diagram showing the treatment schedule for antibody-drug conjugate (2) and / or ALX148_mIgG1. Fig. 20 B is a graph showing tumor volume after vaccination with vehicle, ALX148 monotherapy, antibody-drug conjugate (2) monotherapy, or antibody-drug conjugate (2) and ALX148 combination therapy.

[0248] [Description of implementation plan]

[0249] Hereinafter, a preferred mode for carrying out the present invention is described. The embodiment described below is given only as an example for illustrating a typical embodiment of the present invention and is not intended to limit the scope of the present invention.

[0250] 1.CD47 inhibitors

[0251] CD47 inhibitors are agents that block the binding between CD47 and SIRPα. Tumor cells highly express CD47. When SIRPα expressed on phagocytic cells with phagocytic activity binds to CD47 and they interact, a "don't eat me" signal is transmitted to the phagocytic cells. In this way, tumor cells escape phagocytosis by phagocytes. CD47 inhibitors inhibit the binding between CD47 and SIRPα, thereby inhibiting the transmission of the "don't eat me" signal from tumor cells to phagocytes, thereby enhancing the phagocytosis of tumor cells by phagocytes. As a result, an anti-tumor effect can be exerted. Examples of phagocytic cells with phagocytic activity include macrophages such as M1 and M2 macrophages, and dendritic cells such as immature dendritic cells (imDC).

[0252] In some embodiments of the present invention, the CD47 inhibitor is a polypeptide comprising a 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 with 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 a polypeptide having at least 80%, 90%, 95%, 99% or 100% sequence identity with residues 1 to 149 of SEQ ID NO: 15 or 16.

[0253] In some alternative embodiments, the CD47 inhibitor is a fusion protein and comprises a SIRPα protein or a derivative thereof connected to a further polypeptide at the N-terminus or C-terminus of the SIRPα protein or a derivative thereof. In a preferred embodiment, the further polypeptide comprises a region of the Fc domain. In some variants of this embodiment, the Fc domain is mutated compared to its wild-type form to cancel or reduce binding to Fcγ receptors. In some embodiments comprising a SIRPα protein and an Fc domain, the SIRPα protein is directly conjugated to the Fc domain, while in other embodiments, the SIRPα protein is connected to the Fc domain via a linker (e.g., a spacer) therebetween. In some further embodiments, the CD47 inhibitor is further connected to one or more additional components such as a polymer (e.g., a PEG polymer) to improve its pharmacokinetic properties.

[0254] 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, while the region of residues 150 to 376 of SEQ ID NO: 15 corresponds to the human IgG1 Fc protein. Therefore, in some variations of this embodiment, the CD47 inhibitor comprises a polypeptide having a first region connected to a second region and retaining binding to the CD47 protein, wherein the first region has at least 80%, 90%, 95% or 99% sequence identity with residues 1 to 149 of SEQ ID NO: 15, and the second region has at least 80%, 90%, 95% or 99% sequence identity with residues 150 to 376 of SEQ ID NO: 15.

[0255] 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, while the region of residues 150 to 371 of SEQ ID NO: 16 corresponds to the mouse IgG1 Fc protein. Therefore, in some variations of this embodiment, the CD47 inhibitor comprises a polypeptide having a first region connected to a second region and retaining binding to the CD47 protein, wherein the first region has at least 80%, 90%, 95% or 99% sequence identity with residues 1 to 149 of SEQ ID NO: 16, and the second region has at least 80%, 90%, 95% or 99% sequence identity with residues 150 to 371 of SEQ ID NO: 16.

[0256] In a particularly preferred embodiment, the CD47 inhibitor is Evorpacept, which is also known as ALX148.

[0257] 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.

[0258] In other embodiments of the invention, the CD47 inhibitor is a small molecule agent. In a specific embodiment, the small molecule agent is RRx-001 (J. Med. Chem. 2021, 64, 11, 7261-7271) or IMM-01.

[0259] In some embodiments, the CD47 inhibitor is an anti-CD47 antibody. In such embodiments, the antibody can be obtained in the same manner as described in "2. Antibodies specific for tumor antigens".

[0260] In particular, an anti-CD47 antibody which is a monoclonal antibody can be obtained by immunizing a mammal such as mouse, rat, rabbit, hamster, guinea pig, horse, monkey, dog, pig, cow, goat, sheep with CD47 or a fragment thereof used as an immunogen, fusing spleen cells with myeloma cells to obtain hybridomas, and allowing the hybridomas to produce and secrete antibodies. Hybridomas can be produced by methods known in the art.

[0261] CD47 serving as an immunogen can be chemically synthesized based on the sequence information, or can be obtained as a recombinant protein based on a DNA sequence encoding the protein and produced according to methods known in the art.

[0262] The antibodies can be screened by any method; preferably, by Cell-ELISA using animal cells transfected with DNA encoding CD47.

[0263] In the anti-CD47 antibodies used in the embodiments of the present invention, modified variants of the antibodies are also included. Modified variants refer to variants obtained by subjecting the anti-CD47 antibodies to be used in the present invention to chemical or biological modifications. Examples of chemically modified variants include variants having a chemical group bonded to an amino acid backbone, and variants having a chemical group bonded to an N-linked or O-linked carbohydrate chain. Examples of biologically modified variants include variants obtained by post-translational modification (e.g., N-linked or O-linked glycosylation, N or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants in which methionine residues have been added to the N-terminus by expression using prokaryotic host cells. Further, antibodies such as enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies that are so labeled as to enable detection or separation of the anti-CD47 antibodies used in such embodiments of the present invention are also included in the meaning of modified variants. Such modified variants of the anti-CD47 antibodies used in the present invention can be used, for example, to improve the stability and blood retention of the antibodies, reduce their antigenicity, or detect or separate antibodies.

[0264] It should be noted that the lysine residue at the carboxyl terminus of the antibody heavy chain produced in cultured mammalian cells is known to be deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that the two amino acid residues (glycine and lysine) at the carboxyl terminus of the antibody heavy chain 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 function (e.g., activation of complement, antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the anti-CD47 antibodies used in the embodiments of the present invention also include antibodies and functional fragments thereof subjected to such modifications, as well as deletion variants in which one or two amino acids have been deleted at the carboxyl terminus of the heavy chain, and deletion variants with amidated residues (e.g., a heavy chain in which the carboxyl terminal proline residue has been amidated). It should be noted that the type of deletion variant having a deletion at the carboxyl terminus of the heavy chain of the anti-CD47 antibody used in the embodiment of the present invention is not limited to the above variants, as long as the antigen binding affinity and effector function are preserved. The two heavy chains constituting the anti-CD47 antibody used in the present invention may be one selected from the full-length heavy chain and the above heavy chain having a deletion, or may be two types selected therefrom in combination. The ratio of the amount of each deletion variant may be affected by the type of cultured mammalian cells producing the anti-CD47 antibody used in the present invention and the culture conditions; however, an antibody in which one amino acid residue at the carboxyl terminus has been deleted in both of the two heavy chains in the anti-CD47 antibody used in the present invention can be preferably exemplified.

[0265] The anti-CD47 antibodies used in the present invention include chimeric antibodies and humanized antibodies that have been modified to reduce heterologous antigenicity to humans. Humanized antibodies are also called CDR-grafted antibodies.

[0266] Chimeric antibodies refer to antibodies composed of the light chain variable region and heavy chain variable region of an antibody of a non-human animal, and the light chain constant region and heavy chain constant region of a human antibody. Chimeric antibodies can be prepared by taking out cDNA encoding the light chain variable region and cDNA encoding the heavy chain variable region from a hybridoma producing an anti-CD47 antibody, and 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 a chimeric antibody expression vector, introducing the chimeric antibody expression vector into a host cell and allowing the expression of the antibody.

[0267] It is known that in antibodies produced in cultured mammalian cells, the lysine residue at the carboxyl terminus of the heavy chain is missing (Tsubaki et al., Int. J. Biol. Macromol, 139-147, 2013). However, the deletion of the heavy chain sequence does not affect the antigen binding affinity and effector function (such as activation of complement and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, in the present invention, antibodies lacking the lysine residue at the carboxyl terminus of the heavy chain are included.

[0268] Specific examples of anti-CD47 antibodies include: mololimab (INN RN: 2169232-81-7), leszolimab (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 (lesfalimab) and IMM-0306.

[0269] 2. Antibodies specific for tumor antigens

[0270] The antibody specific for tumor antigen used in the present invention can be derived from any species, and is preferably derived from an antibody of human, rat, mouse or rabbit. In the case where the antibody is derived from a species other than human species, it is preferably chimeric or humanized using well-known techniques. The antibody specific for tumor antigen can be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0271] An antibody specific for a tumor antigen is an antibody having a property capable of targeting cancer cells, and preferably an antibody having, for example, a property of recognizing cancer cells, a property of binding to cancer cells, a property of internalizing in cancer cells, and / or a cytocidal activity against cancer cells.

[0272] The binding activity of antibodies against cancer cells can be confirmed using flow cytometry. Antibody internalization into tumor cells can be confirmed using the following: (1) using a second antibody (fluorescently labeled) combined with a therapeutic antibody to visualize the antibody incorporated into the cell under a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) using a second antibody (fluorescently labeled) combined with a therapeutic antibody to measure the fluorescence intensity of the incorporated cell (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) using a Mab-ZAP assay of an immunotoxin combined with a therapeutic antibody, wherein the toxin is released after being incorporated into the cell to inhibit cell growth (Bio Techniques 28: 162-165, January 2000). As an immunotoxin, a recombinant complex protein of a diphtheria toxin catalytic domain and protein G can be used.

[0273] The anti-tumor activity of the antibody can be confirmed in vitro by determining the inhibitory activity against cell growth. For example, a cancer cell line overexpressing the target protein for the antibody is cultured, and the antibody is added to the culture system at different concentrations to determine the inhibitory activity against lesion formation, colony formation, and spheroid growth. The anti-tumor activity can be confirmed in vivo, for example, by administering the antibody to nude mice with a transplanted cancer cell line that highly expresses the target protein, and determining the changes in the cancer cells.

[0274] It is preferred, but not essential, that the antibody specific for the tumor antigen itself should have an anti-tumor effect. In embodiments where the antibody specific for the tumor antigen is part of an antibody-drug conjugate, an additional consideration is that for the purpose of specifically and selectively exerting the cytotoxic activity of the anti-tumor compound against cancer cells, it is important and also preferred that the antibody should have the property of internalization to migrate into cancer cells.

[0275] Antibodies specific for tumor antigens used in the present invention can be obtained by procedures known in the art. For example, antibodies can be obtained using methods commonly performed in the art, which involve immunizing animals with antigenic polypeptides (i.e., tumor antigens), and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to humans, and animals can be immunized with antigens derived from non-human animals such as mice, rats, etc. In this case, the cross-reactivity of antibodies bound to the obtained heterologous antigens with human antigens can be tested to screen antibodies suitable for human diseases.

[0276] Alternatively, antibody-producing cells that produce antibodies against the 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, NY (1980)) to establish hybridomas from which monoclonal antibodies can in turn be obtained.

[0277] Antigens can be obtained by genetically modifying host cells to produce genes encoding antigenic proteins. Specifically, a vector that allows the expression of antigenic genes is prepared and transferred to host cells so that the genes are expressed. The expressed antigen can therefore be purified. Antibodies can also be obtained by immunizing animals using the above-mentioned genetically modified antigen-expressing cells or cell lines expressing antigens.

[0278] Antibodies specific for tumor antigens are preferably recombinant antibodies obtained by artificial modification for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies or humanized antibodies, or preferably antibodies having only gene sequences derived from human antibodies, i.e. human antibodies. These antibodies can be produced using known methods.

[0279] As chimeric antibodies, there can be exemplified antibodies in which the antibody variable region and constant region are derived from different species, for example, chimeric antibodies in which a mouse- or rat-derived antibody variable region is linked to a human-derived antibody constant region (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0280] Examples of humanized antibodies include antibodies obtained by integrating only the complementarity determining region (CDR) of a heterologous antibody into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies obtained by transplanting part of the amino acid residues of the heterologous antibody framework and the CDR sequence of the heterologous antibody into a human antibody via the CDR transplantation method (WO90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0281] As the human antibody, there can be exemplified an antibody produced by using a human antibody-producing mouse having a human chromosome fragment including heavy chain and light chain genes 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 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), 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, which are selected from human antibody libraries (see Wormstone, IM 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.

[0282] In the antibody specific for tumor antigen, modified variants of the antibody are also included. Modified variants refer to variants obtained by subjecting antibodies specific for tumor antigens to chemical or biological modifications. Examples of chemically modified variants include variants comprising bonding of chemical groups to amino acid backbones, variants comprising bonding of chemical groups to N-linked or O-linked carbohydrate chains, etc. Examples of biologically modified variants include variants obtained by post-translational modification (e.g., N-linked or O-linked glycosylation, N or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants in which methionine residues have been added to the N-terminus by expression in prokaryotic host cells. Further, such labeling so as to enable detection or separation of antibodies according to the present invention or antibodies of antigens, such as enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies are also included in the meaning of modified variants. Such modified variants of antibodies specific for tumor antigens can be used to improve the stability and blood retention of antibodies, reduce their antigenicity, detect or separate antibodies or antigens, and the like.

[0283] Further, by regulating the modification (glycosylation, defucosylation, etc.) of the polysaccharide connected to the antibody specific for the tumor antigen, the cytotoxic activity of the antibody-dependent cells can be enhanced. As a technology for regulating the polysaccharide modification of antibodies, International Publication No. WO 99 / 54342, International Publication No. WO 00 / 61739, International Publication No. WO 02 / 31140, International Publication No. WO 2007 / 133855 and International Publication No. WO 2013 / 120066, etc. are known. However, the technology is not limited to this. In the antibody specific for the tumor antigen, the modified antibody of the polysaccharide is also included.

[0284] It is known that the lysine residue at the carboxyl terminus of the antibody heavy chain produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that the two amino acid residues (glycine and lysine) at the carboxyl terminus of the antibody heavy chain 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 function (activation of complement, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, in the antibody specific for tumor antigens, antibodies and functional fragments of antibodies subjected to such modifications are also included, as well as deletion variants in which one or two amino acids have been deleted at the carboxyl terminus of the heavy chain, deletion variants with amidated residues (e.g., heavy chains in which the carboxyl terminal proline residue has been amidated), and the like. The type of deletion variant having a deletion at the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above variants, as long as the antigen binding affinity and effector function are preserved. The two heavy chains constituting the antibody specific for the tumor antigen may be one type selected from the full-length heavy chain and the above-mentioned deletion variants, or may be two types selected therefrom in combination. The ratio of the amount of each deletion variant may be affected by the type and culture conditions of the cultured mammalian cells producing the antibody according to the present invention; however, antibodies in which one amino acid residue at the carboxyl terminus has been deleted in both of the two heavy chains in the antibody specific for the tumor antigen can be preferably exemplified.

[0285] Examples of isotypes of antibodies specific for tumor antigens include IgG (IgG1, IgG2, IgG3, and IgG4), and preferably IgG1, IgG2, or IgG4.

[0286] Examples of antibodies specific for tumor antigens may include, but are not particularly limited to, anti-HER2 antibodies, anti-HER3 antibodies, anti-TROP2 antibodies, anti-B7-H3 antibodies, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD56 antibodies, anti-CD98 antibodies, anti-DR5 antibodies, anti-EGFR antibodies, anti-EPHA2 antibodies, anti-FGFR2 antibodies, anti-FGFR4 antibodies, anti-FOLR1 antibodies, anti-VEGF antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-C D70 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 preferably anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody and anti-CDH6 antibody can be exemplified.

[0287] In some embodiments, antibodies specific for tumor-associated antigens include multiple antibodies in which each antibody is specific for different tumor antigens. For example, in some embodiments, two antibodies each specific for different tumor-associated antigens are provided. More generally, in some embodiments, multiple antibodies in which each antibody is specific for different tumor antigens are provided, and each antibody is independently selected from the following groups: anti-HER2 antibodies, anti-HER3 antibodies, anti-TROP2 antibodies, anti-B7-H3 antibodies, anti-CD3 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD56 antibodies, anti-CD98 antibodies, anti-DR5 antibodies, anti-EGFR antibodies, anti-EPHA2 antibodies, anti-FGFR2 antibodies, anti-FGFR4 antibodies, anti-FOLR1 antibodies, anti-VEGF antibodies, 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, as well as anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody and anti-CDH6 antibody.

[0288] In the present specification, the term "anti-HER2 antibody" refers to an antibody which specifically binds to HER2 (human epidermal growth factor receptor type 2; ErbB-2) and preferably has an activity of internalizing in a cell expressing HER2 by binding to HER2.

[0289] Examples of the anti-HER2 antibody include trastuzumab (U.S. Patent No. 5821337) and pertuzumab (International Publication No. WO 01 / 00245), and trastuzumab can be preferably exemplified.

[0290] The anti-HER2 antibody is preferably an antibody comprising a heavy chain and a light chain, the heavy chain comprising CDRH1 consisting of an amino acid sequence consisting of amino acid residues 26 to 33 of SEQ ID NO:1, CDRH2 consisting of an amino acid sequence consisting of amino acid residues 51 to 58 of SEQ ID NO:1, and CDRH3 consisting of an amino acid sequence consisting of amino acid residues 97 to 109 of SEQ ID NO:1, the light chain comprising CDRL1 consisting of an amino acid sequence consisting of amino acid residues 27 to 32 of SEQ ID NO:2, CDRL2 consisting of an amino acid sequence consisting of amino acid residues 50 to 52 of SEQ ID NO:2, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 89 to 97 of SEQ ID NO:2, and more preferably an antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 1 to 120 of SEQ ID NO:1, the light chain comprising a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 1 to 107 of SEQ ID NO:2, and even more preferably an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 27 to 32 of SEQ ID NO:2, The antibody is an antibody comprising an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consists of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO:2; or an antibody comprising a heavy chain and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:2.

[0291] In the present specification, the term "anti-HER3 antibody" refers to an antibody which specifically binds to HER3 (human epidermal growth factor receptor type 3; ErbB-3) and preferably has an activity of internalizing in a cell expressing HER3 by binding to HER3.

[0292] Examples of anti-HER3 antibodies include Pertuzumab (U3-1287), U1-59 (International Publication No. WO 2007 / 077028), MM-121 (Ceretuzumab), the anti-ERBB3 antibody described in International Publication No. WO 2008 / 100624, RG-7116 (Rutuzumab) and LJM-716 (Eglinutumab), and Pertuzumab and U1-59 can be preferably exemplified.

[0293] The anti-HER3 antibody is preferably an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of CDRH1 consisting of an amino acid sequence consisting of amino acid residues 26 to 35 of SEQ ID NO:3, CDRH2 consisting of an amino acid sequence consisting of amino acid residues 50 to 65 of SEQ ID NO:3, and CDRH3 consisting of an amino acid sequence consisting of amino acid residues 98 to 106 of SEQ ID NO:3, and the light chain consists of CDRL1 consisting of an amino acid sequence consisting of amino acid residues 24 to 39 of SEQ ID NO:4, CDRL2 consisting of an amino acid sequence consisting of amino acid residues 56 to 62 of SEQ ID NO:4, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 95 to 103 of SEQ ID NO:4;

[0294] More preferably, an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 1 to 117 of SEQ ID NO: 3, and the light chain comprises a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 1 to 113 of SEQ ID NO: 4; and

[0295] Even more preferably, 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 an antibody variant in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0296] In the present specification, the term "anti-TROP2 antibody" refers to an antibody which specifically binds to TROP2 (TACSTD2: tumor calcium signal transducer 2; EGP-1) and preferably has an activity of internalizing in a cell expressing TROP2 by binding to TROP2.

[0297] Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. WO 2015 / 098099).

[0298] The anti-TROP2 antibody is preferably an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of: CDRH1 consisting of an amino acid sequence consisting of amino acid residues 50 to 54 of SEQ ID NO:5, CDRH2 consisting of an amino acid sequence consisting of amino acid residues 69 to 85 of SEQ ID NO:5, and CDRH3 consisting of an amino acid sequence consisting of amino acid residues 118 to 129 of SEQ ID NO:5, and the light chain consists of: CDRL1 consisting of an amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO:6, CDRL2 consisting of an amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO:6, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 109 to 117 of SEQ ID NO:6;

[0299] More preferably, an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO:5, and the light chain comprises a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO:6; and

[0300] Even more preferably, an antibody comprising 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, or an antibody variant in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0301] In the present specification, 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 an activity of internalization in cells expressing B7-H3 by binding to B7-H3.

[0302] Examples of anti-B7-H3 antibodies include M30-H1-L4 (International Publication No. WO 2014 / 057687).

[0303] The anti-B7-H3 antibody is preferably an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of CDRH1 consisting of an amino acid sequence consisting of amino acid residues 50 to 54 of SEQ ID NO:7, CDRH2 consisting of an amino acid sequence consisting of amino acid residues 69 to 85 of SEQ ID NO:7, and CDRH3 consisting of an amino acid sequence consisting of amino acid residues 118 to 130 of SEQ ID NO:7, and the light chain consists of CDRL1 consisting of an amino acid sequence consisting of amino acid residues 44 to 53 of SEQ ID NO:8, CDRL2 consisting of an amino acid sequence consisting of amino acid residues 69 to 75 of SEQ ID NO:8, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 108 to 116 of SEQ ID NO:8;

[0304] More preferably, an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 141 of SEQ ID NO:7, and the light chain comprises a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 21 to 128 of SEQ ID NO:8; and

[0305] Even more preferably, an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:8, or an antibody variant in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0306] In the present specification, the term "anti-GPR20 antibody" refers to an antibody which specifically binds to GPR20 (G protein coupled receptor 20) and preferably has an activity of internalizing in a cell expressing GPR20 by binding to GPR20.

[0307] Examples of anti-GPR20 antibodies include h046-H4e / L7 (International Publication No. WO 2018 / 135501).

[0308] The anti-GPR20 antibody is preferably an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of CDRH1 consisting of an amino acid sequence consisting of amino acid residues 45 to 54 of SEQ ID NO:9, CDRH2 consisting of an amino acid sequence consisting of amino acid residues 69 to 78 of SEQ ID NO:9, and CDRH3 consisting of an amino acid sequence consisting of amino acid residues 118 to 131 of SEQ ID NO:9, and the light chain consists of CDRL1 consisting of an amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO:10, CDRL2 consisting of an amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO:10, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 109 to 117 of SEQ ID NO:10;

[0309] More preferably, an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 142 of SEQ ID NO: 9, and the light chain comprises a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 10; and

[0310] Even more preferably, 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, or an antibody variant in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0311] In the present specification, the term "anti-CDH6 antibody" refers to an antibody which specifically binds to CDH6 (cadherin-6) and preferably has an activity of internalizing in a cell expressing CDH6 by binding to CDH6.

[0312] Examples of anti-CDH6 antibodies include H01L02 (International Publication No. WO 2018 / 212136).

[0313] The anti-CDH6 antibody is preferably an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of: CDRH1 consisting of an amino acid sequence consisting of amino acid residues 45 to 54 of SEQ ID NO: 11, CDRH2 consisting of an amino acid sequence consisting of amino acid residues 69 to 78 of SEQ ID NO: 11, and CDRH3 consisting of an amino acid sequence consisting of amino acid residues 118 to 130 of SEQ ID NO: 11, and the light chain consists of: CDRL1 consisting of an amino acid sequence consisting of amino acid residues 44 to 54 of SEQ ID NO: 12, CDRL2 consisting of an amino acid sequence consisting of amino acid residues 70 to 76 of SEQ ID NO: 12, and CDRL3 consisting of an amino acid sequence consisting of amino acid residues 109 to 116 of SEQ ID NO: 12;

[0314] More preferably, an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 11, and the light chain comprises a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 21 to 128 of SEQ ID NO: 12; and

[0315] Even more preferably, 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, or an antibody variant in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0316] 3. Antibody-drug conjugates

[0317] In some embodiments of the invention, an antibody specific for a tumor antigen is part of an antibody-drug conjugate, which comprises an antibody specific for a tumor antigen connected to a drug via a linker. The partial structure of the antibody-drug conjugate consisting of a linker and a drug is referred to as a "drug-linker".

[0318] It is particularly preferred that the antibody-drug conjugate is one in which a drug-linker represented by the following formula is conjugated to an antibody specific for a tumor antigen via a thioether bond:

[0319] [Formula 1]

[0320]

[0321] Wherein A represents the connection position with the antibody.

[0322] The drug-linker is attached to the thiol groups (in other words, the sulfur atoms of cysteine ​​residues) formed at the interchain disulfide bonding sites in the antibody (two sites between heavy chains, and two sites between heavy and light chains).

[0323] The drug-linker of the embodiment includes exotecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13-dione, (also represented by chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolo[1,2-b]quinoline-10,13(9H,15H)-dione)) which is a topoisomerase I inhibitor as a component. Exotecan is a camptothecin derivative having an antitumor effect represented by the following formula:

[0324] [Formula 2]

[0325]

[0326] The antibody-drug conjugate used in the embodiments of the present invention can also be represented by the following formula:

[0327] [Formula 3]

[0328]

[0329] wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond. n has the same meaning as the average number of so-called conjugated drug molecules (DAR; drug / antibody ratio) and indicates the average number of drug-linker units conjugated per antibody molecule.

[0330] After migrating into cancer cells, the antibody-drug conjugate used in the embodiments of the present invention is cleaved at the linker portion to release a compound represented by the following formula (hereinafter referred to as compound (A)):

[0331] [Formula 4]

[0332]

[0333] The above compounds are inferred to be the original source of at least some of the anti-tumor activity of the antibody-drug conjugates used in such embodiments of the present invention, and have been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15; 22(20): 5097-5108, Epub 2016 Mar 29)

[0334] Topoisomerase I is an enzyme that cuts single strands of DNA and rejoins the cut fragments, and is involved in the conversion of DNA higher-order structures and the synthesis of DNA. Therefore, drugs having a topoisomerase I inhibitory effect inhibit DNA synthesis to terminate cell division during the S phase (DNA synthesis phase) in the cell cycle, and induce apoptosis (cell death) to suppress cancer cell proliferation.

[0335] The antibody-drug conjugates used in the embodiments of the present invention are also known to have a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046).

[0336] The bystander effect is exerted by a process such that the antibody-drug conjugate used in such an embodiment of the present invention is internalized in cancer cells expressing the target, and the above-mentioned compound is released and then also exerts an anti-tumor effect on cancer cells existing in its surroundings and not expressing the target.

[0337] When the antibody-drug conjugate according to such an embodiment of the present invention is used in combination with a CD47 inhibitor, the bystander effect also exerts an excellent anti-tumor effect.

[0338] 4. Production of Antibody-Drug Conjugates

[0339] The drug-linker intermediate used to produce the antibody-drug conjugate according to the present invention is represented by the following formula.

[0340] [Formula 5]

[0341]

[0342] The drug-linker intermediate can be 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]glycylamide, and reference can be made to International Publication No. WO 2014 / 057687, International Publication No. WO 2015 / 098099, International Publication No. WO 2015 / 115091, International Publication No. WO The invention is produced according to the description of International Publication No. WO 2015 / 155998 and International Publication No. WO 2019 / 044947.

[0343] The antibody-drug conjugate used in the embodiments of the present invention can be produced by reacting the above-mentioned drug-linker intermediate with an antibody having a thiol group (alternatively referred to as a sulfhydryl group), the antibody being specific for a tumor antigen.

[0344] Antibodies with sulfhydryl groups can be obtained by methods well known in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, by using 0.3 to 3 molar equivalents of a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) / interchain disulfide bonds within an antibody, and reacting with the antibody in a buffer solution containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an antibody with a sulfhydryl group can be obtained, accompanied by partial or complete reduction of the interchain disulfide bonds within the antibody.

[0345] Further, by using 2 to 20 molar equivalents of the drug-linker intermediate / antibody having a thiol group, an antibody-drug conjugate in which 2 to 8 drug molecules are conjugated per antibody molecule can be produced.

[0346] The average number of conjugated drug molecules per antibody molecule of the produced antibody-drug conjugate can be determined, for example, by a calculation method based on measuring the UV absorbance of the antibody-drug conjugate and its conjugation precursor at two wavelengths of 280 nm and 370 nm (UV method), or by a quantitative calculation method based on HPLC measurement of fragments obtained by treating the antibody-drug conjugate with a reducing agent (HPLC method).

[0347] The conjugation between the antibody and the drug-linker intermediate and the calculation of the average number of conjugated drug molecules per antibody molecule of the antibody-drug conjugate can be performed with reference to the description in International Publication No. WO 2014 / 057687, International Publication No. WO 2015 / 098099, International Publication No. WO 2015 / 115091, International Publication No. WO 2015 / 155998, International Publication No. WO 2018 / 135501 and International Publication No. WO 2018 / 212136, etc.

[0348] In the present invention, "anti-HER2 antibody-drug conjugate" means an antibody-drug conjugate in which the antibody in the antibody-drug conjugate according to the present invention is an anti-HER2 antibody.

[0349] The average number of drug-linker units conjugated per antibody molecule in the anti-HER2 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0350] The anti-HER2 antibody-drug conjugate can be produced with reference to the description in International Publication No. WO 2015 / 115091 and the like.

[0351] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to the present invention is an anti-HER3 antibody.

[0352] The average number of drug-linker units conjugated per antibody molecule in the anti-HER3 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0353] The anti-HER3 antibody-drug conjugate can be produced with reference to the description in International Publication No. WO 2015 / 155998 and the like.

[0354] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to the present invention is an anti-TROP2 antibody.

[0355] The average number of drug-linker units conjugated per antibody molecule in the anti-TROP2 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 5, even more preferably 3.5 to 4.5, and even more preferably about 4.

[0356] The anti-TROP2 antibody-drug conjugate can be produced with reference to the description in International Publication No. WO 2015 / 098099 and the like.

[0357] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to the present invention is an anti-B7-H3 antibody.

[0358] The average number of drug-linker units conjugated per antibody molecule in the anti-B7-H3 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 5, even more preferably 3.5 to 4.5, and even more preferably about 4.

[0359] The anti-B7-H3 antibody-drug conjugate used in the present invention can be produced with reference to the description in International Publication No. WO 2014 / 057687 and the like.

[0360] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to the present invention is an anti-GPR20 antibody.

[0361] The average number of drug-linker units conjugated per antibody molecule in the anti-GPR20 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0362] Anti-GPR20 antibody-drug conjugates can be produced with reference to the description in International Publication No. WO 2018 / 135501 and the like.

[0363] In the present invention, the term "anti-CDH6 antibody-drug conjugate" refers to an antibody-drug conjugate such that the antibody in the antibody-drug conjugate according to the present invention is an anti-CDH6 antibody.

[0364] The average number of drug-linker units conjugated per antibody molecule in the anti-CDH6 antibody-drug conjugate is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0365] Anti-CDH6 antibody-drug conjugates can be produced with reference to the description in International Publication No. WO 2018 / 212136 and the like.

[0366] 5. Relationship between immunogenic cell death and CD47 inhibitors

[0367] Immunogenic cell death (ICD) is 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) to cell death on the cell surface. These are danger signals that activate immune cells. It is reported that ATP is responsible for recruiting and activating dendritic cells (DC) and macrophages; HMGB1 is responsible for enhancing the production of inflammatory cytokines such as type I IFN; and CRT acts as a "eat me signal" and enhances antigen uptake from dead cells (Nature Reviews Immunology.2017, 17, 97-111). In short, ICD of cancer cells occurs, and immunity (anti-tumor immunity) against cancer cells can be induced. As anticancer agents that induce ICD, such as anthracyclines, oxaliplatin and cyclophosphamide are known; however, for example, docetaxel and mitomycin C have not been confirmed as inducing ICD. The presence or absence of ICD effects can be evaluated not only in vitro based on whether danger signals are detected by adding agents, but also in vivo based on vaccination assays. In the latter, cancer cells are treated with drugs and transplanted into immunocompetent mice. A week later, cancer cells that have not been treated with drugs are transplanted to the opposite side. At this point, if immune memory has been formed by cancer cells (which leads to ICD), the implantation and proliferation of transplanted cancer cells are inhibited (Cancer Research, 2017, 77, 2686-2698).

[0368] In order to form immune memory through ICD, it is important for myeloid cells such as dendritic cells and macrophages to absorb cancer antigens. At this time, it is speculated that the "don't eat me signal" is transmitted between myeloid cells and cancer cells through SIRPα-CD47. If a CD47 inhibitor is administered, signal transmission can be inhibited and phagocytosis can be enhanced. As a result, the uptake of cancer antigens can be enhanced. Cancer antigens absorbed by dendritic cells and / or macrophages are processed into 8-30-mer peptide fragments within the cell, which are presented on MHC. MHC has two categories, class I and class II. Antigenic peptides of about 9 polymers presented to MHC class I activate CD8+T cells; while antigenic peptides of about 15 polymers presented to MHC class II activate CD4+T. Generally, foreign antigens are processed and presented to MHC-II class in myeloid cells; however, part of the DC subset presents foreign antigens to MHC-I class and activates CD8+T that exerts cytotoxic activity on cancer cells; in short, part of the DC subset has cross-presentation ability.

[0369] The relationship between antibody-drug conjugates in which drugs with cytocidal activity are integrated and ICD induction has been reported in the past in the case of antibody-drug conjugates in which tubulin inhibitors, pyrrolobenzodiazepines (PBDs) and MMAE are integrated (Cancer Research, 2017, 77, 2686-2698 or ONCOIMMUNOLOGY, 2019, 8 (4), e1565859). Regarding the antibody-drug conjugate containing TpoI inhibitor compound (A) as a payload used in the present invention, only HMGB1 has been reported to be released from cells treated with compound (A) and to exert limited anti-tumor effects through vaccination of therapeutic cells (Clin. Invest. 2020: 130 (1): 374-388). This time, the following was found: 1) Compound (A) not only induces the release of HMGB1 from dying cancer cells, but also induces the release of other danger signals; and 2) Compound (A) activates immune cells present in the microenvironment of cancer and enhances cancer antigen-specific T cell populations to induce anti-tumor immunity.

[0370] The mechanism of action of the present invention will now be explained. First, antibodies specific for tumor antigens bind to cancer cells. The antibody then induces ADCP via nearby immune cells. Secondly, CD47 inhibitors enhance ADCP activity by canceling the "don't eat me signal" presented by cancer cells. Third, in embodiments where antibodies specific for tumor antigens are part of the ADC, the antibody delivers the drug to the cancer cells, and the drug payload thereby induces immunogenic cell death of the cancer cells (to the extent that the cells are not engulfed), and the cancer cells release inflammatory molecules such as ATP and HMGB1. These molecules can activate immune cells. Therefore, due to the immunogenic cell death of the drug payload and in the presence of CD47 inhibitors, the ADCP enhanced by the antibody portion of the antibody-drug conjugate contributes to anti-tumor immunity.

[0371] 6. Treatment, Dosage and Combination

[0372] According to the therapeutic method of the present invention, a CD47 inhibitor and an antibody specific for a tumor antigen are administered in combination.

[0373] In some embodiments of the treatment methods of the present invention, the CD47 inhibitor and the antibody specific for the tumor antigen are contained in different preparations as active ingredients. 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 yet further embodiments, the CD47 inhibitor and the antibody specific for the tumor antigen are contained in a single preparation as active ingredients and are administered together.

[0374] The pharmaceutical composition or treatment method of the present invention can be used to treat cancer, preferably for treating at least one disease selected from the following: breast cancer, gastric cancer (also known as gastric adenocarcinoma), colorectal cancer (also known as colon and rectal cancer, and 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), esophageal gastric junction adenocarcinoma, biliary tract cancer (including bile duct cancer), gallbladder cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumors, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma. More preferably, the invention relates to 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, esophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer and uterine carcinosarcoma; and even more preferably, the invention relates to a method for treating at least one cancer selected from the group consisting of: breast cancer, gastric cancer, lung cancer and ovarian cancer.

[0375] In a preferred embodiment, the specificity of the antibody specific for the tumor antigen is determined by examining the type of cancer and / or tumor markers present. For example, if HER2 expression is found in cancer, anti-HER2 antibodies may be preferably used; if HER3 expression is found in cancer, anti-HER3 antibodies may be preferably used; if TROP2 expression is found in cancer, anti-TROP2 antibodies may be preferably used; if B7-H3 expression is found in cancer, anti-B7-H3 antibodies may be preferably used; if GPR20 expression is found in cancer, anti-GPR20 antibodies may be preferably used; if CDH6 expression is found in cancer, anti-CDH6 antibodies may be preferably used.

[0376] The presence or absence of HER2, HER3, TROP2, B7-H3, GPR20 and CDH6 and other tumor markers can be examined, for example, by collecting tumor tissue from cancer patients and subjecting formalin-fixed paraffin-embedded specimens (FFPE) to examinations at the gene product (protein) level such as immunohistochemistry (IHC), flow cytometry, Western blotting, or at the gene translation level such as in situ hybridization (ISH), quantitative PCR (q-PCR), microarray analysis, alternatively, it can also be examined by collecting cell-free blood circulating tumor DNA (ctDNA) from cancer patients and subjecting it to examination using methods such as next-generation sequencing (NGS).

[0377] The pharmaceutical composition and treatment method of the present invention can be preferably used in mammals, and can be more preferably used in humans.

[0378] The anti-tumor effect of the pharmaceutical composition and treatment method of the present invention can be confirmed, for example, by generating a model in which cancer cells are transplanted into a test animal and measuring the effects of tumor volume reduction and life extension due to the therapeutic pharmaceutical composition and treatment method of the present invention. In addition, comparison with the anti-tumor effect of each of the CD47 inhibitor used in the present invention and the antibody specific for the tumor antigen by single administration can provide confirmation of the combined effect of the CD47 inhibitor used in the present invention and the antibody specific for the tumor antigen.

[0379] In addition, the anti-tumor effects of the pharmaceutical composition and treatment method of the present invention can be confirmed in clinical studies using the solid tumor response evaluation criteria (RECIST) evaluation method, the WHO evaluation method, the Macdonald evaluation method, body weight measurement and other methods; and can be determined by indicators such as complete response (CR), partial response (PR), progressive disease (PD), objective response rate (ORR), duration of response (DoR), progression-free survival (PFS) and overall survival (OS).

[0380] The aforementioned methods can provide confirmation of the advantages of the anti-tumor effects of the pharmaceutical compositions and treatment methods according to the present invention over existing pharmaceutical compositions and treatment methods for cancer treatment.

[0381] The pharmaceutical composition and treatment methods of the present invention can delay cancer cell growth, suppress its proliferation, and can further destroy cancer cells. These effects can allow cancer patients to be free from symptoms caused by cancer or achieve an improvement in the QOL of cancer patients, and obtain therapeutic effects by maintaining the life of cancer patients. Even if the pharmaceutical composition and treatment methods of the present invention do not complete killing of cancer cells, they can also achieve a higher QOL of cancer patients by inhibiting, controlling or preventing the growth of cancer cells, while achieving longer-term survival.

[0382] The pharmaceutical composition of the present invention can exert therapeutic effects by administration to a patient as systemic therapy, and also by local application to cancer tissues.

[0383] The pharmaceutical composition of the present invention containing at least one pharmaceutically suitable ingredient can be administered. In view of the dosage, administration concentration, etc. of the CD47 inhibitor and the antibody specific for the tumor antigen used in the present invention, the pharmaceutically suitable ingredients can be appropriately selected and applied from the formulation additives and the like generally used in the art. For example, the antibody specific for 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 20. The pharmaceutical composition containing the antibody specific for the tumor antigen used in the present invention can be preferably used as an injection, can be more preferably used as an aqueous injection or a lyophilized injection, and can be even more preferably used as a lyophilized injection.

[0384] In the case where the pharmaceutical composition containing an antibody specific for a tumor antigen used in the present invention is an aqueous injection, it may preferably be diluted with a suitable diluent and then administered as an intravenous infusion. For the diluent, dextrose solution, physiological saline, etc. may be exemplified, and dextrose solution may be preferably exemplified, and 5% dextrose solution may be more preferably exemplified.

[0385] In the case where the pharmaceutical composition containing the antibody specific for the tumor antigen used in the present invention is a lyophilized injection, it can be preferably dissolved in water for injection, and then the required amount can be diluted with a suitable diluent and then administered as an intravenous infusion. For the diluent, dextrose solution, physiological saline, etc. can be exemplified, and dextrose solution can be preferably exemplified, and 5% dextrose solution can be more preferably exemplified.

[0386] Examples of administration routes that can be used to administer the pharmaceutical composition of the present invention include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes; and preferably include an intravenous route.

[0387] The antibody-drug conjugate specific for tumor antigens used in the present invention can be administered to a human once at intervals of 1 to 180 days, and can preferably be administered once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks, and can be even more preferably administered once every 3 weeks. In addition, the antibody-drug conjugate specific for tumor antigens used in the present invention can be administered at a dose of about 0.001 to 100 mg / kg, and can preferably be administered at a dose of 0.8 to 12.4 mg / kg. In the case where the antibody-drug conjugate specific for tumor antigens in the present invention is an anti-HER2 antibody-drug conjugate, it can preferably be administered once every 3 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. In the case where the antibody-drug conjugate specific for tumor antigens used in the present invention is an anti-HER3 antibody-drug conjugate, it can preferably be administered once every 3 weeks at a dosage 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. In the case where the antibody-drug conjugate specific for tumor antigens used in the present invention is an anti-TROP2 antibody-drug conjugate, it can preferably be administered once every 3 weeks at a dosage 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 a human once at intervals of 1 to 180 days, and can preferably be administered once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks. In addition, the CD47 inhibitor according to the present invention may be administered at a dose of about 0.001 to 100 mg / kg / dose.

[0388] The pharmaceutical composition and treatment method of the present invention may further contain a cancer therapeutic agent in addition to the CD47 inhibitor according to the present invention and the antibody specific for the tumor antigen. The pharmaceutical composition and treatment method of the present invention may also be administered in combination with another cancer therapeutic agent to enhance the anti-tumor effect. Another cancer therapeutic agent to be used for such purposes may be administered to the subject simultaneously, separately or sequentially with the pharmaceutical composition of the present invention, or may be administered at different dosage intervals. Such cancer therapeutic agents are not limited as long as they are agents having antitumor activity, and can be exemplified by at least one selected from the group consisting of irinotecan (CPT-11), cisplatin, carboplatin, oxaliplatin, fluorouracil (5-FU), gemcitabine, capecitabine, doxorubicin, epirubicin, cyclophosphamide, mitomycin C, tegafur-gimeracil-oxazinic acid combination, panitumumab, bevacizumab, ramucirumab, regorafenib, trifluridine-tipiracil combination, gefitinib, erlotinib, afatinib, methotrexate, pemetrexed, tamoxifen, toremifene, fulvestrant, leuprolide, goserelin, letrozole, anastrozole, progesterone preparations, and lapatinib.

[0389] In the pharmaceutical composition and treatment method of the present invention, in addition to the CD47 inhibitor according to the present invention and the antibody specific for the tumor antigen, an immune checkpoint inhibitor and an antibody-drug that specifically binds to a cancer antigen and has ADCC and / or ADCP activity may be further contained as a cancer therapeutic agent to be used in combination. As immune checkpoint inhibitors, inhibitors of the binding between PD-1 and its ligand PD-L1 or CTLA4 inhibitors are listed. Specific examples thereof include anti-PD-1 antibodies (nivolumab, pembrolizumab, cemiplizumab, spartalizumab, PDR-001 or BI 754091), anti-PD-L1 antibodies (atezolizumab, avelumab or durbarumab) and anti-CTLA4 antibodies (ipilimumab or tremelimumab). Examples of antibody-drugs that specifically bind to cancer antigens and have ADCC and / or ADCP activity include anti-CD20 antibodies (rituximab), anti-HER2 antibodies (trastuzumab or pertuzumab), anti-EGFR antibodies (cetuximab), and anti-CD52 antibodies (alemtuzumab).

[0390] ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) expressing Fcγ receptors recognize antibodies bound to target cells and then cause the lysis of target cells. In NK cells, which are the main cells responsible for 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 phagocytes (e.g., macrophages, neutrophils) expressing Fc receptors recognize antibodies bound to target cells and then engulf target cells intracellularly. In monocytes, which are the main cells responsible for ADCP, FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA are expressed.

[0391] The pharmaceutical compositions and treatment methods of the present invention can also be used in combination with radiotherapy. For example, a cancer patient receives radiotherapy before and / or after or simultaneously with treatment with the pharmaceutical composition of the present invention.

[0392] The pharmaceutical composition and treatment method of the present invention can also be used as adjuvant chemotherapy in combination with surgery. The pharmaceutical composition of the present invention can be administered before the surgical procedure for the purpose of reducing the size of the tumor (referred to as preoperative adjuvant chemotherapy or neoadjuvant therapy), or can be administered after the surgical procedure for the purpose of preventing any tumor recurrence (referred to as postoperative adjuvant chemotherapy or adjuvant therapy).

[0393] In some embodiments, the CD47 inhibitor and / or the antibody specific for the tumor antigen are formulated together with a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, adjuvant and other agents. The "pharmaceutically acceptable carrier" and other agents can be appropriately selected from a wide range according to the type of target disease and the dosage form of the drug. The method for administering the CD47 inhibitor and / or the antibody specific for the tumor antigen according to the present invention can be appropriately selected, 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 perfusate injection. The injection solution can be prepared by using a carrier consisting of a saline solution, a glucose solution, or a mixture of a saline solution and a glucose solution and any buffer. Alternatively, the injection solution can be prepared by preparing a powder preparation and mixing the powder preparation with the above-mentioned liquid carrier (when used).

[0394] Other methods of administration can be appropriately selected together with the development of the preparation. For example, for oral administration, oral liquids, powders, pills, capsules and tablets can be applied. In the case of oral liquids, oral liquid preparations such as suspensions and syrups can be produced 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 parabens; and flavorings such as strawberry flavoring and mint flavoring. 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 glycerol. Tablets and capsules are easy to apply. In this regard, they are preferred unit dosage forms of the compositions of the present invention. When tablets and capsules are produced, solid production carriers are used. [Example]

[0395] The present invention is specifically described in consideration of the embodiments shown below. However, the present invention is not limited to these embodiments. Further, it should never be interpreted in a limiting manner.

[0396] Production Example 1: Production of Antibody-Drug Conjugate (1)

[0397] According to the production method described in International Publication No. WO 2015 / 115091, an antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (1)") was produced using a humanized anti-HER2 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2, hereinafter referred to as "humanized anti-HER2 antibody (1)"), wherein a drug-linker represented by the following formula is conjugated to the anti-HER2 antibody via a thioether bond:

[0398] [Formula 1]

[0399]

[0400] Wherein A represents the linking position to the antibody. The DAR of the antibody-drug conjugate (1) is 7.7 or 7.8.

[0401] Production Example 2: Production of Antibody-Drug Conjugate (2)

[0402] According to the production methods described in International Publication Nos. WO 2015 / 098099 and 2017 / 002776, an antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (2)") was produced using a humanized anti-TROP2 antibody (an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consists 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)), wherein a drug-linker represented by the following formula is conjugated to the anti-TROP2 antibody via a thioether bond:

[0403] [Formula 1]

[0404]

[0405] Wherein A represents the attachment position to the antibody. Although the DAR of the antibody-drug conjugate (2) can be controlled within the range of 0 to 8, antibody-drug conjugates with an average number of conjugated drugs of 3.5 to 4.5 were generated herein.

[0406] Production Example 3: Production of Antibody-Drug Conjugate (3)

[0407] According to the production methods described in International Publication Nos. WO 2014 / 057867 and 2017 / 002776, an antibody-drug conjugate (hereinafter referred to as “antibody-drug conjugate (3)”) was produced using a humanized anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 8, hereinafter referred to as humanized anti-B7-H3 antibody (3)), wherein a drug-linker represented by the following formula was conjugated to the anti-B7-H3 antibody via a thioether bond:

[0408] [Formula 1]

[0409]

[0410] Wherein A represents the attachment position to the antibody. Although the DAR of the antibody-drug conjugate (3) can be controlled within the range of 0 to 8, antibody-drug conjugates with an average conjugated drug number of 3.5 to 4.5 were generated herein.

[0411] Example 1: ADCP activity of the antibody-drug conjugate of compound (A) and anti-CD47 blocker against HER2-positive and TROP2-positive cancer cell lines

[0412] 1-1 Preparation of target cells

[0413] Human gastric cancer cell line AGS (CD47 and TROP2 positive) and human breast cancer cell line JIMT-1 (CD47 and HER2 positive) were collected, washed twice with PBS, and resuspended in PBS. The number of viable cells was counted according to the trypan blue exclusion test. 1 μL / 1×10 6 CellTrace Violet (CTV, Thermo Fisher Scientific) solution was added to a volume of 1×10 cells / mL. The mixture was allowed to stand in the dark at room temperature for 10 minutes. After incubation, the cells were washed twice with 10 mL of RPMI1640 medium (R10) containing 10% FBS. Finally, the cell suspension was prepared so that 1×10 6 cells / mL and were used as target cells.

[0414] 1-2 Preparation of effector cells

[0415] On day 0, Ficoll-Paque PLUS (GE Healthcare) was dispensed into SepMate-50 tubes (STEMCELL Technologies) at 15 mL / tube. Whole blood diluted 2-fold with PBS containing 2% FBS was covered at 33-34 mL / tube. Centrifugation was performed at 1200 g for 10 minutes at room temperature. The supernatant containing PBMCs was collected into a fresh 50 mL tube by decantation, 10 mL of PBS containing 2% FBS was added, and centrifuged at 300 g for 5-8 minutes at room temperature. After removing the supernatant, the PBMCs were washed twice with 10 mL of PBS containing 2% FBS. The number of live PBMCs was counted, and the PBMCs were plated at 5×10 7 The cells / mL were suspended in RoboSep buffer (STEMCELL Technologies). The EasySep human monocyte enrichment mixture in the Human monocyte Enrichment Kit without CD16 depletion (STEMCELL Technologies) was added at 50 μL / 5 x 10 7 After reacting at 4°C for 10 minutes, EasySep magnetic particles (STEMCELL Technologies) were added at 50 μL / 5 x 10 7The amount of PBMC was added to the PBMC suspension. After reacting at 4°C for 5 minutes, RoboSep buffer was added to the PBMC until 2.5 mL. The PBMC was transferred to a 5 mL tube and placed in the magnetic field of EasySep Magnet (STEMCELL Technologies). After 2 minutes and 30 seconds, the supernatant containing human peripheral blood mononuclear cells was collected by decantation and centrifuged. To generate macrophages, the isolated monocytes were suspended in R10 (PEPROTEC) containing 20 ng / mL M-CSF and centrifuged at 225 cm 2 The flasks were inoculated and incubated at 37°C and 5% CO 2 On day 7, the culture supernatant was removed and R10 containing 20 ng / mL M-CSF was added. On day 11, the culture supernatant was removed and R10 (PEPROTEC) containing 20 ng / mL M-CSF and 20 ng / mL IL-10 was added. On day 13, the macrophages were washed once with PBS, TrypLE Express (Thermo Fisher Scientific) was added, and kept at 37°C for 10-30 minutes. After harvesting, the macrophages were cultured at 1×10 6 Cells / mL were suspended in PBS. CellTrace Far Red solution (Thermo Fisher Scientific) was added at 1 μL / 10 6 After incubation at room temperature in the dark for 10 min, the macrophages were washed twice with 10 mL of R10. 6 cells / mL were suspended in R10 and used as effector cells.

[0416] 1-3 Evaluation of ADCP activity

[0417] First, add 100 μL of Fig.17 The indicated agents shown in were added to the cells. Briefly, antibody-drug conjugate (1) or (2) or control antibody-drug conjugate diluted with R10 was added at a final concentration of 5 nM. ALX148_hIgG1 or isotype-matched control Ab diluted with R10 was added at a final concentration of 50 nM. ALX148_hIgG1 was prepared with reference to US publication US2019 / 0169266 A1. The amino acid sequence of ALX148_hIgG1 is set forth in SEQ ID NO: 15 in the sequence table. Next, 50 μL of the above step was added. Prepared effector cells (1×10 6 Finally, 50 μL of the same procedure as above was added. Prepared target cells (1×10 6 Cells / mL) were added to the wells and the cells were mixed by gentle pipetting. The cells were incubated at 37°C and 5% CO 2 Incubate for 4 hours at 510g. After centrifugation for 3 minutes at 510g, remove the supernatant. The cells were washed once with 200 μL of Hank's 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 by BD LSRFortessa X-20 flow cytometer (BD Biosciences). The acquired data were analyzed by Flowjo (TreeStar). Cancer cells were identified as CTV positive (CTV+) cells. Within the cancer cell population, cancer cells engulfed by macrophages are defined as CTV+ and Far Red positive (Far Red+), hereinafter referred to as CTV+Far Red+ cells. ADCP (%) was calculated as follows:

[0418] ADCP (%) = 100 × [(CTV + Far Red + cell count) ÷ (CTV + cell count)]

[0419] Data were analyzed in GraphPad Prism 9.1.0 (GraphPad Software), and statistical analyses were performed in SAS System Release 9.2 (SAS Institute).

[0420] like Fig.17 As shown in , the antibody-drug conjugate (1) or (2) showed ADCP activity against JIMT-1 and AGS cells, respectively, and showed higher ADCP activity in the presence of ALX148_hIgG1 ( Fig.17 A and 17B).

[0421] Example 2: ADCP activity of the antibody-drug conjugate of compound (A) and anti-CD47 blocker against B7-H3 positive cancer cell lines

[0422] 2-1 Preparation of target cells

[0423] Human lung cancer cell line NCI-H322 (CD47 and B7-H3 positive) was collected, washed twice with PBS, and resuspended in PBS. The number of viable cells was counted according to the trypan blue exclusion test. 1 μL / 1×10 6CellTrace Violet (CTV, Thermo Fisher Scientific) solution was added to a volume of 1×10 cells / mL. The mixture was allowed to stand in the dark at room temperature for 10 minutes. After incubation, the cells were washed twice with 10 mL of RPMI1640 medium (R10) containing 10% FBS. Finally, the cell suspension was prepared so that 1×10 6 cells / mL and were used as target cells.

[0424] 2-2 Preparation of effector cells

[0425] On day 0, Ficoll-Paque PLUS (GE healthcare) was dispensed into SepMate-50 tubes (STEMCELL Technologies) at 15 mL / tube. Whole blood diluted 2-fold with PBS containing 2% FBS was overlaid at 33-34 mL / tube. Centrifugation was performed at 1200 g for 10 minutes at room temperature. The supernatant containing PBMCs was collected into a fresh 50 mL tube by decantation, 10 mL of PBS containing 2% FBS was added, and centrifuged at 300 g for 5 minutes at room temperature. After removing the supernatant, PBMCs were washed twice with 10 mL of PBS containing 2% FBS. The number of live PBMCs was counted, and PBMCs were plated at 5×10 7 Cells / mL were suspended in RoboSep buffer (STEMCELL Technologies). EasySep Human Monocyte Enrichment Mix from the Human Monocyte Enrichment Kit without CD16 Depletion (STEMCELL Technologies) was diluted at 50 μL / 5 x 10 7 After reacting at 4°C for 10 minutes, EasySep magnetic particles (STEMCELL Technologies) were added at 50 μL / 5×10 7 The amount of PBMC was added to the PBMC suspension. After reacting at 4°C for 5 minutes, RoboSep buffer was added to the PBMC until 2.5 mL. The PBMC was transferred to a 5 mL tube and placed in the magnetic field of EasySep Magnet (STEMCELL Technologies). After 2 minutes and 30 seconds, the supernatant containing human peripheral blood mononuclear cells was collected by decantation and centrifuged. To generate macrophages, the isolated monocytes were suspended in R10 (PEPROTEC) containing 20 ng / mL M-CSF and centrifuged at 225 cm 2 The flask was inoculated and incubated at 37°C and 5% CO 2On day 7, the culture supernatant was removed, and R10 containing 20 ng / mL M-CSF was added. On day 11, the culture supernatant was removed, and R10 (PEPROTEC) containing 20 ng / mL M-CSF and 20 ng / mL IL-10 was added. On day 13, the macrophages were washed once with PBS, TrypLE Express (Thermo Fisher Scientific) was added, and kept at 37°C for 10 minutes. After harvesting, the macrophages were cultured at 1×10 6 Cells / mL were suspended in PBS. CellTrace Far Red solution (Thermo Fisher Scientific) was added at 1 μL / 10 6 After incubation at room temperature in the dark for 10 min, the macrophages were washed twice with 10 mL of R10. 6 cells / mL were suspended in R10 and used as effector cells.

[0426] 2-3 Evaluation of ADCP activity

[0427] First, 100 μL Fig.18 The indicated agents shown in were each added to a U-bottom 96-well microplate. Briefly, antibody-drug conjugate (3) or control antibody-drug conjugate diluted with R10 was added at a final concentration of 5 nM. ALX148_hIgG1 or isotype-matched control Ab diluted with R10 was added at a final concentration of 50 nM. ALX148_hIgG1 was prepared with reference to US Publication US2019 / 0169266 A1. The amino acid sequence of ALX148_hIgG1 is set forth in SEQ ID NO: 15 in the sequence table. Next, 50 μL of the above step Prepared effector cells (1×10 6 Finally, 50 μL of the same procedure as above was added. Prepared target cells (1×10 6 Cells / mL) were added to the wells and the cells were mixed by gentle pipetting. The cells were incubated at 37°C and 5% CO 2The cells were incubated at 510 g for 4 hours. After centrifugation at 510 g for 3 minutes, the supernatant was removed. The cells were washed once with 200 μL of Hank's balanced salt solution containing 0.1% BSA. The cells were suspended in 50 μL of 1×BD stable fixative (BD Biosciences) and kept at 4°C in the dark until flow cytometry analysis. ADCP was evaluated by BDLSFortessa X-20 flow cytometer (BD Biosciences). The acquired data were analyzed by Flowjo (TreeStar). Cancer cells were identified as CTV positive (CTV+) cells. Within the cancer cell population, cancer cells engulfed by macrophages are defined as CTV+ and Far Red positive (Far Red+), hereinafter referred to as CTV+Far Red+ cells. ADCP (%) was calculated as follows:

[0428] ADCP (%) = 100 × [(CTV + Far Red + cell count) ÷ (CTV + cell count)]

[0429] Data were analyzed in GraphPad Prism 9.1.0 (GraphPad Software), and statistical analyses were performed in SAS System Release 9.2 (SAS Institute).

[0430] like Fig.18 As shown in , the antibody-drug conjugate (3) showed ADCP activity against NCI-H322 cells, and showed higher ADCP activity in the presence of ALX148_hIgG1.

[0431] Example 3: Anti-tumor study (1)

[0432] Mice: Female 6-week-old BALB / c mice (BALB / c AnNCrlCrlj) (The Jackson LaboratoryJapan, INC.) were subjected to the experiment.

[0433] Measurement and calculation formula: The long and short diameters of the tumors were measured twice a week with an electronic digital caliper (Mitutoyo Corp), and the tumor volume (mm) was calculated as follows: 3 ):

[0434] Tumor volume (mm 3 )=0.5×long diameter(mm)×[short diameter(mm)] 2

[0435] The antibody-drug conjugate (1) (drug / antibody ratio: 7.8) was diluted with buffer and intravenously administered to the tail vein at a dose of 10 mL / kg. ALX148_mIgG1 was prepared with reference to US Publication US2019 / 0169266A1. The amino acid sequence of ALX148_mIgG1 is shown in SEQ ID NO: 16 in the sequence table. ALX148_mIgG1 was diluted with saline and administered intraperitoneally at a dose of 10 mL / kg.

[0436] Mouse colon cancer cell line CT26.WT (CRL-2638) was purchased from the American Type Culture Collection and transduced with the human HER2 gene using a retroviral vector. The established CT26.WT-hHER2 was used for the experiment. On the day when mice were inoculated with CT26.WT-hHER2 cells, CT26.WT-hHER2 cells were harvested and cultured at 3.0×10 6 Cells / heads were suspended in PBS (-) and transplanted subcutaneously into the right abdominal area of ​​each animal (day 0). After 6 days, mice were randomly divided into groups based on tumor volume (day 6). Antibody-drug conjugate (1) was intravenously administered into the tail vein at a dose of 10 mg / kg on days 6 and 13 (2 times in total). ALX148_mIgG1 was intraperitoneally administered at a dose of 30 mg / kg on days 7, 10, and 14 (3 times in total). A combination administration group of antibody-drug conjugate (1) and recombinant ALX148_mIgG1 was set up; and a vehicle administration group was set up as a control group. The number of mice in each group was 8. Tumor volume was measured until day 27. Comparisons between each of the single administration group and the combination administration group were performed according to Dunnett's multiple comparisons. A P value of less than 0.05 was defined as statistically significant.

[0437] Results are shown in Fig.19 middle. Fig.19 A shows the treatment schedule for the experiment. Fig.19 B summarizes the number of animals with complete remission at day 27. The combination of ADC(1) and ALX148_mIgG1 induced some animals with complete remission (2 out of 8 mice), whereas monotherapy with ADC(1) or ALX148_mIgG1 did not (0 out of 8 mice).

[0438] Example 4: Anti-tumor study (2)

[0439] Mice: Female 6-week-old B6J mice (C57BL / 6J) (The Jackson Laboratory Japan, INC.) were subjected to the experiment.

[0440] Measurement and calculation formula: The long and short diameters of the tumors were measured twice a week with an electronic digital caliper (Mitutoyo Corp), and the tumor volume [mm 3 ]:

[0441] Tumor volume [mm 3 ]=0.5×long diameter [mm]×[short diameter [mm]] 2 .

[0442] Antibody-drug conjugate (2) (drug / antibody ratio: 3.9) was diluted with ABS and intravenously administered into the tail vein at a dose of 10 mL / kg. Recombinant ALX148_mIgG1 was diluted with PBS and intraperitoneally administered at a dose of 10 mL / kg.

[0443] The mouse colon cancer MC38 cell line was obtained from the National Cancer Institute and transduced 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 the right axilla of each B6J mouse (day 0). Four days later, the mice were randomly divided into groups based on tumor volume (day 4). Antibody-drug conjugate (2) was intravenously administered into the tail vein at a dose of 1 mg / kg on days 4 and 11 (2 times in total). ALX148_mIgG1 was intraperitoneally administered at a dose of 30 mg / kg on days 5, 8, and 12 (3 times in total). A combination administration group of antibody-drug conjugate (2) and ALX148_mIgG1 and a vehicle administration group were set up. The number of mice in each group was 12. Tumor volume was measured until day 18. Comparisons between the single administration group and the combination administration group were performed according to Dunnett's multiple comparisons. A p value of less than 0.05 was defined as statistically significant.

[0444] Results are shown in Fig. 20 middle. Fig. 20 A shows the treatment schedule for the experiment. Fig. 20 B 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) and ALX148_mIgG1 showed superior antitumor effects over ADC (2) and ALX148_mIgG1 monotherapy, respectively ( Fig. 20 B).

[0445] According to the above examples, it was found that the ADC according to the present invention showed an excellent anti-tumor effect if administered in combination with ALX148_mIgG1.

[0446] Since the mechanism of action of ALX148 is based on the blocking activity of the CD47-SIRPα interaction, it is reasonable to expect that other CD47-targeting agents with blocking activity will have the same combinatorial effect.

Claims

1. A pharmaceutical composition comprising: i. CD47 inhibitors; and ii. Antibodies specific for tumor antigens. 2 . The pharmaceutical composition according to claim 1 , wherein the CD47 inhibitor comprises SIRPα or a SIRPα derivative.

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

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

5. The pharmaceutical composition of claim 4, wherein the CD47 inhibitor comprises a polypeptide having a sequence 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 the preceding claims, wherein the CD47 inhibitor is Evorpacept.

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 of claim 1, wherein the CD47 inhibitor comprises an antibody or an antigen-binding fragment thereof, wherein 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: mololimab, leszolimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (lefalimumab) and IMM-0306.

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

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

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

13.

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

14. The pharmaceutical composition of claim 13, wherein the drug-linker is represented by the following formula: [Formula 1] Where A represents the connection position with the antibody, And wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

15. The pharmaceutical composition according to any one of the preceding claims, 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 and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consisting of an amino acid sequence consisting 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 and a light chain, the heavy chain consists of the amino acid sequence represented by SEQ ID NO: 1, and the light chain consists of the amino acid sequence represented by SEQ ID NO:

2.

19. A pharmaceutical composition according to any one of claims 16 to 18 when dependent on claim 14, wherein the average number of drug-linker units conjugated 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 and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:3, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

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

23. A pharmaceutical composition according to any one of claims 20 to 22 when dependent on claim 14, wherein the average number of drug-linker units conjugated 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 a 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 and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:

6.

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

27. A pharmaceutical composition according to any one of claims 24 to 26 when dependent on claim 14, wherein the average number of drug-linker units conjugated 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 a 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 and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

8.

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

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

5.

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

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

10.

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

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

36. The pharmaceutical composition of claim 15, wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody.

37. The pharmaceutical composition according to claim 36, wherein the anti-CDH6 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO:

12.

38. The pharmaceutical composition of claim 37, wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

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

40. A CD47 inhibitor for use in treating or preventing cancer by simultaneous or sequential administration with an antibody specific for a tumor antigen.

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

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

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

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

45. A CD47 inhibitor for use according to any one of claims 40 to 44, wherein the CD47 inhibitor is Evorpacept.

46. ​​The CD47 inhibitor for use according to claim 41, wherein the CD47 inhibitor is 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, wherein 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 mololimab, leszolimab, AO-176, SRF-231, IBI-188, IBI-322, IMC-002, MIL-95, TG-1801, ZL-1201, AK-117 (lefalimumab) and IMM-0306.

49. The CD47 inhibitor for use according to claim 40, wherein the CD47 inhibitor comprises a small molecule agent capable of binding to CD47.

50. The CD47 inhibitor for use according to claim 49, wherein the small molecule agent is selected from 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. A 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, and wherein the antibody-drug conjugate comprises the antibody specific for a tumor antigen linked to a drug via a linker, the linker and the drug forming a drug-linker.

53. The CD47 inhibitor for use according to claim 52, wherein the drug-linker is represented by the following formula: [Formula 1] Where A represents the connection position with the antibody, And 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 is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO:1, and the light chain consisting of an 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 and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:

2.

58. A CD47 inhibitor for use according to any one of claims 55 to 57 when dependent on claim 53, wherein the average number of drug-linker units conjugated 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 a 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 and a light chain, the heavy chain consisting of the amino acid sequence represented by SEQ ID NO:3, and the light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

61. A 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. A CD47 inhibitor for use according to any one of claims 59 to 61 when dependent on claim 53, wherein the average number of drug-linker units conjugated 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 a 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 and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 470 of SEQ ID NO:5, and the light chain consisting of an 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. A CD47 inhibitor for use according to any one of claims 63 to 65 when dependent on claim 53, wherein the average number of drug-linker units conjugated 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 a tumor antigen is an anti-B7-H3 antibody.

68. The CD47 inhibitor for use according to claim 67, wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:7, and the light chain consisting of an 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. A CD47 inhibitor for use according to any one of claims 67 to 69 when dependent on claim 53, wherein the average number of drug-linker units conjugated 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 is an antibody comprising a heavy chain and a light chain, wherein the heavy chain consists of an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO:9, and the light chain consists of an 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. A CD47 inhibitor for use according to any one of claims 71 to 73 when dependent on claim 53, wherein the average number of drug-linker units conjugated 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 is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the light chain consisting of an 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 when dependent on claim 53, wherein the average number of drug-linker units conjugated per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

79. The CD47 inhibitor for use according to any one of claims 40 to 78, wherein the CD47 inhibitor and the antibody specific for a tumor antigen are active ingredients contained in separate preparations.

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

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

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

83. The method of treatment of claim 82, wherein the SIRPα derivative comprises a polypeptide having a sequence 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 method of treatment of claim 81 or claim 82, wherein the CD47 inhibitor is a fusion protein and further comprises an Fc region.

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

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

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

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

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

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

91. The method of treatment of claim 90, wherein the small molecule agent is selected from RRx-001 and IMM-01.

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

13.

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

94. The method of treatment of claim 93, wherein the drug-linker is represented by the formula: [Formula 1] Where A represents the connection position with the antibody, And wherein the drug-linker is conjugated to an antibody specific for a tumor antigen via a thioether bond.

95. The method of any one of claims 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 method of treatment of claim 95, wherein the antibody specific for a tumor antigen is an anti-HER2 antibody.

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

2.

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

2.

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

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

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

4.

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

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

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

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

6.

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

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

5.

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

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

8.

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

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

5.

112. The method of treatment of claim 95, wherein the antibody specific for a tumor antigen is an anti-GPR20 antibody.

113. The method of 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 method of treatment of claim 113, wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

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

116. The method of treatment of claim 95, wherein the antibody specific for a tumor antigen is an anti-CDH6 antibody.

117. The method of claim 116, wherein the anti-CDH6 antibody is an antibody comprising a heavy chain and a light chain, the heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 471 of SEQ ID NO:11, and the 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 of claim 117, wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

119. A method of treatment according to any one of claims 116 to 118 when dependent on claim 94, wherein the average number of drug-linker units conjugated 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 claims 81 to 119, wherein the CD47 inhibitor and the antibody specific for the tumor antigen are active ingredients contained in different preparations.

121. The method of treatment of any one of claims 81 to 120, wherein the method is used to treat at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, esophagogastric junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine carcinosarcoma, head and neck cancer, hepatocellular carcinoma, cervical cancer, brain tumors, gliomas, eye tumors, thyroid cancer, thymic cancer, gallbladder cancer, lymphoma, leukemia, and myelodysplastic syndrome.

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