Combination of antibody-drug conjugate with anti-SIRPα antibody

Abstract

Provided are a pharmaceutical composition and a treatment method that are excellent in terms of antitumor effect and safety. A pharmaceutical composition and a treatment method characterized in that an antibody-drug conjugate, in which a drug linker shown by the formula (in the formula, A represents the binding site with the antibody) and an antibody are bonded by a thioether bond, is administered in combination with an SIRPα-CD47 interaction inhibitor such as an anti-SIRPα antibody, etc. Also, a pharmaceutical composition and a treatment method characterized in that the antibody-drug conjugate and an SIRPα-CD47 interaction inhibitor such as an anti-SIRPα antibody, etc., are administered in combination and used to treat a disease which is improved by the action of activating antitumor immunity.

Description

Combination of antibody-drug conjugate and anti-SIRPα antibody

[0001] The present invention relates to a pharmaceutical composition characterized by administering a combination of a specific antibody-drug conjugate and an anti-SIRPα antibody, and / or a treatment method characterized by administering a combination of a specific antibody-drug conjugate and an anti-SIRPα antibody to an individual.

[0002] SIRPα (SHPS-1) is a single-pass transmembrane molecule of the Ig superfamily present in macrophages, dendritic cells, myeloid cells such as neutrophils, and glial cells (Non-Patent Document 1). The extracellular domain consists of one IgV domain and two IgC domains, and 10 variants of the IgV domain, which is the binding site for CD47, have been reported in humans (Non-Patent Document 2). On the other hand, the intracellular domain contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), and binding to CD47 induces binding to the tyrosine phosphatases SHP-1 and SHP-2, thereby transmitting an inhibitory signal.

[0003] A physiological phenomenon resulting from SIRPα-CD47 interaction has been shown to involve binding of CD47 on red blood cells to SIRPα on macrophages, transmitting a "Don't eat me" signal, thereby preventing unnecessary phagocytosis of red blood cells (Non-Patent Document 3). Meanwhile, it has been suggested that even in the tumor microenvironment, binding of CD47, which is highly expressed on tumor cells, to SIRPα on macrophages and dendritic cells suppresses the phagocytic ability of tumor cells. Suppression of phagocytic ability is expected to subsequently suppress tumor antigen presentation to T cells, and further suppress the tumor immune response. Therefore, the immune phenomenon of tumor cell phagocytosis is thought to be a checkpoint for tumor antigen uptake (entry).

[0004] SIRPα-CD47 is the only phagocytosis-suppressing molecule currently proven, and an inhibitory antibody against this molecule is expected to have potential as a new checkpoint inhibitor against targets other than T cells, and may also be widely effective in patients who are resistant to conventional immune checkpoint inhibitors.

[0005] In recent years, various companies have been reporting patents related to anti-SIRPα antibodies one after another (Patent Documents 1, 2, and 3). The examples of each document show differences in binding to various variants and family molecules (SIRPβ1, SIRPγ, etc.), differences in antibody IgG subclasses, etc. For example, OSE-172 is an IgG4Pro-type antibody that exhibits binding to the V1 type of SIRPα and SIRPβ1, but not to the V2 type of SIRPα and SIRPγ. KWAR23 is an IgG1N279A-type antibody that exhibits binding to all 10 types of SIRPα variants and the family molecules SIRPβ1 and SIRPγ. ADU-1805 is an IgG2-type antibody that exhibits binding to all 10 types of SIRPα variants and SIRPγ. , etc. It is not clear which antibody is most suitable as a pharmaceutical, and efforts to obtain superior antibodies are ongoing. One example of such efforts is the antibody described in Patent Document 4.

[0006] Antibody-drug conjugates (ADCs), which combine a cytotoxic drug with an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into the cells, can selectively deliver the drug to cancer cells, thereby accumulating the drug within the cancer cells and killing the cancer cells. One known antibody-drug conjugate is one that comprises an antibody and a derivative of exatecan, a topoisomerase I inhibitor, as components (Patent Documents 5 to 11, Non-Patent Documents 4 to 7).

[0007] Furthermore, Patent Documents 5 to 11 describe that the above antibody-drug conjugates can be administered together with various cancer therapeutic agents.

[0008] However, no test results have been published showing an excellent combined effect when the above-mentioned antibody-drug conjugate is used in combination with an anti-SIRPα antibody, nor any scientific evidence suggesting such test results.

[0009] International Publication No. WO 2017 / 178653, International Publication No. WO 2018 / 026600, International Publication No. WO 2018 / 190719, International Publication No. WO 2020 / 013170, International Publication No. WO 2014 / 057687, International Publication No. WO 2014 / 061277, International Publication No. WO 2015 / 098099, International Publication No. WO 2015 / 115091, International Publication No. WO 2015 / 146132, International Publication No. WO 2015 / 155976, International Publication No. WO 2015 / 155998

[0010] Matozaki et al. Trends in cell biol. 2009(19) 2, 72-80Takenaka et al. Nat Immunol. 2007(8)12, 1313-1323Matozaki et al. J. Biochem. 2014(155) 6, 335-344Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108.Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046.Doi T, et al., Lancet Oncol 2017; 18: 1512-22.Takegawa N, et al., Int. J. Cancer: 141, 1682-1689 (2017)

[0011] The antibody-drug conjugate used in the present invention (an antibody-drug conjugate containing an exatecan derivative as a component) has been confirmed to exhibit excellent antitumor effects even when used alone. However, it is desirable to develop a treatment method that can demonstrate even more excellent antitumor effects by using it in combination with other anticancer drugs that have different mechanisms of action to comprehensively suppress the proliferation of cancer cells.

[0012] An objective of the present invention is to provide a pharmaceutical composition characterized by administering a combination of a specific antibody-drug conjugate and an anti-SIRPα antibody, and / or a treatment method characterized by administering a combination of a specific antibody-drug conjugate and an anti-SIRPα antibody to an individual.

[0013] The present inventors have conducted extensive research to solve the above problems and have found that administering a specific antibody-drug conjugate in combination with an anti-SIRPα antibody provides an excellent combined effect, thereby completing the present invention.

[0014] That is, the present invention provides the following [1] to

[232] .

[0015] [1] A pharmaceutical composition comprising an antibody-drug conjugate and an anti-SIRPα antibody administered in combination, wherein the antibody-drug conjugate is an antibody represented by the formula

[0016]

[0017] (wherein A represents the binding site to the antibody) and the antibody are linked via a thioether bond. [2] The pharmaceutical composition according to [1], wherein the antibody in the antibody-drug conjugate 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. [3] The pharmaceutical composition according to [2], wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody. [4] The pharmaceutical composition according to [3], wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2. [5] The pharmaceutical composition according to [3], wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2. [6] The pharmaceutical composition according to any one of [3] to [5], wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8. [7] The pharmaceutical composition according to [2], wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody. [8] The pharmaceutical composition according to [7], wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4. [9] The pharmaceutical composition according to [8], wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[10] The pharmaceutical composition according to any one of [7] to [9], wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0018]

[11] The pharmaceutical composition according to [2], wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[12] The pharmaceutical composition according to

[11] , wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[13] The pharmaceutical composition according to

[12] , wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[14] The pharmaceutical composition according to any one of

[11] to

[13] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[15] The pharmaceutical composition according to [2], wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[16] The pharmaceutical composition of

[15] , wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[17] The pharmaceutical composition of

[16] , wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[18] The pharmaceutical composition of any one of

[15] to

[17] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[19] The pharmaceutical composition of [2], wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[20] The pharmaceutical composition of

[19] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 10.

[0019]

[21] The pharmaceutical composition according to

[20] , wherein the anti-GPR20 antibody has a deletion of a lysine residue at the carboxyl terminus of its heavy chain.

[22] The pharmaceutical composition according to any one of

[19] to

[21] , wherein the average number of drug linkers attached per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[23] The pharmaceutical composition according to [2], wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[24] The pharmaceutical composition according to

[23] , wherein the anti-CDH6 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 12.

[25] The pharmaceutical composition according to

[24] , wherein the anti-CDH6 antibody has a deletion of a lysine residue at the carboxyl terminus of its heavy chain.

[26] The pharmaceutical composition according to any one of

[23] to

[25] , wherein the average number of drug linkers attached per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[27] The pharmaceutical composition according to any one of [1] to

[26] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[28] The pharmaceutical composition according to any one of [1] to

[27] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[29] The pharmaceutical composition according to any one of [1] to

[28] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal 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.

[30] A pharmaceutical composition wherein an antibody-drug conjugate and an anti-SIRPα antibody are administered in combination, wherein the antibody-drug conjugate is represented by the formula:

[0020]

[0021] (wherein the drug linker is bound to the antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody)

[0022]

[31] The pharmaceutical composition according to

[30] , wherein the antibody in the antibody-drug conjugate 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.

[32] The pharmaceutical composition according to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[33] The pharmaceutical composition according to

[32] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[34] The pharmaceutical composition according to

[32] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[35] The pharmaceutical composition according to any one of

[32] to

[34] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[36] The pharmaceutical composition according to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[37] The pharmaceutical composition according to

[36] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[38] The pharmaceutical composition according to

[37] , wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[39] The pharmaceutical composition according to any one of

[36] to

[38] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[40] The pharmaceutical composition according to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[0023]

[41] The pharmaceutical composition according to

[40] , wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[42] The pharmaceutical composition according to

[41] , wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[43] The pharmaceutical composition according to any one of

[40] to

[42] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[44] The pharmaceutical composition according to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[45] The pharmaceutical composition according to

[44] , wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[46] The pharmaceutical composition according to

[45] , wherein the anti-B7-H3 antibody has a deleted lysine residue at the carboxyl terminus of its heavy chain.

[47] The pharmaceutical composition according to any one of

[44] to

[46] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[48] The pharmaceutical composition according to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[49] The pharmaceutical composition according to

[48] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10.

[50] The pharmaceutical composition according to

[49] , wherein the anti-GPR20 antibody has a deleted lysine residue at the carboxyl terminus of its heavy chain.

[0024]

[51] The pharmaceutical composition according to any one of

[48] to

[50] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[52] The pharmaceutical composition according to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[53] The pharmaceutical composition according to

[52] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 12.

[54] The pharmaceutical composition according to

[53] , wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[55] The pharmaceutical composition according to any one of

[52] to

[54] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[56] The pharmaceutical composition according to any one of

[30] to

[55] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[57] The pharmaceutical composition according to any one of

[30] to

[56] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[58] The pharmaceutical composition according to any one of

[30] to

[57] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal 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.

[59] A method of treatment comprising administering to an individual in need of treatment a combination of an antibody-drug conjugate and an anti-SIRPα antibody, wherein the antibody-drug conjugate is represented by the formula:

[0025]

[0026] (wherein A represents the binding site to the antibody) and the antibody are linked via a thioether bond.

[60] The treatment method according to

[59] , wherein the antibody in the antibody-drug conjugate 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.

[0027]

[61] The therapeutic method according to

[60] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[62] The therapeutic method according to

[61] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2.

[63] The therapeutic method according to

[61] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[64] The therapeutic method according to any one of

[61] to

[63] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[65] The therapeutic method according to

[60] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[66] The therapeutic method according to

[65] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[67] The method of treatment according to

[66] , wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of its heavy chain.

[68] The method of treatment according to any one of

[65] to

[67] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[69] The method of treatment according to

[60] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[70] The method of treatment according to

[69] , wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO:5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:6.

[0028]

[71] The method of treatment according to

[70] , wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of its heavy chain.

[72] The method of treatment according to any one of

[69] to

[71] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[73] The method of treatment according to

[60] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[74] The method of treatment according to

[73] , wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[75] The method of treatment according to

[74] , wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of its heavy chain.

[76] The method for treatment according to any one of

[73] to

[75] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[77] The method for treatment according to

[60] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[78] The method for treatment according to

[77] , wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10.

[79] The method for treatment according to

[78] , wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[80] The method for treatment according to any one of

[77] to

[79] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0029]

[81] The method of treatment according to

[60] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[82] The method of treatment according to

[81] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 12.

[83] The method of treatment according to

[82] , wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[84] The method of treatment according to any one of

[81] to

[83] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[85] The method of treatment according to any one of

[59] to

[84] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[86] The method of treatment according to any one of (59) to (85), wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[87] The method of treatment according to any one of

[59] to

[86] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal 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.

[88] A method of treatment, characterized in that an antibody-drug conjugate and an anti-SIRPα antibody are administered in combination to an individual in need of treatment, wherein the antibody-drug conjugate is represented by the formula:

[0030]

[0031] (wherein the drug linker is bonded to the antibody via a thioether bond, and n represents the average number of drug linkers bonded per antibody).

[89] The therapeutic method according to

[88] , wherein the antibody in the antibody-drug conjugate 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.

[90] The therapeutic method according to

[89] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0032]

[91] The therapeutic method according to

[90] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2.

[92] The therapeutic method according to

[90] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[93] The therapeutic method according to any one of

[90] to

[92] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[94] The therapeutic method according to

[89] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[95] The therapeutic method according to

[94] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[96] The method of treatment according to

[95] , wherein the anti-HER3 antibody has a deletion of a lysine residue at the carboxyl terminus of its heavy chain.

[97] The method of treatment according to any one of

[94] to

[96] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[98] The method of treatment according to

[89] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[99] The method of treatment according to

[98] , wherein the anti-TROP2 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[100] The method of treatment according to

[99] , wherein the anti-TROP2 antibody has a deletion of a lysine residue at the carboxyl terminus of its heavy chain.

[0033]

[101] The method of treatment according to any one of

[98] to

[100] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[102] The method of treatment according to

[89] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[103] The method of treatment according to

[102] , wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8.

[104] The method of treatment according to

[103] , wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[105] The method of treatment according to any one of

[102] to

[104] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[106] The method of treatment according to

[89] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[107] The method of treatment according to

[106] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10.

[108] The method of treatment according to

[107] , wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[109] The method of treatment according to any one of

[106] to

[108] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[110] The method of treatment according to

[89] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[0034]

[111] The therapeutic method according to

[110] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 12.

[112] The therapeutic method according to

[111] , wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[113] The therapeutic method according to any one of

[110] to

[112] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[114] The method of treatment according to any one of

[88] to

[113] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[115] The method of treatment according to any one of

[88] to

[114] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[116] The method of treatment according to any one of

[88] to

[115] , 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, gastroesophageal 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.

[117] A formula for treating a disease by administering in combination with an anti-SIRPα antibody.

[0035]

[0036] (wherein A represents the binding position to the antibody) and the antibody are bound via a thioether bond.

[118] The antibody-drug conjugate according to

[117] , wherein the antibody in the antibody-drug conjugate 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.

[119] The antibody-drug conjugate according to

[118] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[120] The antibody-drug conjugate according to

[119] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2.

[0037]

[121] The antibody-drug conjugate according to

[119] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[122] The antibody-drug conjugate according to any one of

[119] to

[121] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[123] The antibody-drug conjugate according to

[118] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[124] The antibody-drug conjugate according to

[123] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[125] The antibody-drug conjugate according to

[124] , wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[126] The antibody-drug conjugate according to any one of

[123] to

[125] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[127] The antibody-drug conjugate according to

[118] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[128] The antibody-drug conjugate according to

[127] , wherein the anti-TROP2 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[129] The antibody-drug conjugate according to

[128] , wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-TROP2 antibody is deleted.

[130] The antibody-drug conjugate according to any one of

[127] to

[129] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[0038]

[131] The antibody-drug conjugate according to

[130] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[132] The antibody-drug conjugate according to

[131] , wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[133] The antibody-drug conjugate according to

[132] , wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[134] The antibody-drug conjugate according to any one of

[131] to

[133] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[135] The antibody-drug conjugate according to

[118] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[136] The antibody-drug conjugate according to

[135] , wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 10.

[137] The antibody-drug conjugate according to

[136] , wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[138] The antibody-drug conjugate according to any one of

[135] to

[137] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[139] The antibody-drug conjugate according to

[118] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[140] The antibody-drug conjugate according to

[139] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 12.

[0039]

[141] The antibody-drug conjugate according to

[140] , wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[142] The antibody-drug conjugate according to any one of

[139] to

[141] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[143] The antibody-drug conjugate according to any one of

[117] to

[142] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[144] The antibody-drug conjugate according to any one of

[117] to

[143] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[145] The antibody-drug conjugate according to any one of

[117] to

[144] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal 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.

[146] A formula for treating a disease when administered in combination with an anti-SIRPα antibody.

[0040]

[0041] (wherein the drug linker is bonded to the antibody via a thioether bond, and n represents the average number of drug linkers bonded per antibody).

[147] The antibody-drug conjugate according to

[146] , wherein the antibody in the antibody-drug conjugate 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.

[148] The antibody-drug conjugate according to

[147] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[149] The antibody-drug conjugate according to

[148] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2.

[150] The antibody-drug conjugate according to

[148] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0042]

[151] The antibody-drug conjugate according to any one of

[148] to

[150] , wherein the average number of drug linkers attached per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[152] The antibody-drug conjugate according to

[147] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[153] The antibody-drug conjugate according to

[152] , wherein the anti-HER3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[154] The antibody-drug conjugate according to

[153] , wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[155] The antibody-drug conjugate according to any one of

[152] to

[154] , wherein the average number of drug linkers attached per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[156] The antibody-drug conjugate according to

[147] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[157] The antibody-drug conjugate according to

[156] , wherein the anti-TROP2 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[158] The antibody-drug conjugate according to

[157] , wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[159] The antibody-drug conjugate according to any one of

[156] to

[158] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[160] The antibody-drug conjugate according to

[147] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[0043]

[161] The antibody-drug conjugate according to

[160] , wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[162] The antibody-drug conjugate according to

[161] , wherein the anti-B7-H3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[163] The antibody-drug conjugate according to any one of

[160] to

[162] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[164] The antibody-drug conjugate according to

[147] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[165] The antibody-drug conjugate according to

[164] , wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 of SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 10.

[166] The antibody-drug conjugate according to

[165] , wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[167] The antibody-drug conjugate according to any one of

[164] to

[166] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[168] The antibody-drug conjugate according to

[147] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[169] The antibody-drug conjugate according to

[168] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 12.

[170] The antibody-drug conjugate according to

[169] , wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-CDH6 antibody is deleted.

[0044]

[171] The antibody-drug conjugate according to any one of

[168] to

[170] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[172] The antibody-drug conjugate according to any one of

[146] to

[171] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[173] The antibody-drug conjugate according to any one of

[146] to

[172] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[174] The antibody-drug conjugate according to any one of

[146] to

[173] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal 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.

[175] A compound according to the present invention for the preparation of a medicament for treating a disease by administering the compound in combination with an anti-SIRPα antibody.

[0045]

[0046] (wherein A represents the binding site to the antibody) and an antibody are linked via a thioether bond.

[176] The use according to

[175] , wherein the antibody in the antibody-drug conjugate 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.

[177] The use according to

[176] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[178] The use according to

[177] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[179] The use according to

[177] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[180] The use according to any one of

[177] to

[179] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0047]

[181] The use according to

[176] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[182] The use according to

[181] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[183] ​​The use according to

[182] , wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[184] The use according to any one of

[181] to

[183] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[185] The use according to

[176] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[186] The use according to

[185] , wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 6.

[187] The use according to

[186] , wherein the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[188] The use according to any one of

[185] to

[187] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[189] The use according to

[176] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[190] The use according to

[189] , wherein the anti-B7-H3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[0048]

[191] The use according to

[190] , wherein the anti-B7-H3 antibody has a deleted lysine residue at the carboxyl terminus of its heavy chain.

[192] The use according to any one of

[189] to

[191] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[193] The use according to

[176] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[194] The use according to

[193] , wherein the anti-GPR20 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10.

[195] The use according to

[194] , wherein the anti-GPR20 antibody has a deleted lysine residue at the carboxyl terminus of its heavy chain.

[196] The use according to any one of

[193] to

[195] , wherein the average number of drug linkers attached per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[197] The use according to

[176] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[198] The use according to

[197] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 12.

[199] The use according to

[198] , wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[200] The use according to any one of

[197] to

[199] , wherein the average number of drug linkers attached per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0049]

[201] The use according to any one of

[175] to

[200] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[202] The use according to any one of

[175] to

[201] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[203] The use according to any one of

[175] to

[202] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal 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.

[204] A compound according to the present invention for the preparation of a medicament for treating a disease by administering the compound in combination with an anti-SIRPα antibody.

[0050]

[0051] (wherein the drug linker is bonded to the antibody via a thioether bond, and n represents the average number of drug linkers bonded per antibody).

[205] The use according to

[204] , wherein the antibody in the antibody-drug conjugate 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.

[206] The use according to

[205] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[207] The use according to

[206] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2.

[208] The use according to

[206] , wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[209] The use according to any one of

[206] to

[208] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[210] The use according to

[205] , wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

[0052]

[211] The use according to

[210] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[212] The use according to

[211] , wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[213] The use according to any one of

[210] to

[212] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[214] The use according to

[205] , wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

[215] The use according to

[214] , wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6.

[216] The use according to

[215] , wherein the anti-TROP2 antibody has a deletion of a lysine residue at the carboxyl terminus of its heavy chain.

[217] The use according to any one of

[214] to

[216] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[218] The use according to

[205] , wherein the antibody in the antibody-drug conjugate is an anti-B7-H3 antibody.

[219] The use according to

[218] , wherein the anti-B7-H3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 8.

[220] The use according to

[219] , wherein the anti-B7-H3 antibody has a deletion of a lysine residue at the carboxyl terminus of its heavy chain.

[0053]

[221] The use according to any one of

[218] to

[220] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 3.5 to 4.5.

[222] The use according to

[205] , wherein the antibody in the antibody-drug conjugate is an anti-GPR20 antibody.

[223] The use according to

[222] , wherein the anti-GPR20 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10.

[224] The use according to

[223] , wherein the anti-GPR20 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[225] The use according to any one of

[222] to

[224] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[226] The use according to

[205] , wherein the antibody in the antibody-drug conjugate is an anti-CDH6 antibody.

[227] The use according to

[226] , wherein the anti-CDH6 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO: 12.

[228] The use according to

[227] , wherein the anti-CDH6 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[229] The use according to any one of

[226] to

[228] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[230] The use according to any one of

[204] to

[229] , wherein the anti-SIRPα antibody is an antibody according to any one of the following (1) to (5): (1) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 13 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 466 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16; and (5) An antibody of any one of (1) to (4) in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0054]

[231] The use according to any one of

[204] to

[230] , wherein the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in different formulations and administered simultaneously or at different times.

[232] The use according to any one of

[204] to

[231] , for the treatment of at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, and 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.

[0055] The present invention can provide a pharmaceutical composition characterized by administering a specific antibody-drug conjugate and an anti-SIRPα antibody in combination, and / or a treatment method characterized by administering a specific antibody-drug conjugate and an anti-SIRPα antibody in combination to an individual.

[0056]

[0023] Figure 1 shows the amino acid sequence of an anti-HER2 antibody heavy chain (SEQ ID NO: 1). Figure 2 shows the amino acid sequence of an anti-HER2 antibody light chain (SEQ ID NO: 2). Figure 3 shows the amino acid sequence of an anti-HER3 antibody heavy chain (SEQ ID NO: 3). Figure 4 shows the amino acid sequence of an anti-HER3 antibody light chain (SEQ ID NO: 4). Figure 5 shows the amino acid sequence of an anti-TROP2 antibody heavy chain (SEQ ID NO: 5). Figure 6 shows the amino acid sequence of an anti-TROP2 antibody light chain (SEQ ID NO: 6). Figure 7 shows the amino acid sequence of an anti-B7-H3 antibody heavy chain (SEQ ID NO: 7). Figure 8 shows the amino acid sequence of an anti-B7-H3 antibody light chain (SEQ ID NO: 8). Figure 9 shows the amino acid sequence of an anti-GPR20 antibody heavy chain (SEQ ID NO: 9). Figure 10 shows the amino acid sequence of an anti-GPR20 antibody light chain (SEQ ID NO: 10). Figure 11 shows the amino acid sequence of an anti-CDH6 antibody heavy chain (SEQ ID NO: 11). Figure 12 shows the amino acid sequence of an anti-CDH6 antibody light chain (SEQ ID NO: 12).

[0023] Figure 1 shows the amino acid sequence (SEQ ID NO: 13) of the hH1 heavy chain of humanized anti-SIRPα antibody D13. Figure 2 shows the amino acid sequence (SEQ ID NO: 14) of the hH2 heavy chain of humanized anti-SIRPα antibody D13. Figure 3 shows the amino acid sequence (SEQ ID NO: 15) of the hL2 light chain of humanized anti-SIRPα antibody D13. Figure 4 shows the amino acid sequence (SEQ ID NO: 16) of the hL3 light chain of humanized anti-SIRPα antibody D13. Figure 5 shows the amino acid sequence (SEQ ID NO: 17) of the hL4 light chain of humanized anti-SIRPα antibody D13. Figure 6 shows the CDR sequences (SEQ ID NOs: 18 to 23) of anti-SIRPα antibody cD13. Figure 7 shows the amino acid sequence (SEQ ID NO: 24) of the OSE-172 antibody heavy chain (OSE-172_hG4Pro) and the amino acid sequence (SEQ ID NO: 25) of the light chain (OSE-172_hK).

[0023] Figure 1 shows the amino acid sequence (SEQ ID NO: 26) of the KWAR23 antibody heavy chain (KWAR23_hG4Pro) and the amino acid sequence (SEQ ID NO: 27) of the light chain (KWAR23_hK).

[0024] Figure 1 shows the amino acid sequence (SEQ ID NO: 28) of the ADU-1805 antibody heavy chain (ADU-1805_hG2) and the amino acid sequence (SEQ ID NO: 29) of the light chain (ADU-1805_hK).

[0025] Figure 1 shows the amino acid sequence (SEQ ID NO: 30) of the 5C12 anti-mouse SIRPα antibody heavy chain and the amino acid sequence (SEQ ID NO: 31) of the light chain.27 shows the amino acid sequence of the heavy chain (SEQ ID NO: 32) and light chain (SEQ ID NO: 33) of the YW243.55S70 anti-mouse / human anti-PD-L1 antibody. This figure shows the release of ATP and HMGB1 during in vitro ICD induction by compound (A). This figure shows the expression of calreticulin (CRT) on the cell surface during in vitro ICD induction by compound (A). This figure shows the release of HMGB1 during in vitro ICD induction by compound (A). Mouse colon cancer cells in which ICD was induced were transplanted into mice in the test of FIG. 27. This figure shows the formation of immune memory against tumors (vaccination effect) through in vivo ICD induction by compound (A). This figure shows the number of IFNγ-producing splenocytes in spleens isolated from mice transplanted with mouse colon cancer cells in the test of FIG. 27.

[0046] Figure 31A shows the results of population analysis of T cells in spleens isolated from mice transplanted with mouse colon cancer cells and administered PBS in the test of Figure 27. Figure 31B shows the ADCP activity of anti-SIRPα antibodies and / or antibody-drug conjugates (2) against human gastric cancer cells. Figure 31C shows the anti-SIRPα antibody concentration-dependent enhancement of ADCP activity of anti-SIRPα antibodies and antibody-drug conjugates (1) against human gastric cancer cells. (Figure 31D) A figure showing the anti-SIRPα antibody concentration-dependent enhancement of ADCP activity by anti-SIRPα antibody and antibody-drug conjugate (2) against human gastric cancer cells. A figure showing the anti-tumor effect of anti-SIRPα antibody, anti-PD-L1 antibody and / or antibody-drug conjugate (1) in mice transplanted with HER2-expressing mouse breast cancer cells. A figure showing the anti-tumor effect of anti-SIRPα antibody and / or antibody-drug conjugate (2) in mice transplanted with TROP2-expressing mouse colon cancer cells.FIG. 1 shows the antitumor effect of anti-SIRPα antibody and / or antibody-drug conjugate (3) in mice transplanted with HER3-expressing mouse colon cancer cells.

[0057] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiment described below shows one example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0058] 1. Antibody-drug conjugates The antibody-drug conjugates used in the present invention are represented by the formula

[0059]

[0060] (wherein A represents the binding site to the antibody) and the antibody are bound via a thioether bond.

[0061] In the present invention, a partial structure of an antibody-drug conjugate consisting of a linker and a drug is referred to as a “drug linker.” This drug linker is bound to a thiol group (in other words, a sulfur atom of a cysteine ​​residue) generated at an interchain disulfide bond site of the antibody (two sites between heavy chains and two sites between heavy chains and light chains).

[0062] The drug linker of the present invention is based on the topoisomerase I inhibitor exatecan (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]indolizino[1,2-b]quinoline-10,13-dione, (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]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione)). Exatecan has the formula

[0063]

[0064] It is a camptothecin derivative having an antitumor effect, represented by the formula:

[0065] The antibody-drug conjugate used in the present invention can also be represented by the following formula:

[0066]

[0067] Here, the drug linker is bound to the antibody via a thioether bond, and n is synonymous with the so-called average drug binding number (DAR; Drug-to-Antibody Ratio), which indicates the average number of drug linkers bound per antibody.

[0068] The antibody-drug conjugate used in the present invention is translocated into cancer cells, and then the linker moiety is cleaved, resulting in the formation of a drug conjugate of the formula

[0069]

[0070] (hereinafter referred to as compound (A)).

[0071] The above compound is believed to be the basis of the antitumor activity of the antibody-drug conjugate used in the present invention, and has been confirmed to have topoisomerase I inhibitory activity (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0072] Topoisomerase I is an enzyme involved in DNA synthesis, which changes the higher-order structure of DNA by breaking and recombining single strands of DNA. Therefore, drugs with topoisomerase I inhibitory activity can inhibit DNA synthesis, thereby stopping cell division at the S phase (DNA synthesis phase) of the cell cycle and inducing cell death by apoptosis, thereby suppressing the proliferation of cancer cells.

[0073] The antibody-drug conjugate used in the present invention is also known to have a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046).

[0074] This bystander effect is exerted when the antibody-drug conjugate used in the present invention is internalized into target-expressing cancer cells, and the compound is then released to exert an antitumor effect on nearby cancer cells that do not express the target.

[0075] This bystander effect is also exhibited as an excellent antitumor effect when the antibody-drug conjugate of the present invention is used in combination with an anti-SIRPα antibody.

[0076] 2. Antibodies in Antibody-Drug Conjugates The antibodies in the antibody-drug conjugates used in the present invention may be derived from any species, but are preferably derived from humans, rats, mice, and rabbits. When the antibodies are derived from species other than humans, they are preferably chimerized or humanized using well-known techniques. The antibodies of the present invention may be polyclonal or monoclonal antibodies, but are preferably monoclonal antibodies.

[0077] The antibody in the antibody-drug conjugate used in the present invention preferably has the property of being able to target cancer cells, and preferably has the ability to recognize cancer cells, the ability to bind to cancer cells, the ability to be taken up and internalized in cancer cells, and / or cytocidal activity against cancer cells.

[0078] The binding of antibodies to cancer cells can be confirmed using flow cytometry. Antibody uptake into cancer cells can be confirmed using (1) an assay in which a secondary antibody (fluorescently labeled) that binds to a therapeutic antibody is used to visualize the antibody taken up into the cells using a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which a secondary antibody (fluorescently labeled) that binds to a therapeutic antibody is used to measure the amount of fluorescence taken up into the cells (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay in which an immunotoxin that binds to a therapeutic antibody is released upon intracellular uptake, thereby inhibiting cell proliferation (BioTechniques 28:162-165, January 2000). A recombinant conjugate protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as the immunotoxin.

[0079] The antitumor activity of an antibody can be confirmed in vitro by measuring its inhibitory activity against cell proliferation. For example, a cancer cell line overexpressing the antibody's target protein can be cultured, and the antibody can be added to the culture system at various concentrations to measure its inhibitory activity against focus formation, colony formation, and spheroid growth. In vivo, the antitumor activity can be confirmed, for example, by administering the antibody to nude mice transplanted with a cancer cell line overexpressing the target protein and measuring changes in the cancer cells.

[0080] Although it is preferable that the antibody itself has an antitumor effect, since the antibody-drug conjugate is bound to a compound that exerts an antitumor effect, the antibody itself does not necessarily have an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxicity of the antitumor compound on cancer cells, it is important and preferable that the antibody has the property of being internalized and transported into cancer cells.

[0081] The antibody in the antibody-drug conjugate used in the present invention can be obtained by known means. For example, it can be obtained by immunizing an animal with a polypeptide that serves as an antigen, and collecting and purifying the antibody produced in the body, using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibody that binds to the heterologous antigen with human antigens.

[0082] Alternatively, a monoclonal antibody can be obtained by fusing antibody-producing cells that produce an antibody against an antigen with myeloma cells to establish a hybridoma according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0083] Antigens can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce it. Specifically, a vector capable of expressing the antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above-mentioned genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[0084] The antibody in the antibody-drug conjugate used in the present invention is preferably a genetically engineered antibody that has been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as a chimeric antibody or a humanized antibody, or is preferably an antibody having only the genetic sequence of a human-derived antibody, i.e., a human antibody. These antibodies can be produced using known methods.

[0085] Chimeric antibodies include antibodies whose variable and constant regions are heterologous, such as chimeric antibodies in which the variable region of a mouse- or rat-derived antibody is joined to a human-derived constant region (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0086] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody has been incorporated into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies in which not only the CDR sequence of a heterologous antibody but also some framework amino acid residues of the heterologous antibody have been grafted onto a human antibody by CDR grafting (WO 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0087] Examples of human antibodies include antibodies produced using human antibody-producing mice carrying human chromosomal fragments containing human antibody heavy and light chain genes (see, for example, 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). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be used (see, for example, Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427-431).

[0088] The antibody in the antibody-drug conjugate used in the present invention also includes modified antibodies. The modified antibody refers to an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include those having a chemical moiety attached to the amino acid backbone or an N- or O-linked carbohydrate chain. Biological modifications include those that have undergone post-translational modification (e.g., addition of an N- or O-linked sugar chain, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, etc.), and those in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Also included within the meaning of such modified antibodies are those labeled to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme-labeled, fluorescent-labeled, or affinity-labeled antibodies. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens, etc.

[0089] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the antibody of the present invention. Techniques for modulating antibody sugar chain modification are known, including, but not limited to, those described in International Publication Nos. 99 / 54342, 00 / 61739, 02 / 31140, 2007 / 133855, and 2013 / 120066. Antibodies of the present invention also include antibodies with modified sugar chain modifications.

[0090] It is known that antibodies produced in cultured mammalian cells have deletions of lysine residues at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (e.g., complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the antibodies of the present invention also include antibodies and functional fragments of such antibodies that have undergone such modifications, including deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and amidated deletions (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody of the present invention are not limited to the above types. The two heavy chains constituting the antibody of the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above-mentioned deletions, or a combination of any two types. The quantitative ratio of each deletion may be affected by the type and culture conditions of the cultured mammalian cells producing the antibody of the present invention, but preferred examples of the antibody of the present invention include those in which one amino acid residue is deleted at the carboxyl terminus of each of the two heavy chains.

[0091] The isotype of the antibody according to the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1, IgG2, or IgG4.

[0092] The antibody in the antibody-drug conjugate used in the present invention is not particularly limited, and examples thereof include an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-CD3 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD56 antibody, an anti-CD98 antibody, an anti-DR5 antibody, an anti-EGFR antibody, an anti-EPHA2 antibody, an anti-FGFR2 antibody, an anti-FGFR4 antibody, an anti-FOLR1 antibody, an anti-VEGF antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD70 antibody, an anti-PS antibody, ...PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti-PS antibody, an anti Examples of the antibody include an MA antibody, an anti-CEA antibody, an anti-mesothelin antibody, an anti-A33 antibody, an anti-CanAg antibody, an anti-Cripto antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-tenascin-C antibody, an anti-SLC44A4 antibody, an anti-GPR20 antibody, and an anti-CDH6 antibody, and preferred examples include an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, an anti-GPR20 antibody, and an anti-CDH6 antibody.

[0093] In the present invention, the term "anti-HER2 antibody" refers to an antibody that specifically binds to HER2 (Human Epidermal Growth Factor Receptor Type 2; ErbB-2), and preferably has the activity of being internalized into HER2-expressing cells upon binding to HER2.

[0094] Examples of anti-HER2 antibodies include trastuzumab (US Pat. No. 5,821,337) and pertuzumab (WO 01 / 00245), with trastuzumab being preferred.

[0095] In the present invention, the term "anti-HER3 antibody" refers to an antibody that specifically binds to HER3 (Human Epidermal Growth Factor Receptor Type 3; ErbB-3), and preferably has the activity of being internalized into HER3-expressing cells upon binding to HER3.

[0096] Examples of anti-HER3 antibodies include patritumab (U3-1287), U1-59 (WO 2007 / 077028), MM-121 (seribantumab), the anti-ERBB3 antibodies described in WO 2008 / 100624, RG-7116 (lumretuzumab), and LJM-716 (elgemtumab), and preferred examples include patritumab and U1-59.

[0097] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2; EGP-1), and preferably has the activity of being internalized into TROP2-expressing cells upon binding to TROP2.

[0098] An example of an anti-TROP2 antibody is hTINA1-H1L1 (WO 2015 / 098099).

[0099] In the present invention, 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 the activity of being internalized into B7-H3-expressing cells upon binding to B7-H3.

[0100] An example of an anti-B7-H3 antibody is M30-H1-L4 (WO 2014 / 057687).

[0101] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that specifically binds to GPR20 (G protein-coupled receptor 20), and preferably has the activity of being internalized into GPR20-expressing cells upon binding to GPR20.

[0102] An example of an anti-GPR20 antibody is h046-H4e / L7 (WO 2018 / 135501).

[0103] In the present invention, the term "anti-CDH6 antibody" refers to an antibody that specifically binds to CDH6 (cadherin-6), and preferably has the activity of being internalized into CDH6-expressing cells upon binding to CDH6.

[0104] An example of an anti-CDH6 antibody is H01L02 (WO 2018 / 212136).

[0105] 3. Preparation of Antibody-Drug Conjugates The drug linker intermediate used in the preparation of the antibody-drug conjugates of the present invention is represented by the following formula:

[0106]

[0107] The drug linker intermediate shown above is N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4': 6,7] indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl] glycinamide, and can be produced with reference to the descriptions in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, and WO 2019 / 044947.

[0108] The antibody-drug conjugate used in the present invention can be produced by reacting the aforementioned drug linker intermediate with an antibody having a thiol group (also called a sulfhydryl group).

[0109] Antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, G. T., Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, antibodies having sulfhydryl groups in which intra-chain disulfides have been partially or completely reduced can be obtained by reacting an antibody with a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in an amount of 0.3 to 3 molar equivalents per intra-chain disulfide in the antibody in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA).

[0110] Furthermore, antibody-drug conjugates with 2 to 8 drugs bound per antibody can be prepared using 2 to 20 molar equivalents of drug linker intermediate per antibody having a sulfhydryl group.

[0111] The average number of drugs bound per antibody molecule in the produced antibody-drug conjugate can be calculated, for example, by a method in which the UV absorbance of the antibody-drug conjugate and its conjugation precursor is measured at two wavelengths, 280 nm and 370 nm (UV method), or by a method in which the antibody-drug conjugate is treated with a reducing agent, and each of the resulting fragments is quantified by HPLC measurement and then calculated (HPLC method).

[0112] Conjugation of an antibody and a drug linker intermediate, and calculation of the average number of drugs bound per antibody molecule in an antibody-drug conjugate can be carried out with reference to the descriptions in WO 2014 / 057687, WO 2015 / 098099, WO 2015 / 115091, WO 2015 / 155998, WO 2018 / 135501, WO 2018 / 212136, and the like.

[0113] In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention in which the antibody is an anti-HER2 antibody.

[0114] The anti-HER2 antibody preferably comprises a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 26 to 33 of SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 51 to 58 of SEQ ID NO: 1, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 97 to 109 of SEQ ID NO: 1, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 27 to 32 of SEQ ID NO: 2, a CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 50 to 52 of SEQ ID NO: 2, and a CDRL3 consisting of the amino acid sequence set forth in amino acid numbers 89 to 97 of SEQ ID NO: 2; and more preferably, an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 120 of SEQ ID NO: 1, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 107 of SEQ ID NO: 2; and even more preferably, an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2, or an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 of SEQ ID NO: 2.

[0115] The average number of drug linkers bound per antibody 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.

[0116] Anti-HER2 antibody-drug conjugates can be produced with reference to the descriptions in WO 2015 / 115091 and the like.

[0117] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention in which the antibody is an anti-HER3 antibody.

[0118] The anti-HER3 antibody is preferably an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 26 to 35 of SEQ ID NO: 3, a CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 50 to 65 of SEQ ID NO: 3, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 98 to 106 of SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 24 to 39 of SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 56 to 62 of SEQ ID NO: 4, and a CDRL3 consisting of the amino acid sequence set forth in amino acid numbers 95 to 103 of SEQ ID NO: 4; more preferably an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 117 of SEQ ID NO: 3, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 1 to 113 of SEQ ID NO: 4; and even more preferably an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted.

[0119] The average number of drug linkers bound per antibody 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.

[0120] Anti-HER3 antibody-drug conjugates can be produced with reference to the descriptions in WO 2015 / 155998 and the like.

[0121] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention in which the antibody is an anti-TROP2 antibody.

[0122] The anti-TROP2 antibody preferably comprises a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 50 to 54 of SEQ ID NO:5, a CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 85 of SEQ ID NO:5, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 118 to 129 of SEQ ID NO:5; and a CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 of SEQ ID NO:6, a CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 of SEQ ID NO:6, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 109 to 117 of SEQ ID NO:6. and a light chain comprising a CDRL3 consisting of: a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 of SEQ ID NO:5; and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 of SEQ ID NO:6; and even more preferably, an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO:5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:6; or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody has been deleted.

[0123] The average number of drug linkers bound per antibody 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.

[0124] Anti-TROP2 antibody-drug conjugates can be produced with reference to the descriptions in WO 2015 / 098099 and the like.

[0125] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention in which the antibody is an anti-B7-H3 antibody.

[0126] The anti-B7-H3 antibody preferably comprises a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 50 to 54 of SEQ ID NO:7, a CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 85 of SEQ ID NO:7, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 118 to 130 of SEQ ID NO:7, and a CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 53 of SEQ ID NO:8, a CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 75 of SEQ ID NO:8, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 108 to 116 of SEQ ID NO:8. and a light chain comprising a CDRL3 consisting of: a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 141 of SEQ ID NO:7; and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 128 of SEQ ID NO:8; and even more preferably, an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 of SEQ ID NO:7 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:8; or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody has been deleted.

[0127] The average number of drug linkers bound per antibody 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.

[0128] The anti-B7-H3 antibody-drug conjugate used in the present invention can be produced with reference to the descriptions in WO 2014 / 057687 and the like.

[0129] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention in which the antibody is an anti-GPR20 antibody.

[0130] The anti-GPR20 antibody preferably comprises a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 45 to 54 of SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 78 of SEQ ID NO: 9, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 118 to 131 of SEQ ID NO: 9, and a CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 of SEQ ID NO: 10, a CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 of SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 109 to 117 of SEQ ID NO: 10. and a light chain comprising a CDRL3 consisting of:

[0131] The average number of drug linkers bound per antibody 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.

[0132] Anti-GPR20 antibody-drug conjugates can be produced with reference to the descriptions in WO 2018 / 135501 and the like.

[0133] In the present invention, the term "anti-CDH6 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention in which the antibody is an anti-CDH6 antibody.

[0134] The anti-CDH6 antibody preferably comprises a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in amino acid numbers 45 to 54 of SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 78 of SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 118 to 130 of SEQ ID NO: 11; and a CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 of SEQ ID NO: 12, a CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 of SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in amino acid numbers 109 to 116 of SEQ ID NO: 12. and a light chain comprising a CDRL3 consisting of the amino acid sequence of SEQ ID NO: 11, more preferably a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 11, and a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 12, and even more preferably an antibody comprising a heavy chain consisting of the amino acid sequence of amino acids 20 to 471 in SEQ ID NO: 11 and a light chain consisting of the amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 12, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody has been deleted.

[0135] The average number of drug linkers bound per antibody 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.

[0136] Anti-CDH6 antibody-drug conjugates can be produced with reference to the descriptions in WO 2018 / 212136 and the like.

[0137] 4. Anti-SIRPα Antibodies SIRPα (signal regulatory protein α) is a single-pass transmembrane molecule of the Ig superfamily present in macrophages, dendritic cells, myeloid cells such as neutrophils, and glial cells. Its extracellular region consists of one IgV domain and two IgC domains, and ten variants, V1 to V10, of the IgV domain, which is the binding site for CD47, have been reported in humans. The extracellular IgV domain of the SIRPα protein is one of three extracellular Ig-like domains that constitute the SIRPα protein. Of these, V1 and V2 are major variants, and the anti-SIRPα antibodies of the present invention bind to all variants, including the major variants V1 and V2. In the present invention, "SIRPα" may also be referred to as "SIRPA."

[0138] The amino acid sequence of human SIRPα protein is disclosed in GenBank Accession No.: NP_001035111.

[0139] The anti-SIRPα antibody used in the present invention can be obtained by a method similar to the method described in "2. Antibodies in antibody-drug conjugates."

[0140] The monoclonal antibody used in the present invention can be obtained by immunizing a mammal such as a mouse, rat, rabbit, hamster, guinea pig, horse, monkey, dog, pig, cow, goat, or sheep with SIRPα or a fragment thereof as an immunogen, fusing the spleen cells or the like with myeloma cells to prepare a hybridoma, and then producing and secreting the antibody from the hybridoma by a known method.

[0141] SIRPα as an immunogen can be chemically synthesized based on sequence information, or can be obtained as a recombinant protein by known methods based on DNA sequence information encoding the protein.

[0142] Antibodies can be screened by any method, but preferably by Cell-ELISA using animal cells transfected with DNA encoding SIRPα.

[0143] The anti-SIRPα antibody used in the present invention inhibits the binding of SIRPα to CD47.

[0144] Tumor cells highly express CD47, and SIRPα expressed on phagocytes with phagocytic ability binds to and interacts with CD47, thereby transmitting a "Don't eat me" signal to the phagocytes and allowing the tumor cells to escape phagocytosis by the phagocytes. By inhibiting the binding of SIRPα to CD47, anti-SIRPα antibodies inhibit 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 phagocytes with phagocytic ability include macrophages such as M1-type and M2-type macrophages, and dendritic cells such as imDCs (immature dendritic cells).

[0145] In this case, when the anti-SIRPα antibody has an effector function and binds to an Fc receptor such as an Fcγ receptor on phagocytes such as macrophages or effector cells such as natural killer cells or T cells, the antibody attacks its own effector cells such as peripheral blood mononuclear cells (PBMCs) and macrophages through ADCC (antibody-dependent cellular cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis).

[0146] To avoid attacking self-cells, the anti-SIRPα antibody used in the present invention has a reduced effector function. As a result, the anti-SIRPα antibody used in the present invention only has the effect of inhibiting the binding of SIRPα to CD47, and does not bind to the Fc receptor of effector cells, and therefore does not exert its effector function.

[0147] The anti-SIRPα antibody used in the present invention does not attack autologous immune cells and can therefore be used safely as a medicine without side effects.

[0148] However, since the anti-SIRPα antibody used in the present invention has a reduced effector function, it does not exert a sufficient antitumor effect when used alone, and therefore is used in combination with other antitumor agents.

[0149] In order to reduce effector function, it is necessary that the Fc portion of the anti-SIRPα antibody does not bind to Fc receptors on macrophages or T cells. For this reason, the subclass of the anti-SIRPα antibody used in the present invention is substituted with one derived from IgG4. Among human IgG subclasses, IgG4 is generally known as a subclass with low effector functions such as ADCC activity, CDC activity, and / or ADCP activity (Bruggemann et al., J. Exp. Med., 1351-1361, 1987). When therapeutic antibodies are used to target molecules expressed in normal organs, IgG4 is used as one of the IgG formats to avoid toxicity due to effector function-mediated cytotoxicity (e.g., Opdivo). However, even though the effector function of the IgG4 subclass is low, it does not mean that it is completely absent. Therefore, the anti-SIRPα antibody used in the present invention has a mutation introduced into the heavy chain constant region that further reduces effector function, i.e., a mutation such as the substitution of one or more amino acids that reduces ADCC and / or ADCP activity. Such mutations include a substitution of phenylalanine at position 234 with alanine (F234A) and a substitution of leucine at position 235 with alanine (L235A) as indicated by the EU index by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 1991 Fifth edition) (Parekh et al., mAbs, 310-318, 2012). Such antibody mutations are referred to as FALA mutations.

[0150] Furthermore, since IgG4 does not have stable disulfide bond formation between antibody heavy chains, a mutation that promotes disulfide bond formation between antibody heavy chains is introduced to enhance stability. An example of such a mutation is the substitution of serine with proline at position 228 (S228P) as indicated by the EU index by Kabat et al. (ANGAL et al., Molecular Immunology, 105-108, 1993). This antibody mutation is called a PRO mutation.

[0151] The above-mentioned FALA mutation and PRO mutation may be simultaneously introduced into the constant region of the anti-SIRPα antibody used in the present invention (Vafa et al., Methods, 65, 114-126, 2014). An IgG4 heavy chain having both the FALA mutation and the Pro mutation is also referred to as an "IgG4proFALA"-type heavy chain, an "IgG4PFALA"-type heavy chain, or an "IgG4pf"-type heavy chain.

[0152] Among human IgG subclasses, human IgG1 has very strong effector functions, such as CDC activity mediated by complement fixation and antibody-dependent cytotoxicity (Bruggemann et al., J. Exp. Med., 1351-1361, 1987). When therapeutic antibodies target molecules highly expressed in cancer, they are used as an IgG format that exhibits therapeutic effects by promoting the induction of cancer cell death through effector-mediated cytotoxicity (trastuzumab, rituximab, etc.). When IgG1 is the isotype of the anti-SIRPα antibody used in the present invention, the effector function can be adjusted by substituting a portion of the amino acid residues in the constant region (see WO88 / 007089, WO94 / 28027, WO94 / 29351). Examples of IgG1 mutants with attenuated effector functions include IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), etc. IgG1 heavy chain constant regions into which these mutations have been introduced can also be used as the constant region of the anti-SIRPα antibody used in the present invention.

[0153] Among human IgG subclasses, human IgG2 has very weak effector functions such as CDC activity mediated by complement fixation and antibody-dependent cytotoxicity (Bruggemann et al., J. Exp. Med., 1351-1361, 1987), and is used as one of the IgG formats to avoid toxicity due to effector function-mediated cytotoxicity when therapeutic antibodies target molecules expressed in normal organs (denosumab, evolocumab, brodalumab, etc.). The IgG2 heavy chain constant region can also be used as the constant region of the anti-SIRPα antibody used in the present invention.

[0154] The anti-SIRPα antibodies used in the present invention also include modified antibodies. The term "modified" refers to an anti-SIRPα antibody used in the present invention that has been chemically or biologically modified. Chemical modifications include those in which a chemical moiety is attached to the amino acid backbone, or a chemical moiety is attached to an N- or O-linked carbohydrate chain. Biological modifications include those that have undergone post-translational modification (e.g., addition of an N- or O-linked glycan, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, etc.), and those in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the anti-SIRPα antibody or antigen used in the present invention, such as enzyme-labeled, fluorescent-labeled, or affinity-labeled antibodies. Such modified anti-SIRPα antibodies used in the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, detecting or isolating antibodies or antigens, and the like.

[0155] It is known that antibodies produced in cultured mammalian cells lose the lysine residue at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)), and that two amino acid residues, glycine and lysine, are also deleted from the carboxyl terminus of the heavy chain, and a proline residue newly positioned at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the anti-SIRPα antibodies used in the present invention include antibodies that have undergone such modifications and functional fragments of such antibodies, as well as deletions in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain and amidated deletions (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletions at the carboxyl termini of the heavy chains of the anti-SIRPα antibodies used in the present invention are not limited to the above types. The two heavy chains constituting the anti-SIRPα antibodies used in the present invention may be any one type of heavy chain selected from the group consisting of full-length heavy chains and the above-mentioned deletions, or a combination of any two types. The quantitative ratio of each deletion may be affected by the type and culture conditions of mammalian cultured cells that produce the anti-SIRPα antibodies used in the present invention, but the anti-SIRPα antibodies used in the present invention preferably include those in which one amino acid residue is deleted at the carboxyl terminus of each of the two heavy chains.

[0156] The anti-SIRPα antibodies used in the present invention also include chimeric antibodies and humanized antibodies that have been modified to reduce heterologous antigenicity to humans. Humanized antibodies are also called CDR-grafted antibodies.

[0157] A chimeric antibody is an antibody consisting of the light chain variable region and heavy chain variable region of an antibody of an animal other than human and the light chain constant region and heavy chain constant region of a human antibody. A chimeric antibody can be produced by collecting cDNA encoding the light chain variable region and cDNA encoding the heavy chain variable region from a hybridoma producing an anti-SIRPα antibody, inserting them into an expression vector containing cDNA encoding the light chain constant region and heavy chain constant region of a human antibody to construct a chimeric antibody expression vector, and introducing the vector into a host cell for expression.

[0158] The heavy chain constant region consists of three domains: Ｈ 1. C Ｈ 2 and C Ｈ In the anti-SIRPα antibody used in the present invention, the human heavy chain constant region of the chimeric antibody is a heavy chain constant region of the IgG4 subclass, IgG4proFALA, which has a Pro mutation and a FALA mutation. The light chain constant region may be a κ or λ constant region as long as it belongs to human Ig.

[0159] An example of a chimeric anti-SIRPα antibody used in the present invention is antibody cD13, a chimeric antibody having the variable region of the rat anti-human SIRPα monoclonal antibody D13 described in Patent Document 4 (WO 2020 / 013170). The cD13 antibody has high binding affinity to human SIRPα and high inhibitory activity against the binding of SIRPα to CD47.

[0160] The antibody cD13 comprises, as CDRs (complementarity determining regions) of its light chain variable region, a CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 21 (GASKSVRTYMH), a CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 22 (SASNLEA), and a CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 23 (QQSNEPPYT). Furthermore, the antibody cD13 comprises, as CDRs of its heavy chain variable region, a CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 18 (GFTFSDYGMI), a CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 19 (SISSSSSSYIY), and a CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 20 (RYYGFNYPFDY) ( Figure 18 ).

[0161] That is, the anti-SIRPα antibody used in the present invention is an antibody that comprises a CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 21, a CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 22, and a CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 23, and further comprises, as CDRs of the heavy chain variable region, a CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 18, a CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 19, and a CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 20.

[0162] Each of the above CDRs also includes CDRs consisting of amino acid sequences in which one or several, preferably one or two, and more preferably one amino acid is deleted, substituted or added in the amino acid sequence represented by each CDR.

[0163] A humanized antibody (CDR-grafted antibody) is an antibody in which the amino acid sequences of the CDRs of the light chain variable region and heavy chain variable region of an antibody of a non-human animal have been grafted into appropriate positions in the light chain variable region and heavy chain variable region of a human antibody.

[0164] The humanized anti-SIRPα antibody of the present invention can be produced by constructing cDNA encoding variable regions in which the amino acid sequences of the CDRs of the light chain variable region and heavy chain variable region of an antibody of a non-human animal, produced by a hybridoma that produces a monoclonal antibody that binds to human SIRPα and inhibits the binding of SIRPα to CD47, thereby enhancing the phagocytic ability of macrophages, are grafted into the framework (FR) regions of the light chain variable region and heavy chain variable region of any human antibody, and inserting the cDNA into an expression vector for animal cells that contains genes encoding the light chain constant region and heavy chain constant region of a human antibody to construct a humanized antibody expression vector, which can then be expressed and produced by introducing the vector into animal cells.

[0165] Specifically, a DNA sequence designed to link the CDRs of antibody cD13 with the framework regions of a human antibody can be synthesized. The framework regions of the human antibody linked via the CDRs are selected so that the CDRs form a good antigen-binding site. Furthermore, if necessary, amino acids in the framework regions of the antibody variable regions can be substituted so that the CDRs of the humanized antibody form a suitable antigen-binding site. Humanized antibodies can be produced by grafting CDRs using known CDR grafting techniques.

[0166] Using the above method, examples of heavy chains of humanized antibodies having the CDRs of the heavy and light chain variable regions of antibody cD13 (six CDRs consisting of the amino acids set forth in SEQ ID NOS: 18 to 23), in which some amino acids in the framework regions of the variable regions have been substituted, include the humanized antibody heavy chain hH1 and humanized antibody heavy chain hH2 described in Patent Document 4 (WO 2020 / 013170). Furthermore, examples of light chains of humanized antibodies having the CDRs of the light chain variable region of antibody D13, in which some amino acids in the framework regions of the variable regions have been substituted, include the humanized antibody light chain hL2, humanized antibody light chain hL3, and humanized antibody light chain hL4, also described in Patent Document 4 (WO 2020 / 013170).

[0167] The full-length amino acid sequence of the humanized antibody heavy chain hH1 is shown in SEQ ID NO: 13. The full-length amino acid sequence of the humanized antibody heavy chain hH2 is shown in SEQ ID NO: 14. In SEQ ID NOs: 13 and 14, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 139 is a variable region, and the amino acid sequence consisting of amino acid residues 140 to 466 is a constant region.

[0168] The anti-SIRPα antibodies used in the present invention include antibodies having a heavy chain variable region consisting of amino acid residues 20 to 139 of SEQ ID NO: 13 or 14 and a heavy chain constant region consisting of amino acid residues 140 to 466 of SEQ ID NO: 13 or 14.

[0169] The full-length amino acid sequence of the humanized antibody light chain hL2 is shown in SEQ ID NO: 15. The full-length amino acid sequence of the humanized antibody light chain hL3 is shown in SEQ ID NO: 16. The full-length amino acid sequence of the humanized antibody light chain hL4 is shown in SEQ ID NO: 17. In SEQ ID NOs: 15, 16, and 17, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues 21 to 127 is a variable region, and the amino acid sequence consisting of amino acid residues 128 to 234 is an amino acid sequence of a constant region.

[0170] The anti-SIRPα antibodies used in the present invention include antibodies having a variable region consisting of amino acid residues 21 to 127 of SEQ ID NOs: 15, 16, and 17 and a light chain constant region consisting of amino acid residues 128 to 234.

[0171] The heavy chain constant region of the humanized antibody is a heavy chain constant region of the IgG4 subclass, IgG4proFALA, having Pro mutation and FALA mutation.

[0172] Examples of antibodies that have high binding affinity to human SIRPα and high inhibitory activity against the binding of SIRPα to CD47 include an antibody consisting of a humanized antibody heavy chain hH1 and a humanized antibody light chain hL3 (hD13_H1L3 antibody), an antibody consisting of a humanized antibody heavy chain hH1 and a humanized antibody light chain hL4 (hD13_H1L4 antibody), an antibody consisting of a humanized antibody heavy chain hH2 and a humanized antibody light chain hL2 (hD13_H2L2 antibody), and an antibody consisting of a humanized antibody heavy chain hH2 and a humanized antibody light chain hL3 (hD13_H2L3 antibody).

[0173] The hD13_H1L3 antibody has a heavy chain consisting of amino acid residues 20 to 466 of SEQ ID NO:13 and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:16.

[0174] The hD13_H1L4 antibody has a heavy chain consisting of amino acid residues 20 to 466 of SEQ ID NO:13 and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:17.

[0175] The hD13_H2L2 antibody has a heavy chain consisting of amino acid residues 20 to 466 of SEQ ID NO:14 and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:15.

[0176] The hD13_H2L3 antibody has a heavy chain consisting of amino acid residues 20 to 466 of SEQ ID NO:14 and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:16.

[0177] Other anti-SIRPα antibodies that can be used in the present invention include the antibodies described in Patent Documents 1 to 3.

[0178] An example of the antibody described in Patent Document 1 (WO 2017 / 178653) is OSE-172. The amino acid sequence of the heavy chain of OSE-172 is shown in SEQ ID NO: 24 in the Sequence Listing, and the amino acid sequence of the light chain of OSE-172 is shown in SEQ ID NO: 25. OSE-172 is an antibody having a heavy chain consisting of amino acid residues 20 to 466 of SEQ ID NO: 24 and a light chain consisting of amino acid residues 21 to 239 of SEQ ID NO: 25.

[0179] An example of the antibody described in Patent Document 2 (WO 2018 / 026600) is KWAR23. The amino acid sequence of the heavy chain of KWAR23 is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain of KWAR23 is shown in SEQ ID NO: 27. KWAR23 is an antibody having a heavy chain consisting of amino acid residues 20 to 459 of SEQ ID NO: 26 and a light chain consisting of amino acid residues 21 to 235 of SEQ ID NO: 27.

[0180] An example of the antibody described in Patent Document 3 (WO 2018 / 190719) is ADU-1805. The amino acid sequence of the heavy chain of ADU-1805 is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain of ADU-1805 is shown in SEQ ID NO: 29. ADU-1805 is an antibody having a heavy chain consisting of amino acid residues 20 to 467 of SEQ ID NO: 28 and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO: 29.

[0181] It is known that antibodies produced in cultured mammalian cells have deletions of lysine residues at the carboxyl termini of their heavy chains (Tsubaki et al., Int. J. Biol. Macromol, 139-147, 2013). However, this deletion of the heavy chain sequence does not affect the antigen-binding ability or effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the present invention also includes antibodies in which the lysine residue at the carboxyl terminus of the heavy chain is deleted.

[0182] The cancer therapeutic agent used in the present invention may contain a therapeutically effective amount of an anti-SIRPα antibody and pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, adjuvants, etc. The "pharmaceutically acceptable carrier" etc. may be appropriately selected from a wide range depending on the type of target disease and the dosage form of the drug. The administration method of the antitumor agent of the present invention may be appropriately selected, and may be, for example, injection, including local injection, intraperitoneal injection, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusion injection, etc. Furthermore, solutions for injection may be formulated using carriers such as salt solutions, glucose solutions, mixtures of salt water and glucose solutions, various buffer solutions, etc. Alternatively, the agent may be formulated in a powder form and mixed with the liquid carrier at the time of use to prepare an injection solution.

[0183] Other administration methods can also be appropriately selected based on the development of the formulation. For example, oral administration can be performed in the form of oral liquids, powders, pills, capsules, tablets, etc. Oral liquids, such as suspensions and syrups, can be prepared 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 parahydroxybenzoate, and flavors such as strawberry flavor and peppermint. Powders, pills, capsules, and tablets can be formulated using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin. Tablets and capsules are preferred unit dosage forms for the composition of the present invention because of their ease of administration. When tablets or capsules are made, solid manufacturing carriers are used.

[0184] 5. Relationship between immunogenic cell death and anti-SIRPα antibodies Immunogenic cell death (ICD) is a type of cell death characterized by the massive release of intracellular molecules such as ATP and HMGB1 (High-mobility group box 1 protein) and the cell surface exposure of calreticulin (CRT). These danger signals activate immune cells. It has been reported that ATP recruits and activates dendritic cells (DCs) and macrophages, HMGB1 enhances the production of inflammatory cytokines such as Type I IFN, and CRT enhances antigen uptake from dead cells as an eat-me-signal (Nature Reviews Immunology. 2017, 17, 97-111). In other words, when cancer cells cause ICD, immunity against the cancer cells (anti-tumor immunity) can be induced. Anticancer drugs known to induce ICD include anthracyclines, oxaliplatin, and cyclophosphamide, but docetaxel, mitomycin C, and the like have not been shown to induce ICD. The presence or absence of ICD effects can be assessed by in vitro detection of the Danger signal following drug addition, as well as by in vivo vaccination assays. In the latter, drug-treated cancer cells are transplanted into immunocompetent mice, and then drug-untreated cancer cells are transplanted into the opposite side one week later. If immunological memory is established by the ICD-inducing cancer cells, the engraftment and proliferation of the transplanted cancer cells are inhibited (Cancer Research, 2017, 77, 2686-2698).

[0185] The uptake of cancer antigens by myeloid cells such as dendritic cells and macrophages is important for the establishment of immune memory through ICD. It is believed that a "Don't eat me" signal via SIRPα-CD47 acts between myeloid cells and cancer cells. Administration of anti-SIRPα antibodies inhibits this signal, enhancing phagocytic activity and ultimately enhancing the uptake of cancer antigens. Cancer antigens taken up by dendritic cells and macrophages are processed intracellularly into 8-30-mer peptide fragments, which are presented on MHC. There are two types of MHC: class I and class II. Antigen peptides of approximately 9-mer presented on MHC-class I activate CD8+ T cells, while antigen peptides of approximately 15-mer presented on MHC-class II activate CD4+ T cells. Generally, foreign antigens are processed within myeloid cells and then presented on MHC-class II, but some DC subsets present foreign antigens on MHC-class I and have the ability to cross-present, activating CD8+ T cells that exhibit cytotoxic activity against cancer cells. DCs exist as subsets called cDC1 and cDC2; cDC1 is SIRPα-negative and has cross-presentation ability, while cDC2 is SIRPα-positive and does not have cross-presentation ability. Recent academic reports have shown that in anti-tumor studies using a mouse syngeneic model, administration of an anti-SIRPα antibody increases the cDC1 subset, and that the addition of an anti-SIRPα antibody to a co-culture system of cancer cells, DCs, and T cells enhances cross-presentation ability to CD8+ T cells. These findings suggest that administration of anti-SIRPα antibodies not only promotes phagocytosis of cancer cells and uptake of cancer antigens, but also enhances cross-presentation ability, ultimately enhancing the induction of CD8+ T cells, which are essential for tumor immunity.

[0186] The relationship between antibody-drug conjugates loaded with cytocidal drugs and ICD induction has been reported for antibody-drug conjugates loaded with tubulysin, pyrrolobenzodiazepine (PBD), and MMAE (Cancer Research, 2017, 77, 2686-2698 or ONCOIMMUNOLOGY, 2019, 8 (4), e1565859). However, for the antibody-drug conjugates used in the present invention that are payloaded with Compound (A), a TpoI inhibitor, only reports have been made of HMGB1 release from Compound (A)-treated cells and limited antitumor effects due to vaccination of the treated cells (Clin. Invest. 2020:130(1):374-388). In this study, it was revealed that 1) compound (A) induces the release of not only HMGB1 but also other Danger signals from dying cancer cells, 2) compound (A) activates immune cells present in the cancer microenvironment and enhances the cancer antigen-specific T cell population, thereby inducing anti-tumor immunity, and 3) anti-SIRPα antibodies enhance the immune response induced by compound (A).

[0187] 6. Pharmaceuticals Hereinafter, a pharmaceutical composition and a method of treatment characterized by administering a combination of the antibody-drug conjugate according to the present invention and an anti-SIRPα antibody will be described.

[0188] The pharmaceutical composition and treatment method of the present invention may be characterized in that the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in separate formulations and administered simultaneously or at different times, or may be characterized in that the antibody-drug conjugate and the anti-SIRPα antibody are contained as active ingredients in a single formulation and administered.

[0189] The pharmaceutical composition and treatment method of the present invention can be used for the treatment of cancer, and preferably includes breast cancer, gastric cancer (sometimes called gastric adenocarcinoma), colon cancer (sometimes called colorectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, head and neck cancer (including salivary gland cancer and pharyngeal cancer), gastroesophageal junction adenocarcinoma, biliary tract cancer (including bile duct cancer), gallbladder cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, uterine cancer, kidney cancer, vulvar cancer, thyroid cancer, thymic cancer, penile cancer, The composition may be used to treat at least one cancer selected from the group consisting of leukemia, lymphoma, malignant lymphoma, plasmacytoma, myeloma, myelodysplastic syndrome, brain tumor, glioma, glioblastoma multiforme, osteosarcoma, and melanoma, more preferably at least one cancer selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, and uterine carcinosarcoma, and even more preferably at least one cancer selected from the group consisting of breast cancer, gastric cancer, lung cancer, and ovarian cancer.

[0190] Among the antibody-drug conjugates used in the present invention, which antibody is particularly suitable for the antibody-drug conjugate can be determined by examining the type of cancer and tumor markers. For example, if HER2 expression is confirmed in the cancer, an anti-HER2 antibody-drug conjugate can be preferably used; if HER3 expression is confirmed in the cancer, an anti-HER3 antibody-drug conjugate can be preferably used; if TROP2 expression is confirmed in the cancer, an anti-TROP2 antibody-drug conjugate can be preferably used; if B7-H3 expression is confirmed in the cancer, an anti-B7-H3 antibody-drug conjugate can be preferably used; if GPR20 expression is confirmed in the cancer, an anti-GPR20 antibody-drug conjugate can be preferably used; and if CDH6 expression is confirmed in the cancer, an anti-CDH6 antibody-drug conjugate can be preferably used.

[0191] The presence or absence of HER2, HER3, TROP2, B7-H3, GPR20, and CDH6, as well as other tumor markers, can be confirmed, for example, by collecting tumor tissue from a cancer patient and examining the formalin-fixed, paraffin-embedded (FFPE) specimen at the gene product (protein) level using immunohistochemistry (IHC), a flow cytometer, Western blot analysis, or the like, or by examining the gene transcription level using in situ hybridization (ISH), quantitative PCR (q-PCR), microarray analysis, or the like. Alternatively, cell-free circulating tumor DNA (ctDNA) in the blood can be collected from a cancer patient and confirmed by testing using a method such as next-generation sequencing (NGS).

[0192] The pharmaceutical composition and treatment method of the present invention can be preferably used in mammals, and more preferably in humans.

[0193] The anti-tumor effect of the pharmaceutical composition and treatment method of the present invention can be confirmed, for example, by creating a model in which cancer cells are transplanted into a test animal and measuring the reduction in tumor volume and the life-prolonging effect of administering the pharmaceutical composition and treatment method of the present invention. The combined effect of the antibody-drug conjugate and anti-SIRPα antibody used in the present invention can then be confirmed by comparing the anti-tumor effect with that of the antibody-drug conjugate and anti-SIRPα antibody used in the present invention when administered alone.

[0194] The antitumor effects of the pharmaceutical composition and treatment method of the present invention can be confirmed in clinical trials by the Response Evaluation Criteria in Solid Tumors (RECIST) evaluation method, the WHO evaluation method, the Macdonald evaluation method, weight measurement, and other methods, and the results can be evaluated for complete response (CR), partial response (PR), progressive disease (PD), objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), and other important parameters. The survival rate can be determined by an index such as OS (Optical Survival Rate).

[0195] By the above-mentioned method, it is possible to confirm the superiority of the antitumor effect of the pharmaceutical composition and treatment method of the present invention over existing pharmaceutical compositions and treatment methods for cancer treatment.

[0196] The pharmaceutical composition and treatment method of the present invention can slow the growth of cancer cells, suppress their proliferation, and even destroy them. These actions can relieve cancer patients from cancer-related symptoms and improve their quality of life, thereby achieving therapeutic effects while preserving the lives of cancer patients. Even if cancer cells are not destroyed, the inhibition and control of cancer cell proliferation can enable cancer patients to achieve a higher quality of life and longer survival.

[0197] The pharmaceutical composition of the present invention can be applied to patients as a systemic therapy, and can also be applied locally to cancer tissue to achieve therapeutic effects.

[0198] The pharmaceutical compositions of the present invention can be administered containing one or more pharmaceutically compatible ingredients. The pharmaceutically compatible ingredients can be appropriately selected from formulation additives and other ingredients commonly used in this field depending on the dose and administration concentration of the antibody-drug conjugate and anti-SIRPα antibody used in the present invention. For example, the antibody-drug conjugate 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-drug conjugate used in the present invention can be preferably used as an injection, more preferably as an aqueous injection or a lyophilized injection, and even more preferably as a lyophilized injection.

[0199] When the pharmaceutical composition containing the antibody-drug conjugate used in the present invention is an aqueous injection, it can be preferably administered intravenously by infusion after diluting with an appropriate diluent. Examples of the diluent include glucose solution and physiological saline, preferably glucose solution, and more preferably 5% glucose solution.

[0200] When the pharmaceutical composition containing the antibody-drug conjugate used in the present invention is in the form of a lyophilized injection, it is preferably dissolved in water for injection, diluted with an appropriate diluent at the required amount, and then administered intravenously by infusion. Examples of the diluent include glucose solution and physiological saline, preferably glucose solution, and more preferably 5% glucose solution.

[0201] Examples of introduction 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 the intravenous route.

[0202] The antibody-drug conjugate used in the present invention can be administered to humans at intervals of once every 1 to 180 days, preferably once every 1, 2, 3, or 4 weeks, and even more preferably once every 3 weeks. Furthermore, the antibody-drug conjugate used in the present invention can be administered at a single dose of approximately 0.001 to 100 mg / kg, preferably at a single dose of 0.8 to 12.4 mg / kg. When the antibody-drug conjugate used in the present invention is an anti-HER2 antibody-drug conjugate, it can be preferably administered at a single 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, once every 3 weeks. When the antibody-drug conjugate used in the present invention is an anti-HER3 antibody-drug conjugate, it can be preferably administered at a single dose of 1.6 mg / kg, 3.2 mg / kg, 4.8 mg / kg, 5.6 mg / kg, 6.4 mg / kg, 8.0 mg / kg, 9.6 mg / kg, or 12.8 mg / kg, once every three weeks. When the antibody-drug conjugate used in the present invention is an anti-TROP2 antibody-drug conjugate, it can be preferably administered at a single dose of 0.27 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6.0 mg / kg, 8.0 mg / kg, or 10.0 mg / kg, once every three weeks. The anti-SIRPα antibody of the present invention can be administered to a human at intervals of once every 1 to 180 days, preferably once every 1, 2, 3, or 4 weeks. The anti-SIRPα antibody of the present invention can be administered at a single dose of approximately 0.001 to 100 mg / kg.

[0203] The pharmaceutical composition and treatment method of the present invention may further comprise a cancer therapeutic agent other than the antibody-drug conjugate and anti-SIRPα antibody of the present invention. The pharmaceutical composition and treatment method of the present invention may also be administered in combination with other cancer therapeutic agents, thereby enhancing the anti-tumor effect. The other cancer therapeutic agents used for such purposes may be administered to an individual simultaneously with the pharmaceutical composition of the present invention, separately, or consecutively, or may be administered at different administration intervals. Such cancer therapeutic agents are not limited as long as they are drugs having antitumor activity, and examples thereof include irinotecan (CPT-11), cisplatin, carboplatin, oxaliplatin, fluorouracil (5-FU), gemcitabine, capecitabine, doxorubicin, epirubicin, cyclophosphamide, and mitomycin C. C), Tegafur / Gimeracil / Oteracil combination drug, Panitumumab, Bevacizumab, Ramucirumab, Regorafenib, Trifluridine / Tipiracil combination drug, Gefitinib, Erlotinib, Afatinib, At least one selected from the group consisting of afatinib, methotrexate, pemetrexed, tamoxifen, toremifene, fulvestrant, leuprorelin, goserelin, letrozole, anastrozole, a progesterone formulation, and lapatinib can be mentioned.

[0204] The pharmaceutical composition and treatment method of the present invention may further comprise, as a cancer therapeutic agent in addition to the antibody-drug conjugate and anti-SIRPα antibody of the present invention, an immune checkpoint inhibitor or an antibody drug that specifically binds to a cancer antigen and has ADCC and / or ADCP activity. Examples of immune checkpoint inhibitors include inhibitors of the binding between PD-1 and its ligand PD-L1, or CTLA4 inhibitors, and specific examples include anti-PD-1 antibodies (nivolumab, pembrolizumab, cemiplimab, spartalizumab, PDR-001, or BI 754091), anti-PD-L1 antibodies (atezolizumab, avelumab, or durbarumab), and anti-CTLA4 antibodies (ipilimumab or tremelimumab). Furthermore, examples of antibody drugs that specifically react with 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 (Alemutuzumab).

[0205] ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fcγ receptors (e.g., NK cells, neutrophils, macrophages, etc.) recognize antibodies bound to target cells, followed by lysis of the target cells. NK cells, the primary cells responsible for ADCC, express FcγRIIC and FcγRIIIA, while monocytes express FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA. On the other hand, ADCP refers to a cell-mediated reaction in which phagocytes expressing Fc receptors (e.g., macrophages, neutrophils, etc.) recognize antibodies bound to target cells, followed by phagocytosis of the target cells. Monocytes, the primary cells responsible for ADCP, express FcγRI, FcγRIIA, FcγRIIC, and FcγRIIIA.

[0206] The pharmaceutical compositions and treatment methods of the present invention can also be used in combination with radiation therapy. For example, a cancer patient may receive radiation therapy before and / or after, or simultaneously with, treatment with the pharmaceutical composition of the present invention.

[0207] 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 may be administered before surgery to reduce tumor size (referred to as neoadjuvant chemotherapy or neoadjuvant therapy), or after surgery to prevent tumor recurrence (referred to as postoperative adjuvant chemotherapy or adjuvant therapy).

[0208] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples. Furthermore, these examples should not be construed as limiting in any sense.

[0209] Production Example 1: Production of antibody-drug conjugate (1) According to the production method described in WO 2015 / 115091, a humanized anti-HER2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2; hereinafter referred to as "humanized anti-HER2 antibody (1)") was used to produce a conjugate of the formula

[0210]

[0211] An antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (1)") was produced in which an anti-HER2 antibody was linked to a drug linker represented by the formula: (wherein A represents the binding site to the antibody) via a thioether bond. The DAR of antibody-drug conjugate (1) was 7.7 or 7.8.

[0212] Production Example 2: Production of antibody-drug conjugate (2) According to the production method described in WO 2015 / 098099 and WO 2017 / 002776, a humanized anti-TROP2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 5 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6; hereinafter referred to as humanized anti-TROP2 antibody (1)) was used to produce a conjugate of the formula

[0213]

[0214] An antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (2)") was prepared in which an anti-TROP2 antibody was linked via a thioether bond to a drug linker represented by the formula: (wherein A represents the binding site to the antibody). The DAR of antibody-drug conjugate (2) can be adjusted within the range of 0 to 8, but in this study, antibody-drug conjugates with an average drug binding number of 3.5 to 4.5 were prepared.

[0215] Preparation Example 3: Preparation of Compound (A) Compound (A) of the formula

[0216]

[0217] A compound represented by the formula (compound (A)) was prepared.

[0218] Production Example 4: Preparation of anti-mouse SIRPα antibody (clone: ​​5C12) Anti-mouse SIRPα antibody 5C12 was established by the following method. Female WKY / Izm or Wistar rats (Japan SLC) were used for immunization. A mixture of mouse SIRPα protein (Sino Biological) and Freund's Complete Adjuvant (Wako Pure Chemical Industries, Ltd.) was subcutaneously administered to the base of the tail of the rats, and lymph nodes were collected and used to produce hybridomas.

[0219] Lymph node cells and mouse myeloma SP2 / 0-ag14 cells (ATCC: CRL-1581) were fused using the polyethylene glycol method, and the resulting cells were suspended in HAT selective medium and cultured by limiting dilution. Monoclonal hybridomas were produced by recovering the hybridoma colonies that emerged. Each recovered hybridoma colony was cultured, and antibody-producing hybridomas were screened using the resulting hybridoma culture supernatants based on their binding to mouse SIRPα protein, and 5C12 was selected.

[0220] To amplify the cDNA encoding the 5C12 variable region, total RNA was prepared from the 5C12-producing hybridoma using TRIzol Reagent (Ambion). Based on this, cDNA encoding the heavy and light chain variable regions was amplified using SMARTer RACE 5' / 3' Kit (Clontech). The cDNA encoding the heavy and light chain variable regions amplified by 5'-RACE PCR was cloned into a plasmid, and the nucleotide sequences of the cDNA encoding the heavy and light chain variable regions were then analyzed. The 5C12 heavy and light chain amino acid sequences are shown in SEQ ID NOs: 30 and 31.

[0221] The homology between humans and mice of SIRPα is low, at about 60%, and it has been reported that there are 10 types of variants in the IgV domain of SIRPα, which is the site of interaction with CD47, in humans and at least four types in mice due to differences in genetic background. For this reason, it is presumed to be difficult to obtain functional antibodies that are cross-reactive with human and mouse orthologues and can inhibit the SIRPα-CD47 interaction, and in fact, no functional antibodies cross-reactive with mice have been found in rats immunized with human antigens.

[0222] SIRPα is a molecule present in myeloid cells such as macrophages and dendritic cells. Drugs that exhibit direct anticancer activity can generally be evaluated using xenograft models in which human cancer cell lines are transplanted into immunodeficient mice. However, to evaluate the antitumor effect of target molecules expressed in host immune cells, such as SIRPα, one of the following methods must be used: (1) a model in which a genetically matched mouse cancer cell line is transplanted into immunocompetent mice, and a surrogate antibody cross-reactive with mouse SIRPα is used; (2) a model in which a human SIRPα gene is introduced into immunodeficient mice and a human cancer cell line is transplanted, and an anti-human SIRPα antibody is used; or (3) a mouse model in which a mouse cancer cell line expressing the human CD47 gene is transplanted into immunocompetent mice introduced with a SIRPα target gene, and an anti-human SIRPα antibody is used. Of these, for (2), immunodeficient mice lacking T cells are used, making it impossible to evaluate the impact of SIRPα inhibition on the adaptive immune system (immune responses centered on T cells). Furthermore, for (3), production takes time, making it difficult to predict the extent to which regulation of SIRPα gene expression levels, etc., will affect the anti-tumor effect. On the other hand, for (1), a surrogate antibody is used, but evaluation is possible using a general mouse model. Furthermore, the combined effects of clones such as MY-1 and P84 with anti-cancer antibodies and immune checkpoint antibodies have been reported (WO 2017 / 178653, or Yanagita et al. JCI Insight, 2017 (2) 1, 1-15). Therefore, in this experiment, the 5C12 clone was obtained as an anti-mouse SIRPα antibody, and the anti-tumor effect was evaluated. It has been confirmed that 5C12 has high binding affinity for the mouse SIRPα antigen, and that this affinity is comparable to the binding affinity for the human SIRPα antigen of the anti-human SIRPα antibody hD13_H1L3. Additionally, several PD-1 antibodies that have been marketed to date have also been evaluated using surrogate antibodies in non-clinical pharmacological studies, and their efficacy has subsequently been confirmed in clinical trials. Based on the above, the results of immunological evaluations using anti-SIRPα surrogate antibodies and evaluation results such as antitumor tests can be extrapolated to results in humans.

[0223] Production Example 5: Preparation of humanized anti-SIRPα antibody (clone: ​​hD13_H1L3) A humanized anti-SIRPα antibody (clone: ​​hD13_H1L3) was produced according to the production method described in Patent Document 4 (WO 2020 / 013170).

[0224] Production Example 6: Production of antibody-drug conjugate (3) According to the production method described in WO 2015 / 155998, a humanized anti-HER3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4; hereinafter referred to as "humanized anti-HER3 antibody (1)") was used to produce a conjugate of the formula

[0225]

[0226] An antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (3)") was prepared in which an anti-HER3 antibody was linked to a drug linker represented by the formula: (wherein A represents the binding site to the antibody) via a thioether bond. The DAR of antibody-drug conjugate (3) was 7.7 or 7.8.

[0227] Example 1: In Vitro ICD Induction Evaluation: Measurement of ATP / HMGB1 in Culture Supernatants. The expression levels of ATP and HMGB1, indicators of immunogenic cell death (ICD), were measured when cancer cells were treated with various compounds. Mouse colon cancer cell line CT26.WT cells were seeded into a 6-well plate and cultured overnight. The supernatant was removed and the cells were washed twice with PBS. Compound (A) was dissolved in RPMI 1640 culture medium (R10) containing 10% FBS to prepare solutions with final concentrations of 0, 1, 4, and 16 μM. As a positive control, mitoxantrone (MTX) was dissolved in DMSO and added to final concentrations of 0.4, 1.5, and 6 μM. The control (0 μM) contained the same amount of DMSO as when compound (A) was added (R10-DMSO). After 24 hours, the culture supernatant was collected, and ATP and HMGB1 were measured. ATP was measured by adding the culture supernatant to a 96-well white plate, followed by the addition of an equal volume of Backtiter-Glo (Promega), and measuring the luminescence level using a plate reader. HMGB1 was detected by sandwich ELISA using an HMGB1 ELISA kit (Shinotest). After adding test buffer to the provided plate, an HMGB1 standard solution diluted with test buffer and the culture supernatant were added, and the plate was left to stand at 37°C for 24 hours. The supernatant was removed, and the plate was washed five times with 200 μL / well of PBS / Tween 20. The provided secondary antibody solution was then added at 100 μL / well, and the plate was left to stand at room temperature for 2 hours. The supernatant was removed, and the plate was washed five times with 200 μL / well of PBS / Tween 20. The accompanying color-developing solution was added at 100 μL / well and allowed to stand at room temperature for 30 minutes. A stop solution was added at 100 μL / well, and after stirring, the absorbance at 405 nm was measured using a plate reader. Comparisons between the control group and the compound (A) group were performed using Dunnett's test. P values ​​were reported to four decimal places, and results meeting P<0.05 (two-sided test) were considered significant.

[0228] The results are shown in Figures 24A-D. In the MTX-treated group, a maximum of approximately 4-fold increase in ATP and approximately 3-fold increase in HMGB1 were observed in the culture supernatant after 24 hours compared to the control (0 µM) group (P<0.0001). In addition, in the compound (A)-treated group, a maximum of approximately 4.6-fold increase in ATP and HMGB1 was observed compared to R10 or R10-DMSO (P<0.0001).

[0229] From the above, it was suggested that compound (A) significantly increases the release of ATP and HMGB1, which are indices of ICD induction in vitro, from cancer cells.

[0230] Example 2: In vitro ICD induction evaluation: Measurement of calreticulin (CRT) on the cell surface. The expression level of calreticulin (CRT), an indicator of immunogenic cell death (ICD), was measured when cancer cells were treated with various compounds. Mouse colon cancer cell line CT26.WT cells were seeded in a 6-well plate and cultured overnight. The supernatant was removed and the cells were washed twice with PBS. Compound (A) was added at 4 μM or mitoxantrone (MTX) at 1 μM to RPMI1640 culture medium (R10) containing 10% FBS. As a control, DMSO was added in the same amount as compound (A) (R10-DMSO). After drug treatment, CT26.WT cells were collected, suspended in PBS, and then diluted to 1 × 10 ６The cells were seeded into a 96-well round-bottom plate at 100 μL / well. After centrifugation at 1200 rpm for 3 minutes, the supernatant was removed, and 100 μL / well of LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific) diluted 1 / 1000 with PBS and Mouse FcγR Blocker (Biolegend) diluted 1 / 50 were added. After allowing to stand at room temperature for 30 minutes, the plate was centrifuged at 1200 rpm for 3 minutes. The supernatant was removed, and 100 μL / well of mildform (Wako) was added, followed by incubation at 37°C for 10 minutes. After centrifugation, the supernatant was removed and the plate was washed once with 200 μL / well of FACS buffer (1 mM EDTA, 5% FBS). After centrifugation, the supernatant was removed and 50 μL / well of primary antibody solution (PE or AF647-labeled anti-CRT: both manufactured by Abcam) diluted with FACS buffer was added. After standing at 4°C for 25 minutes in the dark, the plate was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. The plate was washed twice with 200 μL / well of FACS buffer and centrifuged again, after which the supernatant was removed. The plate was suspended in Stabilizing Fixative (manufactured by Becton Dickinson) and left to stand at room temperature for 10 minutes. The expression level of CRT on cancer cells was measured using flow cytometry (FACS Canto II: manufactured by Becton Dickinson). FlowJo (manufactured by TreeStar) was used for data analysis. LIVE / DEAD Fixable Violet-positive cells were considered dead cells and excluded from the analysis. The mean fluorescence intensity (MFI) of CRT was calculated, and the adjusted MFI was calculated by subtracting the MFI of cells treated with the isotype control from the MFI of stained cells. The experiment was performed in triplicate. Comparison between the control group (0 μM) and each drug group was performed using Dunnett's test. P values ​​are reported to four decimal places, and P<0.05 (two-tailed test) was considered significant (***: P<0.001, **: P<0.01).

[0231] The results are shown in Figure 25. After 24 hours, a significant increase in CRT expression was observed on the surface of CT26.WT cells, approximately 2.6-fold (P<0.0001) in the MTX treatment and approximately 12.7-fold (P<0.0001) in the compound (A) treatment, compared to the respective control groups.

[0232] From the above, it was demonstrated that compound (A) promotes ICD induction in cancer cells in vitro by exposing CRT to the cell surface.

[0233] Example 3: In vivo ICD induction evaluation. Mouse colon cancer cell line CT26.WT cells were seeded in a cell culture flask and cultured overnight. The supernatant was removed and the cells were washed twice with PBS. To induce ICD, Compound (A) was added at 4 μM or MTX at 1 μM to RPMI 1640 culture medium (R10) containing 10% FBS. After 24 hours, HMGB1 in the culture supernatant was measured to confirm ICD induction using the method described in Example 1.

[0234] As shown in Figure 26, HMGB1 release was observed in the culture supernatant after 24 hours in each drug-treated group compared to R10. For in vivo ICD evaluation, the culture supernatant was removed from each drug-treated culture flask, washed twice with PBS, and then the cells were collected. After removing the supernatant and washing once with PBS, 5.0 x 10 cells were collected. ６ As a control, CT26.WT cells were cultured in RPMI1640 medium containing 10% FBS without any drug, and the suspension was prepared at 5.0 × 10 cells / mL. ６ After centrifugation and removal of the supernatant, the cells were subjected to freeze-thaw three times and resuspended in PBS to prepare necrotic cell death (NCD) cells.

[0235] 5.0 × 10 each of the aforementioned cells ６The cells were subcutaneously transplanted into the right axilla of 6-week-old female BALB / c mice (BALB / c AnNCrlCrlj) (Charles River Japan) (transplantation, Day 0). Anti-SIRPα antibody (clone: ​​5C12) or anti-CD47 antibody (clone: ​​MIAP410, Bio X Cell) was intraperitoneally administered at a dose of 10 mg / kg twice on Days 1 and 5. As a control, the same amount of PBS was administered. After 7 days, 3.0 × 10 untreated CT26.WT cells were transplanted. ６ The cells were subcutaneously transplanted into the left axilla of each mouse (retransplantation, Day 0), and the tumor volume was measured on Day 14 to assess the vaccination effect. Each group consisted of six mice. Comparisons between the NCD-PBS group and each group were performed using Dunnett's test, with P values ​​reported to four decimal places and P<0.05 (two-sided test) considered significant.

[0236] Figure 27A shows an overview of in vivo ICD-guided evaluation. Figure 27B shows tumor volume (mm ３ ), the horizontal axis indicates the name of each group. The MTX-treated group was set as a positive control, and the NCD-treated group was set as a negative control. Figure 27C shows an overview of each treatment group and the number of CR individuals. As shown in Figure 27B, when CT26.WT cells were re-implanted into the NCD group, tumor growth was observed. On the other hand, when CT26.WT cells were re-implanted into the MTX-PBS-administered group, tumor engraftment was rejected in 2 / 6 cases (CR). Furthermore, the number of CR individuals in the MTX-anti-SIRPα antibody-administered group was significantly higher (4 / 6) than in the NCD-administered group (P = 0.0486). Furthermore, when CT26.WT cells were re-implanted into the compound (A)-PBS group, tumor engraftment was rejected in 2 / 6 cases (CR). When WT cells were retransplanted, 5 / 6 cases achieved CR (P=0.0362), and all cases in the compound (A)-anti-SIRPα antibody administration group or the anti-CD47 antibody administration group achieved CR (P=0.0325 for both), indicating a significant increase in the number of CR individuals compared to NCD.

[0237] These results demonstrate that treatment of CT26.WT cells with compound (A) or MTX resulted in the release of HMGB1, an indicator of ICD, from the cells. Furthermore, transplantation of ICD-induced cells with these drugs into mice resulted in the formation of immune memory against tumors, demonstrating a vaccination effect. Furthermore, it was demonstrated that this vaccination effect was enhanced by administration of an anti-SIRPα antibody or an anti-CD47 antibody.

[0238] Example 4: ELISPOT analysis The number of CT26.WT-reactive T cells in mouse spleen cells was quantified by measuring the number of IFNγ spots produced by each spleen cell using Murine IFNγ Single-Color Enzymatic ELISPOT Assay (CELLULAR TECHNOLOGY). Spleens were removed from the mice used in Example 3, and spleen cells were diluted at 1.0 x 10 using CTL test medium. ６ CT26.WT cells were treated with 10 μg / mL mitomycin C for 2 hours, washed, and then collected and diluted to 1.0 × 10 cells / mL with CTL test medium. ６ The antigen was prepared at 100 μL / well of spleen cells and antigen, and co-cultured at 37°C for 24 hours. The plate was then stained with the antibody and coloring reagent provided, and the number of IFNγ-producing spleen cells was measured. Comparisons between the NCD cell transplant group and the compound (A) or MTX-treated group were performed using the Wilcoxon rank-sum test. P values ​​are reported to four decimal places, and P<0.05 (two-sided test) was considered significant.

[0239] The results are shown in Figure 28. Spleen cells from Compound (A)-treated CT26.WT cell transplanted-PBS administration group [Compound (A)_PBS] mice showed a tendency for an increased number of IFNγ-producing spleen cells compared to spleen cells from the NCD cell transplanted-PBS administration group [NCD_PBS]. Furthermore, spleen cells from Compound (A)_anti-SIRPα antibody mice showed a significant increase in the number of IFNγ-producing spleen cells compared to spleen cells from the NCD_PBS administration group (P=0.0043). A comparison between the Compound (A)_PBS and Compound (A)_αSIRPα groups also showed a significant increase in the number of IFNγ-producing spleen cells (P=0.013).

[0240] These results suggest that T cells that recognize antigens derived from CT26.WT cells are induced in compound (A)-treated mice, and this induction was significantly enhanced by administration of anti-SIRPα antibody. Furthermore, the induction of antigen-specific T cells tended to be more enhanced in the compound (A)-treated group than in the MTX-treated group.

[0241] Example 5: FCM analysis of spleen cells Spleens were removed from the mice used in Example 3, and spleen cells were prepared using PBS. ６The cells were seeded into a 96-well round-bottom plate at 100 μL / well. After centrifugation at 1200 rpm for 3 minutes, the supernatant was removed, and human FcγR Blocker (Biolegend) diluted 1 / 20 with PBS was added to the plate at 100 μL / well. The plate was left to stand at room temperature for 30 minutes, and then centrifuged at 1200 rpm for 3 minutes. The supernatant was removed, and 50 μL / well of primary antibody solution (FITC anti-CD3ε antibody, PerCP anti-CD4 antibody, PE / Cy7 anti-CD8α antibody, APC anti-CD62L antibody, and APC / Cy7 anti-CD44 antibody; all manufactured by Biolegend) diluted with FACS buffer (1 mM EDTA, 5% FBS) was added. After allowing to stand at 4°C for 25 minutes in the dark, the plate was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. The plate was then washed twice with 200 μL / well of FACS buffer. After centrifugation, the supernatant was removed, and the cells were suspended in Stabilizing Fixative (Becton Dickinson) and allowed to stand at room temperature for 10 minutes. Measurement was performed using flow cytometry (FACS Canto II: Becton Dickinson). Data analysis was performed using FlowJo (TreeStar). Comparisons between the NCD group and each drug treatment group, or comparisons between the PBS-administered group of each cell treatment group and the anti-SIRPα antibody group or anti-CD47 antibody group were performed using Dunnett's multiple comparison test, with P values ​​reported to four decimal places and P<0.05 (two-sided test) considered significant. Identification of each population of CD4-positive T cells and CD8-positive T cells was performed under the following conditions. Tcm: central memory T cells [CD44(+) CD62L(+)]

[0242] The results are shown in Figures 29 and 30. As shown in Figure 29A, the proportion of Tcm among CD4-positive T cells was significantly increased in the MTX-treated group (P = 0.0005) or compound (A)-treated group (P < 0.0001) compared to the NCD-treated group.

[0243] As shown in Figure 29B, the proportion of Tcm among CD8-positive T cells was significantly increased in the MTX-treated group (P = 0.0004) or compound (A)-treated group (P < 0.0001) compared to the NCD-treated group.

[0244] As shown in Figure 30A, the proportion of Tcm among CD4-positive T cells tended to increase in the MTX-anti-SIRPα antibody group and the MTX-anti-CD47 antibody group compared to the MTX-PBS group. Furthermore, compared to the Compound (A)-PBS group, the proportion tended to increase in the Compound (A)-anti-SIRPα antibody group, but was significantly decreased in the Compound (A)-anti-CD47 antibody group (P = 0.0002).

[0245] As shown in Figure 30B, the proportion of Tcm among CD8-positive T cells was significantly increased in the MTX-anti-SIRPα antibody group compared to the MTX-PBS group (P = 0.035), and tended to increase in the MTX-anti-CD47 antibody group. Furthermore, compared to the Compound (A)-PBS group, the Compound (A)-anti-SIRPα antibody group showed almost the same value, but was significantly decreased in the Compound (A)-anti-CD47 antibody group (P = 0.0017).

[0246] As shown in Figure 30C, the proportion of CD8-positive T cells among all cells was significantly increased in the MTX-treated anti-SIRPα antibody-administered group (P = 0.015) and tended to increase in the Compound (A)-anti-SIRPα antibody-administered group. Furthermore, the proportion of CD4-positive T cells among all cells tended to increase in the MTX-treated anti-SIRPα antibody-administered group and was significantly increased in the Compound (A)-treated anti-SIRPα antibody-administered group (P = 0.029).

[0247] When naive CD4+ T cells / CD8+ T cells are activated by antigen-specific recognition of cancer cells, they differentiate into CD4+ Teff cells, which are the command center of a broad immune response, or CD8+ Teff cells, which have the ability to kill target cells. Tcm cells are CD4 / CD8+ T cells that are formed from some Teff cells that maintain the ability to react to target cells and have acquired the ability to survive for a long period of time. These cells remember specific antigens they have encountered once, and when exposed to the same antigen again, they induce a rapid and efficient immune response.

[0248] The results of this study showed that treatment with MTX or compound (A) significantly increased long-term viable CD4 / CD8-positive Tcm (Figure 29), indicating the induction of a T cell population capable of maintaining anti-tumor immunity in vivo for a long period of time.

[0249] Furthermore, these effects were enhanced by administration of an anti-SIRPα antibody, demonstrating that the anti-SIRPα antibody, when used in combination with compound (A), has an advantageous effect in terms of inducing anti-tumor immunity.

[0250] The results of Examples 3, 4, and 5 demonstrated that compound (A) used in the present invention acts on tumor cells to induce ICD accompanied by increased production of HMGB1, ATP, etc., and increased cell surface expression of CRT, and that the induction of these ICD molecules promotes immune memory formation in vivo. Furthermore, these effects were enhanced by administration of anti-CD47 antibody and anti-SIRPα antibody, and the enhancement by administration of anti-SIRPα antibody was particularly significant, demonstrating that anti-SIRPα antibody, when used in combination with compound (A), has an advantageous effect in terms of inducing anti-tumor immunity.

[0251] Example 6: ADCP activity of antibody-drug conjugate of compound (A) and anti-human SIRPα antibody against cancer cell lines 6-1 Preparation of target cells CD47, HER2, TROP2-positive human gastric cancer cell line AGS cells were collected, washed twice with PBS, and then resuspended in PBS. The number of viable cells was counted by trypan blue dye exclusion test. 1 × 10 ６ 1 μL of CellTrace Far Red (Thermo Fisher Scientific) solution was added per 1 mL of cells / mL, and the mixture was left to stand at room temperature for 10 minutes. 20 mL of RPMI 1640 medium containing 10% FBS (R10 / Thermo Fisher Scientific) was added, and the mixture was left to stand for 5 minutes. 20 mL of R10 was added, and the mixture was washed twice, and then 1 × 10 ６ The cells were resuspended to 1000 cells / mL and used as target cells.

[0252] 6-2 Preparation of Effector Cells Ficoll-Paque PLUS (GE) was dispensed into SepMate-50 tubes (STEMCELL) at 15 mL / tube, and whole blood diluted 2-fold with 2% FBS-containing PBS was layered onto the tube at 17 mL / tube. The cells were centrifuged at 1200 g for 10 minutes at room temperature. The supernatant containing PBMCs was decanted into a new 50 mL tube and centrifuged at 1200 rpm for 8 minutes at room temperature. After removing the supernatant, the cells were washed twice by adding 25 mL of 2% FBS-containing PBS and centrifuging at 300 g for 8 minutes at room temperature. The cells were suspended in 1 mL of Robosep buffer (STEMCELL), and the viable PBMC count was measured using a trypan blue exclusion test. RoboSep buffer (STEMCELL) 5 × 10 ７ The PBMC cell suspension was adjusted to 100 cells / mL, and 50 μL of EasySep human monocyte enrichment cocktail included in the Human Monocyte Enrichment Kit Without CD16 Depletion (manufactured by STEMCELL) was added per mL of PBMC cell suspension. After incubation at 4°C for 10 minutes, 50 μL of EasySep Magnetic Particles were added per mL of PBMC cell suspension. After incubation at 4°C for 5 minutes, RoboSep buffer (manufactured by STEMCELL) was added to make the total volume 2.5 mL, and the mixture was placed in an EasySep Magnet. After 2 minutes and 30 seconds, the supernatant was collected and centrifuged at 1200 rpm for 5 minutes to separate the monocyte fraction. After washing once with R10, R10 containing 20 ng / mL M-CSF (manufactured by PEPROTEC) was added, and the cells were separated into 225 cm floatation tubes. ２ The cells were seeded in flasks (manufactured by Sumitomo Bakelite Co., Ltd.) at 37°C and 5% CO ２ The cells were cultured for 11 days under the conditions of 1. The culture supernatant was removed, and R10 containing 20 ng / mL IL-10 and 20 ng / mL M-CSF (PEPROTEC) was added, followed by further culture for 2 days. After 13 days, the differentiation-induced macrophages were added with TrypLE Express (Life Technology), incubated at 37°C for 15 minutes, and then detached. 25 mL of R10 was added and the cells were collected. After washing twice with PBS, 1 x 10 ６The cells were resuspended in PBS to a concentration of 1 μL / 10 cells / mL. ６ 1 × 10 cells / mL CFSE solution (ThermoFisher) was added and left to stand at room temperature for 10 minutes. After washing twice with 20 ml of R10, 1 × 10 cells / mL CFSE solution (ThermoFisher) was added and left to stand at room temperature for 10 minutes. ６ The cells were resuspended to 1000 cells / mL and used as effector cells.

[0253] 6-3 Evaluation of ADCP Activity Target cells prepared by the method described in 6-1 were added at 50 μL / well to a 96-well U-bottom microplate with an ultra-low attachment surface (Sumitomo Bakelite Co., Ltd.). Antibody-drug conjugate (1) or (2), humanized anti-HER2 (1) antibody, humanized anti-TROP2 antibody (1), or various control antibodies diluted with R10 to a final concentration of 0 to 1000 ng / mL were added at 50 μL / well. R10 was added at 50 μL / well to the single-agent treatment group, and humanized anti-SIRPα antibody (clone: ​​hD13_H1L3) diluted with R10 to a final concentration of 2000 ng / mL was added at 50 μL / well to the combination treatment group. In another example, antibody-drug conjugate (1) or (2), anti-HER2 antibody or anti-TROP2 antibody, and various control antibodies diluted with R10 to a final concentration of 1000 ng / mL were added at 50 μL / well. 50 μL of R10 was added to the single agent group, and 50 μL of anti-SIRPα antibody diluted with R10 to a final concentration of 0 to 2000 ng / mL was added to the combination group. 1 × 10 ６ After adding effector cells at 50 μL / well, the cells were incubated at 37°C, 5% CO ２The cells were then left to stand for 4 hours under these conditions. After centrifugation at 1200 rpm for 5 minutes at 4°C and removal of the supernatant, the cells were washed with 200 μL / well of FACS buffer. The cells were suspended in 50 μL / well of 1×BD Stabilizing Fixative (Becton Dickinson) and left to stand at room temperature for 10 minutes. Measurement was performed using flow cytometry (AttuneNxT: Thermo Fisher Scientific). Data analysis was performed using FlowJo (TreeStar). After development using FSC (forward scattered light) / SSC (side scattered light), the number of cells that were APC-positive (A) and APC- and FITC-positive (B) was calculated. Cells that were positive for both APC and FITC (B) were considered to have been target cells phagocytosed by effector cells. The cell phagocytosis rate due to ADCP activity was calculated using the following formula:

[0254] Cell phagocytosis rate (%)=B / (A+B)×100

[0255] As shown in Figures 31A-D, antibody-drug conjugates (1) and (2) exhibited ADCP activity against CD47-, HER2-, and TROP2-positive human gastric cancer cell line AGS cells that was dependent on the antibody concentration added (Figures 31A and 31B), and showed higher ADCP activity when used in combination with humanized anti-SIRPα antibody hD13_H1L3 (Figures 31A and 31B). Furthermore, these effects were not observed with the Control antibody-drug conjugate, indicating that both effects are target-specifically induced. Furthermore, the ADCP activity was comparable to or greater than that of the parent antibodies, humanized anti-HER2 antibody (1) or humanized anti-TROP2 antibody (1), which are not drug-conjugated, suggesting that there is no negative effect of the drug conjugate. On the other hand, as shown in Figures 31C and 31D, even when the concentration of the antibody-drug conjugate (1) or (2) was fixed and the concentration of the humanized anti-SIRPα antibody hD13_H1L3 was examined, a concentration-dependent enhancement of ADCP activity of the added anti-SIRPα antibody was observed.

[0256] Example 7: Antitumor test (1) Mice: Six-week-old female BALB / c mice (BALB / c AnNCrlCrlj) (Charles River Japan) were used in the experiment.

[0257] Measurement and calculation formula: The major and minor diameters of the tumor were measured twice a week using an electronic digital caliper (CD15-CX, manufactured by Mitutoyo Corporation), and the tumor volume (mm ３ ) was calculated using the following formula:

[0258] Tumor volume (mm ３ ) = 0.5 × major axis (mm) × [minor axis (mm)] ２

[0259] Tumor volume 3000 mm ３ Any individuals exceeding this limit were euthanized from the standpoint of animal experiment ethics.

[0260] The antibody-drug conjugate (1) (drug-to-antibody ratio: 7.6) was diluted with PBS and administered intravenously at a dose of 10 mL / kg. The anti-mouse SIRPα antibody (clone: ​​5C12) and anti-PD-L1 antibody (clone: ​​YW243.55S70, prepared according to U.S. Patent Publication US 2013 / 0045201 A1) were diluted with PBS and administered intraperitoneally at a dose of 10 mL / kg.

[0261] 4T1-hmHER2 cells were used, which were prepared by introducing the human / mouse chimeric HER2 gene into the mouse breast cancer cell line 4T1 (CRL-2539) purchased from American Type Culture Collection using a lentiviral vector. These cells express the human / mouse chimeric HER2 protein on the cell membrane. 4T1-hmHER2 cells were suspended in PBS and cultured at a concentration of 1.0 x 10 ６The cells were subcutaneously transplanted into the right axilla of BALB / c mice (Day 0), and four days later, mice were randomly assigned to groups (Day 4). Antibody-drug conjugate (1) was administered twice, at a dose of 10 mg / kg, via the tail vein on Days 4 and 11. Anti-SIRPα antibody (clone: ​​5C12) or anti-PD-L1 antibody (clone: ​​YW243.55S70) was administered intraperitoneally three times, at a dose of 10 mg / kg, on Days 5, 8, and 12. A group receiving a combination of antibody-drug conjugate (1), anti-SIRPα antibody, and anti-PD-L1 antibody, and a control group receiving PBS were also included. Each group contained five mice, and tumor volume was measured until Day 21. Comparisons between the control group and each single agent or combination administration group, or between the antibody-drug conjugate (1) group and each combination group were performed using Dunnett's multiple comparison method, and P values ​​were reported to four decimal places, with P<0.05 (two-sided test) considered significant.

[0262] The results are shown in Figure 32. Figure 32A shows an outline of the antitumor test. Figure 32B shows the tumor growth curves for each administration group, with the vertical axis plotting tumor volume (mm ３ ), the horizontal axis indicates the number of days from the date of tumor implantation. Figure 32C shows an overview of each treatment group, tumor growth inhibition (TGI / %), and the number of CR cases. At day 21, compared to the control group, the antibody-drug conjugate (1) group (P<0.0001) and the anti-SIRPα antibody group (P=0.0087) showed significantly superior anti-tumor effects. Compared to the control group, the antibody-drug conjugate (1) + anti-SIRPα antibody combination group and the triple-drug combination group showed significantly superior anti-tumor effects (both P<0.0001). Furthermore, compared to the antibody-drug conjugate (1) group, the antibody-drug conjugate (1) + anti-SIRPα antibody combination group (P=0.0497) and the triple-drug combination group (P=0.0237) showed significantly superior anti-tumor effects. Furthermore, no weight loss was observed in mice in any of the groups in this study. From the above, the single administration of the antibody-drug conjugate (1) and the anti-SIRPα antibody demonstrated antitumor effects, and the combined use of these agents significantly enhanced these effects.

[0263] Example 8: Antitumor test (2) Mice: Six-week-old female BALB / c mice (BALB / c AnNCrlCrlj) (Charles River Japan) were used in the experiment.

[0264] Measurement and calculation formula: The major and minor diameters of the tumor were measured twice a week using an electronic digital caliper (CD15-CX, manufactured by Mitutoyo Corporation), and the tumor volume (mm ３ ) was calculated using the following formula:

[0265] Tumor volume (mm ３ ) = 0.5 × major axis (mm) × [minor axis (mm)] ２

[0266] Tumor volume 3000 mm ３ Any animals exceeding this number were euthanized from the standpoint of animal experiment ethics.

[0267] The antibody-drug conjugate (2) (drug-to-antibody ratio: 4) was diluted with PBS and administered intravenously at a dose of 10 mL / kg. The anti-SIRPα antibody (5C12) was diluted with PBS and administered intraperitoneally at a dose of 10 mL / kg.

[0268] CT26.WT-hTROP2 cells were prepared by introducing the human TROP2 gene into the mouse colon cancer cell line CT26.WT (CRL2638) purchased from American Type Culture Collection using a lentiviral vector. These cells express human TROP2 protein on the cell membrane. CT26.WT-hTROP2 cells were suspended in saline and cultured at 2.0 x 10 ６Cells were subcutaneously transplanted into the right axilla of BALB / c mice (Day 0), and seven days later, mice were randomly assigned to groups (Day 7). Antibody-drug conjugate (2) was administered twice via the tail vein at a dose of 10 mg / kg on Days 7 and 12. Anti-SIRPα antibody (clone: ​​5C12) was administered intraperitoneally three times at a dose of 10 mg / kg on Days 8, 11, and 13. A group administered a combination of antibody-drug conjugate (2) and anti-SIRPα antibody, and a control group administered PBS, were also included. Each group contained six mice, and tumor volumes were measured up to Day 18. Comparisons between the control group and each single-agent and combination group were performed using Dunnett's multiple comparison method. P values ​​are reported to four decimal places, and P<0.05 (two-tailed test) was considered significant.

[0269] The results are shown in Figure 33. Figure 33A shows an outline of the antitumor test. Figure 33B shows the tumor growth curves for each administration group, with the vertical axis plotting tumor volume (mm ３ The horizontal axis indicates the number of days from the day of tumor implantation. Figure 33C shows an overview of each treatment group, tumor growth inhibition (TGI / %). The vertical axis indicates tumor volume (mm ３ ), the horizontal axis indicates the number of days from the first administration. At Day 18, compared to the control group, the antibody-drug conjugate (2) group and the anti-SIRPα antibody group showed partial antitumor effects. Furthermore, the combination group showed significantly superior antitumor effects (P = 0.0007). Furthermore, no weight loss was observed in mice in any of the groups in this study. From the above, the antitumor effects of both drugs were observed when administered alone, and the effects were enhanced by the combined use of both drugs.

[0270] Example 9: Antitumor test (3) Mice: Six-week-old female BALB / c mice (BALB / c AnNCrlCrlj) (Charles River Japan) were used in the experiment.

[0271] Measurement and calculation formula: The major and minor diameters of the tumor were measured twice a week using an electronic digital caliper (CD15-CX, manufactured by Mitutoyo Corporation), and the tumor volume (mm ３ ) was calculated using the following formula:

[0272] Tumor volume (mm ３) = 0.5 × major axis (mm) × [minor axis (mm)] ２

[0273] Tumor volume 3000 mm ３ Any animals exceeding this number were euthanized from the standpoint of animal experiment ethics.

[0274] The antibody-drug conjugate (3) (drug-to-antibody ratio: 8) and the anti-SIRPα antibody (5C12) were diluted with PBS and used for administration.

[0275] CT26.WT-hHER3 cells were used, which were prepared by introducing the human HER3 gene into the mouse colon cancer cell line CT26.WT (CRL2638) purchased from American Type Culture Collection using a lentiviral vector. These cells express human HER3 protein on the cell membrane. CT26.WT-hHER3 cells were suspended in physiological saline and cultured at 2.0 x 10 ６ Cells were subcutaneously transplanted into the right axilla of BALB / c mice (Day 0), and 7 days later, mice were randomly assigned to groups (Day 7). Antibody-drug conjugate (3) was administered twice via the tail vein at a dose of 10 mg / kg on Days 7 and 14. Anti-SIRPα antibody (clone: ​​5C12) was administered intraperitoneally at a dose of 10 mg / kg three times on Days 8, 11, and 15. A group receiving a combination of antibody-drug conjugate (3) and an anti-SIRPα antibody and a control group (PBS) were also included. Each group contained six mice, and tumor volumes were measured up to Day 18. Comparisons between the control group and each single-agent and combination group were performed using Dunnett's multiple comparison method. P values ​​are reported to four decimal places, and P<0.05 (two-sided test) was considered significant.

[0276] The results are shown in Figure 34. Figure 34A shows an outline of the antitumor test. Figure 34B shows the tumor growth curves for each administration group, with the vertical axis plotting tumor volume (mm ３), the horizontal axis indicates the number of days from the date of tumor implantation. Figure 34C shows an overview of each treatment group, showing tumor growth inhibition (TGI / %). At Day 18, compared to the control group, the anti-SIRPα antibody single-agent group showed no efficacy, while the antibody-drug conjugate (3) group showed a partial anti-tumor effect. Furthermore, the combined use of both drugs showed a significantly enhanced anti-tumor effect compared to the anti-SIRPα antibody single-agent group (P = 0.046) and showed a tendency toward enhanced anti-tumor effect compared to the antibody-drug conjugate (3) group. Furthermore, no weight loss was observed in mice in any of the groups in this study.

[0277] From the above experimental results, it was found that the antibody-drug conjugate according to the present invention exhibits excellent anti-tumor effects when administered in combination with an anti-SIRPα antibody.

[0278] SEQ ID NO: 1: Amino acid sequence of anti-HER2 antibody heavy chain SEQ ID NO: 2: Amino acid sequence of anti-HER2 antibody light chain SEQ ID NO: 3: Amino acid sequence of anti-HER3 antibody heavy chain SEQ ID NO: 4: Amino acid sequence of anti-HER3 antibody light chain SEQ ID NO: 5: Amino acid sequence of anti-TROP2 antibody heavy chain SEQ ID NO: 6: Amino acid sequence of anti-TROP2 antibody light chain SEQ ID NO: 7: Amino acid sequence of anti-B7-H3 antibody heavy chain SEQ ID NO: 8: Amino acid sequence of anti-B7-H3 antibody light chain SEQ ID NO: 9: Amino acid sequence of anti-GPR20 antibody heavy chain SEQ ID NO: 10: Amino acid sequence of anti-GPR20 antibody light chain SEQ ID NO: 11: Amino acid sequence of anti-CDH6 antibody heavy chain SEQ ID NO: 12: Amino acid sequence of anti-CDH6 antibody light chain SEQ ID NO: 13: Amino acid sequence of hH1 heavy chain of humanized anti-SIRPα antibody hD13 SEQ ID NO: 14: Amino acid sequence of hH2 heavy chain of humanized anti-SIRPα antibody hD13 SEQ ID NO: 15: Amino acid sequence of the hL2 light chain of humanized anti-SIRPα antibody hD13 SEQ ID NO: 16: Amino acid sequence of the hL3 light chain of humanized anti-SIRPα antibody hD13 SEQ ID NO: 17: Amino acid sequence of the hL4 light chain of humanized anti-SIRPα antibody hD13 SEQ ID NO: 18: Amino acid sequence of CDR-H1 of chimeric SIRPα antibody cD13 SEQ ID NO: 19: Amino acid sequence of CDR-H2 of chimeric SIRPα antibody cD13 SEQ ID NO: 20: Amino acid sequence of CDR-H3 of chimeric SIRPα antibody cD13 SEQ ID NO: 21: Amino acid sequence of CDR-L1 of chimeric SIRPα antibody cD13 SEQ ID NO: 22: Amino acid sequence of CDR-L2 of chimeric SIRPα antibody cD13 SEQ ID NO: 23: Amino acid sequence of CDR-L3 of chimeric SIRPα antibody cD13 SEQ ID NO: 24: Amino acid sequence of OSE-172 antibody heavy chain (OSE-172_hG4Pro) SEQ ID NO: 25: Amino acid sequence of OSE-172 antibody light chain (OSE-172_hK) SEQ ID NO: 26: Amino acid sequence of KWAR23 antibody heavy chain (KWAR23_hG4Pro) SEQ ID NO: 27: Amino acid sequence of KWAR23 antibody light chain (KWAR23_hK) SEQ ID NO: 28: Amino acid sequence of ADU-1805 antibody heavy chain (ADU-1805_hG2) SEQ ID NO: 29: Amino acid sequence of ADU-1805 antibody light chain (ADU-1805_hK) SEQ ID NO: 30: Amino acid sequence of 5C12 anti-mouse SIRPα antibody heavy chain SEQ ID NO: 31: Amino acid sequence of 5C12 anti-mouse SIRPα antibody light chainSEQ ID NO: 32: Amino acid sequence of the heavy chain of the YW243.55S70 anti-mouse / human anti-PD-L1 antibody SEQ ID NO: 33: Amino acid sequence of the light chain of the YW243.55S70 anti-mouse / human anti-PD-L1 antibody

Claims

1. A pharmaceutical combination containing an antibody-drug conjugate and an anti-SIRPα antibody, in which An antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker compound represented by the following formula is conjugated to the antibody via a thioether bond: where A represents the binding position of the antibody, and wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, or an anti-TROP2 antibody.

2. The pharmaceutical combination according to claim 1, wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

3. The pharmaceutical combination according to claim 2, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1, and a light chain consisting of an amino acid sequence consisting of amino acid residues 1-214 of SEQ ID NO:

2.

4. The pharmaceutical combination according to claim 2, wherein the anti-HER2 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence presented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence presented by SEQ ID NO:

2.

5. The pharmaceutical combination according to any one of claims 2 to 4, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

6. The pharmaceutical combination according to claim 1, wherein the antibody in the antibody-drug conjugate is an anti-HER3 antibody.

7. The pharmaceutical combination according to claim 6, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence presented by SEQ ID NO: 3 and a light chain consisting of the amino acid sequence presented by SEQ ID NO:

4.

8. The pharmaceutical combination according to claim 6, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1-446 of SEQ ID NO: 3, and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

4.

9. The pharmaceutical combination according to any one of claims 6 to 8, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 7 to 8.

10. The pharmaceutical combination of claim 1, wherein the antibody in the antibody-drug conjugate is an anti-TROP2 antibody.

11. The pharmaceutical combination according to claim 10, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-470 of SEQ ID NO: 5, and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO:

6.

12. The pharmaceutical combination according to claim 10, wherein the anti-TROP2 antibody is an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-469 of SEQ ID NO: 5, and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO:

6.

13. The pharmaceutical combination according to any one of claims 10 to 12, wherein the average number of conjugated drug-linker units per antibody molecule in the antibody-drug conjugate is in the range of 3.5 to 4.

5.

14. The pharmaceutical combination according to any one of claims 1 to 13, wherein the anti-SIRPα antibody is any of the following antibodies (1) to (5): (1) an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 13 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 16; (2) an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 13 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 17; (3) an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 14 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 15; (4) an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 14 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 16; and (5) an antibody according to any one of (1) to (4) that does not contain a lysine residue at the carboxyl terminus of the heavy chain.

15. The pharmaceutical combination according to any one of claims 1 to 13, wherein the anti-SIRPα antibody is any of the following antibodies (1) and (2): (1) an antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 13 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21-234 of SEQ ID NO: 16; and (2) an antibody according to (1) that does not contain a lysine residue at the carboxyl terminus of the heavy chain.

16. A pharmaceutical combination according to any one of claims 1 to 15, wherein the antibody-drug conjugate and the anti-SIRPα antibody are separately contained as active components in different compositions.

17. A pharmaceutical combination according to any one of claims 1 to 15, wherein the antibody-drug conjugate and the anti-SIRPα antibody are separately contained as active components in a single composition.

18. Use of an antibody-drug conjugate for the preparation of a medicament for the treatment of cancer, wherein the antibody-drug conjugate and the anti-SIRPα antibody are intended to be administered in combination, and the antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker compound represented by the following formula is conjugated to the antibody via a thioether bond: where A represents the binding position of the antibody, and wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, or an anti-TROP2 antibody.

19. The use according to claim 18, wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody as defined in any of claims 2 to 13.

20. The use according to claim 18 or 19, wherein the anti-SIRPα antibody is an antibody as defined in claim 14 or 15.

21. The use according to any one of claims 18 to 20, wherein the antibody-drug conjugate and the anti-SIRPα antibody are separately contained as active components in different compositions and are administered simultaneously or at different times.

22. The use according to any one of claims 18 to 20, wherein the antibody-drug conjugate and the anti-SIRPα antibody are separately contained as active components in a single composition.

23. The use according to any one of claims 18 to 22, wherein the use is for the treatment of at least one selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, biliary tract cancer, Paget's disease, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, uterine cancer, sarcoma, head and neck cancer, hepatocellular cancer, cervical cancer, brain tumor, glioma, eye tumor, thyroid cancer, thymus cancer, gallbladder cancer, lymphoma, leukemia and myelodysplastic syndrome.

24. The use according to any one of claims 18 to 22, wherein the cancer is at least one selected from the group consisting of breast cancer, gastric cancer and colorectal cancer.

25. A method of treating cancer, comprising administering an antibody-drug conjugate and an anti-SIRPα antibody in combination to a subject in need of treatment, wherein the antibody-drug conjugate is an antibody-drug conjugate in which a drug-linker compound represented by the following formula is conjugated to the antibody via a thioether bond: where A represents the binding position of the antibody, and where the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, or an anti-TROP2 antibody.

26. The method of claim 25, wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody as defined in any of claims 2-13.

27. The method according to claim 25 or 26, wherein the anti-SIRPα antibody is an antibody as defined in claim 14 or 15.

28. The method according to any one of claims 25-27, wherein the antibody-drug conjugate and the anti-SIRPα antibody are separately contained as active components in different compositions and are administered simultaneously or at different times.

29. The method according to any one of claims 25-27, wherein the antibody-drug conjugate and the anti-SIRPα antibody are separately contained as active components in a single composition.

30. The method according to any one of claims 25 to 29, wherein the cancer is a cancer as defined in claim 23 or 24.

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