Combination of antibody-drug conjugates and immune checkpoint inhibitors

Abstract

To provide a pharmaceutical composition and a treatment method having excellent antitumor effect and safety.SOLUTION: The present invention provides a pharmaceutical composition and a treatment method, characterized in that an antibody-drug conjugate in which a drug linker represented by the following formula (where A represents the antibody binding site) is bound to an antibody via a thioether bond and an immune checkpoint inhibitor are administered in combination; as well as a pharmaceutical composition and a method of treatment, characterized in that the antibody-drug conjugate is contained or used for treating a disease that is improved by the action of activating anti-tumor immunity.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition and a therapeutic method characterized by the administration of a combination of a specific antibody-drug conjugate and an immune checkpoint inhibitor, and to a pharmaceutical composition and a therapeutic method characterized by the use of a specific antibody-drug conjugate in the treatment of a disease that is improved by activating antitumor immunity. [Background technology]

[0002] Antibody-drug conjugates (ADCs), which involve attaching a cytotoxic drug to an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized within the cell, are expected to selectively deliver the drug to cancer cells, thereby accumulating the drug within the cancer cells and ultimately killing them (Non-patent documents 1-5).

[0003] One type of antibody-drug conjugate is one in which an antibody and the topoisomerase I inhibitor exatecan are components (Patent Documents 1-7). Of these, anti-HER2 antibody-drug conjugates (Non-Patent Documents 6, 7), which have particularly excellent antitumor effects and safety, are currently undergoing clinical trials.

[0004] Immune checkpoint inhibitors are drugs that inhibit the immunosuppressive system and activate anti-tumor immunity (Non-Patent Documents 8-10). Known immune checkpoint inhibitors include the anti-PD-1 antibodies nivolumab (Patent Document 8) and pembrolizumab (Patent Document 9), the anti-PD-L1 antibodies atezolizumab (Patent Document 10), durvalumab (Patent Document 11), and avelumab (Patent Document 12), and the anti-CTLA-4 antibodies ipilimumab (Patent Document 13) and tremelimumab (Patent Document 14).

[0005] An example of a combined administration of antibody-drug conjugates and immune checkpoint inhibitors is the study on the combined use of trastuzumab emtansine (T-DM1) and an anti-CTLA-4 / PD-1 antibody (Non-Patent Literature 11). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2014 / 057687 [Patent Document 2] International Publication No. 2014 / 061277 [Patent Document 3] International Publication No. 2015 / 098099 [Patent Document 4] International Publication No. 2015 / 115091 [Patent Document 5] International Publication No. 2015 / 146132 [Patent Document 6] International Publication No. 2015 / 155976 [Patent Document 7] International Publication No. 2015 / 155998 [Patent Document 8] International Publication No. 2006 / 121168 [Patent Document 9] International Publication No. 2008 / 156712 [Patent Document 10] International Publication No. 2010 / 077634 [Patent Document 11] International Publication No. 2011 / 066389 [Patent Document 12] International Publication No. 2013 / 079174 [Patent Document 13] International Publication No. 2001 / 014424 [Patent Document 14] International Publication No. 2000 / 037504 [Non-patent literature]

[0007]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0008] The present invention aims to provide a pharmaceutical composition and treatment method that have excellent antitumor effects and safety when an antibody-drug conjugate and an immune checkpoint inhibitor are administered in combination. Furthermore, the present invention aims to provide a pharmaceutical composition and treatment method characterized by its use in the treatment of diseases that are improved by activating antitumor immunity, and which contains a specific antibody-drug conjugate. [Means for solving the problem]

[0009] The inventors have discovered that administering a combination of a specific antibody-drug conjugate and an immune checkpoint inhibitor exhibits excellent antitumor effects. Furthermore, they have found that this antibody-drug conjugate has the effect of activating antitumor immunity.

[0010] In other words, the present invention is [1] A pharmaceutical composition characterized by the administration of an antibody-drug conjugate and an immune checkpoint inhibitor in combination, The antibody-drug conjugate is,

[0011] [ka]

[0012] (In the formula, A indicates the binding site with the antibody.) A pharmaceutical composition comprising an antibody-drug conjugate in which a drug linker, represented by [the symbol], and an antibody are linked by 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, or an anti-B7-H3 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 [2] or [3], wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214. [5] The pharmaceutical composition according to [2] or [3], wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2. [6] A pharmaceutical composition according to any one of claims [1] to [5], wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8. [7] A pharmaceutical composition according to any one of claims [1] 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. [8] A pharmaceutical composition according to any one of claims [1] to [5], wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8. [9] A pharmaceutical composition according to any one of [1] to [8], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[10] The pharmaceutical composition according to [9], wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

[11] The pharmaceutical composition according to [9], wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[12] The pharmaceutical composition according to [9], wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[13] A pharmaceutical composition according to any one of [1] to

[12] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are each contained as active ingredients in separate formulations and administered simultaneously or at different times.

[14] A pharmaceutical composition according to any one of [1] to

[12] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are contained as active ingredients in a single formulation and administered.

[15] A pharmaceutical composition according to any one of items [1] to

[14] for the treatment of cancer.

[16] The pharmaceutical composition according to

[15] , wherein the cancer is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[17] The pharmaceutical composition according to

[16] , wherein the cancer is colorectal cancer.

[18] The pharmaceutical composition according to

[16] , wherein the cancer is breast cancer.

[19] A pharmaceutical composition according to any one of [1] to

[18] , wherein the antibody-drug conjugate has the effect of activating antitumor immunity.

[20] Antibody-drug conjugates, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, A pharmaceutical composition according to any one of claims [1] to

[19] , having at least one action selected from the group consisting of the following. [twenty one] A pharmaceutical composition according to any one of [1] to

[20] , wherein the antibody-drug conjugate has the effect of promoting the formation of immunological memory against a tumor. [twenty two] The pharmaceutical composition according to

[21] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate. [twenty three] The pharmaceutical composition according to

[21] , wherein a portion of the tumor does not express the antigen for the antibody in the antibody-drug conjugate. [twenty four] Antibody-drug conjugates, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A pharmaceutical composition according to any one of [1] to

[23] , having at least one action selected from the group consisting of the following. [twenty five] The pharmaceutical composition according to any one of [1] to

[24] , characterized in that the antibody-drug conjugate exhibits a stronger antitumor effect when an immune checkpoint inhibitor cancels an immunosuppressive signal generated by the antibody-drug conjugate promoting an increase in PD-L1 expression on cancer cells.

[26] formula

[0013] [ka]

[0014] (In the formula, A indicates the binding site with the antibody.) A pharmaceutical composition characterized by containing an antibody-drug conjugate in which a drug linker and an antibody are linked by a thioether bond, and used for the treatment of diseases that are improved by activating antitumor immunity.

[27] Antibody-drug conjugates, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, A pharmaceutical composition according to

[26] having at least one action selected from the group consisting of the following.

[28] The pharmaceutical composition according to

[26] or

[27] , wherein the antibody-drug conjugate has the effect of promoting the formation of immunological memory against tumors.

[29] The pharmaceutical composition according to claim

[28] , wherein the tumor expresses an antigen for the antibody in the antibody-drug conjugate.

[30] The pharmaceutical composition according to

[28] , wherein a portion of the tumor does not express the antigen for the antibody in the antibody-drug conjugate.

[31] Antibody-drug conjugates, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A pharmaceutical composition according to any one of claims

[26] to

[30] , having at least one action selected from the group consisting of the following.

[32] A pharmaceutical composition according to any one of

[26] to

[31] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[33] The pharmaceutical composition according to

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

[34] The pharmaceutical composition according to

[32] or

[33] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[35] The pharmaceutical composition according to

[32] or

[33] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[36] A pharmaceutical composition according to any one of

[26] to

[35] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[37] A pharmaceutical composition according to any one of

[26] to

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

[38] A pharmaceutical composition according to any one of

[26] to

[35] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[39] A pharmaceutical composition according to any one of

[26] to

[38] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[40] The pharmaceutical composition described in

[39] , wherein the disease is colorectal cancer.

[41] The pharmaceutical composition described in

[39] , wherein the disease is breast cancer.

[42] formula

[0015] [ka]

[0016] A pharmaceutical composition characterized by its use in the treatment of diseases that are improved by releasing a compound represented by within a tumor and activating anti-tumor immunity.

[43] The compound, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, The pharmaceutical composition according to

[42] , having at least one action selected from the group consisting of the following.

[44] The pharmaceutical composition according to

[42] or

[43] , wherein the compound has the effect of promoting the formation of immunological memory against tumors.

[45] The compound, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A pharmaceutical composition according to any one of claims

[42] to

[44] , having at least one action selected from the group consisting of the following.

[46] A pharmaceutical composition according to any one of

[42] to

[45] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[47] A therapeutic method characterized by the administration of an antibody-drug conjugate and an immune checkpoint inhibitor in combination, The antibody-drug conjugate is,

[0017] [ka]

[0018] (In the formula, A indicates the binding site with the antibody.) A therapeutic method in which a drug linker, indicated by [the symbol], and an antibody are linked by a thioether bond to form an antibody-drug conjugate.

[48] The therapeutic method according to

[47] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[49] The therapeutic method described in

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

[50] The therapeutic method according to

[48] or

[49] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[51] The therapeutic method according to

[48] or

[49] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[52] A therapeutic method according to any one of the articles

[47] to

[51] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[53] A therapeutic method according to any one of paragraphs

[47] to

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

[54] A therapeutic method according to any one of paragraphs

[47] to

[51] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[55] The treatment method according to any one of paragraphs

[47] to

[54] , wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[56] The treatment method described in

[55] , wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

[57] The treatment method described in

[55] , wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[58] The treatment method described in

[55] , wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[59] A therapeutic method according to any one of paragraphs

[47] to

[58] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are each contained as active ingredients in separate formulations and administered simultaneously or at different times.

[60] A therapeutic method according to any one of

[47] to

[58] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are contained as active ingredients in a single formulation and administered.

[61] A treatment method for cancer as described in any one of paragraphs

[47] to

[60] .

[62] The treatment method according to

[61] , wherein the cancer is selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[63] The cancer is colorectal cancer, and the treatment method is as described in

[62] .

[64] The cancer is breast cancer, and the treatment method is as described in

[62] .

[65] A therapeutic method according to any one of paragraphs

[47] to

[64] , wherein the antibody-drug conjugate has the effect of activating antitumor immunity.

[66] Antibody-drug conjugates, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, A pharmaceutical composition according to any one of the claims

[47] to

[65] , having at least one action selected from the group consisting of the following.

[67] A therapeutic method according to any one of paragraphs

[47] to

[66] , wherein the antibody-drug conjugate has the effect of promoting the formation of immunological memory against a tumor.

[68] The treatment method described in

[67] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate.

[69] A treatment method according to

[67] in which a portion of the tumor does not express the antigen for the antibody in the antibody-drug conjugate.

[70] Antibody-drug conjugates, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A treatment method according to any one of the items

[47] to

[69] , having at least one action selected from the group consisting of the following.

[71] A therapeutic method according to any one of

[47] to

[70] , characterized in that the antibody-drug conjugate exhibits a stronger antitumor effect when an immune checkpoint inhibitor cancels an immunosuppressive signal generated by the antibody-drug conjugate promoting an increase in PD-L1 expression on cancer cells.

[72] formula

[0019] [ka]

[0020] (In the formula, A indicates the binding site with the antibody.) A treatment method characterized by administering an antibody-drug conjugate, in which a drug linker and an antibody are linked by a thioether bond, for diseases that are improved by activating antitumor immunity.

[73] Antibody-drug conjugates, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, A therapeutic method according to

[72] , having at least one action selected from the group consisting of the following.

[74] The therapeutic method according to

[72] or

[73] , wherein the antibody-drug conjugate has the effect of promoting the formation of immunological memory against a tumor.

[75] The treatment method described in

[74] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate.

[76] A treatment method described in

[74] in which a portion of the tumor does not express the antigen for the antibody in the antibody-drug conjugate.

[77] Antibody-drug conjugates, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A treatment method according to any one of the items

[72] to

[76] , having at least one action selected from the group consisting of the following.

[78] The treatment method according to any one of paragraphs

[72] to

[77] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[79] The therapeutic method described in

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

[80] The therapeutic method according to

[78] or

[79] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[81] The therapeutic method according to

[78] or

[79] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[82] A therapeutic method according to any one of paragraphs

[72] to

[81] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[83] A therapeutic method according to any one of paragraphs

[72] to

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

[84] A therapeutic method according to any one of paragraphs

[72] to

[81] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[85] A treatment method according to any one of paragraphs

[72] to

[84] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[86] The disease is colorectal cancer, and the treatment method is as described in

[85] .

[87] The disease is breast cancer, and the treatment method is as described in

[85] .

[88] formula

[0021] [ka]

[0022] A therapeutic method characterized by using a compound represented by [the specified compound] for diseases that are improved by its effect of releasing it within the tumor and activating anti-tumor immunity.

[89] The compound, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, A therapeutic method according to

[88] , having at least one action selected from the group consisting of the following.

[90] The therapeutic method according to

[88] or

[89] , wherein the compound has the effect of promoting the formation of immunological memory against tumors.

[91] The compound, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A treatment method according to any one of the items

[88] to

[90] , having at least one action selected from the group consisting of the following.

[92] A treatment method according to any one of paragraphs

[88] to

[91] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[93] When administered in combination with immune checkpoint inhibitors, it is used to treat diseases. formula

[0023] [ka]

[0024] (In the formula, A indicates the binding site with the antibody.) An antibody-drug conjugate in which a drug linker, indicated by [the symbol], and an antibody are linked by a thioether bond.

[94] The antibody-drug conjugate according to

[93] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[95] The antibody-drug conjugate described in

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

[96] The antibody-drug conjugate according to

[94] or

[95] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[97] The antibody-drug conjugate according to

[94] or

[95] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[98] An antibody-drug conjugate according to any one of the paragraphs

[93] to

[97] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[99] An antibody-drug conjugate according to any one of the paragraphs

[93] to

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

[0100] An antibody-drug conjugate according to any one of the paragraphs

[93] to

[97] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0101] An antibody-drug conjugate according to any one of paragraphs

[93] to

[0100] , wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0102] The antibody-drug conjugate described in

[0101] is an immune checkpoint inhibitor that is an anti-PD-1 antibody.

[0103] The antibody-drug conjugate described in

[0101] is an immune checkpoint inhibitor that is an anti-PD-L1 antibody.

[0104] The antibody-drug conjugate described in

[0101] is an immune checkpoint inhibitor that is an anti-CTLA-4 antibody.

[0105] An antibody-drug conjugate according to any one of

[93] to

[0104] , characterized in that the antibody-drug conjugate and the immune checkpoint inhibitor are each contained as active ingredients in separate formulations and are administered simultaneously or at different times.

[0106] An antibody-drug conjugate according to any one of

[93] to

[0104] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are contained as active ingredients in a single formulation and administered.

[0107] An antibody-drug conjugate as described in any one of paragraphs

[93] to

[0106] for the treatment of cancer.

[0108] The antibody-drug conjugate described in

[0107] , wherein the cancer is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0109] The cancer is colorectal cancer, and the antibody-drug conjugate is as described in

[0108] .

[0110] The cancer is breast cancer, and the antibody-drug conjugate is as described in

[0108] .

[0111] An antibody-drug conjugate according to any one of items

[93] to

[0110] , which has the effect of activating antitumor immunity.

[0112] (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, An antibody-drug conjugate according to any one of the items

[93] to

[0111] , having at least one action selected from the group consisting of the following.

[0113] An antibody-drug conjugate according to any one of

[93] to

[0112] , which has the effect of promoting the formation of immunological memory against tumors.

[0114] The antibody-drug conjugate described in

[0113] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate.

[0115] The antibody-drug conjugate described in

[0113] , wherein a portion of the tumor does not express the antigen against the antibody in the antibody-drug conjugate.

[0116] (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, An antibody-drug conjugate according to any one of the items

[93] to

[0115] , having at least one action selected from the group consisting of the following.

[0117] The antibody-drug conjugate according to any one of

[93] to

[0116] , characterized in that the antibody-drug conjugate exhibits a stronger antitumor effect by releasing immunosuppressive signals generated by promoting increased PD-L1 expression on cancer cells with an immune checkpoint inhibitor.

[0118] For use in the treatment of diseases that are improved by activating antitumor immunity. formula

[0025] [ka]

[0026] (In the formula, A indicates the binding site with the antibody.) An antibody-drug conjugate in which a drug linker, indicated by [the symbol], and an antibody are linked by a thioether bond.

[0119] (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, An antibody-drug conjugate according to

[0118] having at least one action selected from the group consisting of the following.

[0120] An antibody-drug conjugate according to

[0118] or

[0119] that has the effect of promoting the formation of immunological memory against tumors.

[0121] The antibody-drug conjugate according to claim

[0120] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate.

[0122] The antibody-drug conjugate described in

[0120] , wherein a portion of the tumor does not express the antigen against the antibody in the antibody-drug conjugate.

[0123] (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, An antibody-drug conjugate according to any one of the items

[0118] to

[0122] for use in the treatment of a disease which is improved by at least one action selected from the group consisting of the following.

[0124] An antibody-drug conjugate according to any one of items

[0118] to

[0123] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[0125] The antibody-drug conjugate described in

[0124] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0126] The antibody-drug conjugate according to

[0124] or

[0125] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[0127] The antibody-drug conjugate according to

[0124] or

[0125] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[0128] An antibody-drug conjugate according to any one of items

[0118] to

[0127] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[0129] An antibody-drug conjugate according to any one of items

[0118] to

[0127] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0130] An antibody-drug conjugate according to any one of items

[0118] to

[0127] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0131] An antibody-drug conjugate according to any one of paragraphs

[0118] to

[0130] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0132] The disease is colorectal cancer, and the antibody-drug conjugate is as described in

[0131] .

[0133] The disease is breast cancer, and the antibody-drug conjugate is as described in

[0131] .

[0134] For use in the treatment of diseases that are improved by activating anti-tumor immunity through release within tumors. formula

[0027] [ka]

[0028] The compound shown by [this symbol].

[0135] (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, A compound according to

[0134] having at least one action selected from the group consisting of the above.

[0136] The compound described in

[0134] or

[0135] , which has the effect of promoting the formation of immunological memory against tumors.

[0137] (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, A compound according to any one of the items

[0134] to

[0136] , having at least one action selected from the group consisting of the above.

[0138] A compound according to any one of claims

[0134] to

[0137] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0139] For the manufacture of pharmaceuticals that, when administered in combination with immune checkpoint inhibitors, treat diseases. formula

[0029] [ka]

[0030] (In the formula, A indicates the binding site with the antibody.) The use of an antibody-drug conjugate in which a drug linker, as shown, and an antibody are linked by a thioether bond.

[0140] The use described in

[0139] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[0141] The use described in

[0140] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0142] The use described in

[0140] or

[0141] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[0143] The use described in

[0140] or

[0141] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[0144] The use described in any one of paragraphs

[0139] to

[0143] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[0145] The use described in any one of the paragraphs

[0139] to

[0143] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0146] The use described in any one of paragraphs

[0139] to

[0143] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0147] The use described in any one of paragraphs

[0139] to

[0146] , wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0148] The use described in

[0147] , wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0149] The use described in

[0147] , where the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0150] The use described in

[0147] , where the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0151] The use described in any one of paragraphs

[0139] to

[0150] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are each contained as active ingredients in separate formulations and administered simultaneously or at different times.

[0152] The use according to any one of items

[0139] to

[0150] , characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are contained as active ingredients in a single formulation and administered.

[0153] Uses described in any one of paragraphs

[0139] to

[0152] for the treatment of cancer.

[0154] The use described in

[0153] , wherein the cancer is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0155] Use as described in

[0154] , where the cancer is colorectal cancer.

[0156] Use as described in

[0154] , where the cancer is breast cancer.

[0157] The use described in any one of paragraphs

[0139] to

[0156] , wherein the antibody-drug conjugate has the effect of activating antitumor immunity.

[0158] Antibody-drug conjugates, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, The use according to any one of the items

[0139] to

[0157] , having at least one action selected from the group consisting of the above.

[0159] The use described in any one of paragraphs

[0139] to

[0158] , wherein the antibody-drug conjugate has the effect of promoting the formation of immunological memory against tumors.

[0160] The use described in

[0159] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate.

[0161] Use as described in

[0159] , wherein a portion of the tumor does not express the antigen for the antibody in the antibody-drug conjugate.

[0162] Antibody-drug conjugates, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, The use according to any one of the items

[0139] to

[0161] , having at least one action selected from the group consisting of the above.

[0163] The use according to any one of items

[0139] to

[0162] , characterized in that the antibody-drug conjugate exhibits a stronger antitumor effect by having an immune checkpoint inhibitor release the immunosuppressive signal generated by the antibody-drug conjugate promoting an increase in PD-L1 expression on cancer cells.

[0164] For the manufacture of pharmaceuticals characterized by their use in treating diseases that are improved by activating antitumor immunity. formula

[0031] [ka]

[0032] (In the formula, A indicates the binding site with the antibody.) The use of an antibody-drug conjugate in which a drug linker, as shown, and an antibody are linked by a thioether bond.

[0165] Antibody-drug conjugates, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, The use described in

[0164] , having at least one action selected from the group consisting of the following.

[0166] The use described in

[0164] or

[0165] , wherein the antibody-drug conjugate has the effect of promoting the formation of immunological memory against tumors.

[0167] The use according to claim

[0166] , wherein the tumor expresses an antigen against the antibody in the antibody-drug conjugate.

[0168] Use as described in

[0166] , wherein a portion of the tumor does not express the antigen for the antibody in the antibody-drug conjugate.

[0169] (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, Use according to any one of paragraphs

[0164] to

[0168] for the manufacture of a pharmaceutical product for use in the treatment of a disease which is improved by at least one action selected from the group consisting of the following.

[0170] The use described in any one of paragraphs

[0164] to

[0169] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, or an anti-B7-H3 antibody.

[0171] The use described in

[0170] , wherein the antibody in the antibody-drug conjugate is an anti-HER2 antibody.

[0172] The use described in

[0170] or

[0171] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

[0173] The use described in

[0170] or

[0171] , wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[0174] The use described in any one of paragraphs

[0164] to

[0173] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

[0175] The use described in any one of paragraphs

[0164] to

[0173] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7 to 8.

[0176] The use described in any one of paragraphs

[0164] to

[0173] , wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

[0177] Uses according to any one of paragraphs

[0164] to

[0176] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0178] Use as described in

[0177] when the disease is colorectal cancer.

[0179] Use as described in

[0177] when the disease is breast cancer.

[0180] formula

[0033] [ka]

[0034] The use of the compound for the manufacture of a pharmaceutical product, characterized in that the compound is released within a tumor and is used to treat a disease that is improved by activating anti-tumor immunity.

[0181] The compound, (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, The use described in

[0180] , having at least one action selected from the group consisting of the following.

[0182] The use described in

[0180] or

[0181] , wherein the compound has the effect of promoting the formation of immunological memory against tumors.

[0183] The compound, (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, The use according to any one of the items

[0180] to

[0182] , having at least one action selected from the group consisting of the above.

[0184] Uses described in any one of paragraphs

[0180] to

[0183] , wherein the disease is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0035] Regarding. [Effects of the Invention]

[0036] The present invention provides a pharmaceutical composition and treatment method that exhibits superior antitumor efficacy and safety when administered in combination with a specific antibody-drug conjugate and an immune checkpoint inhibitor. Furthermore, the present invention provides a pharmaceutical composition and treatment method for treating diseases that are improved by promoting the formation of immune memory against tumors, characterized by containing a specific antibody-drug conjugate. [Brief explanation of the drawing]

[0037] [Figure 1] The amino acid sequence (SEQ ID NO: 1) of the humanized anti-HER2 antibody heavy chain is shown. [Figure 2] The amino acid sequence (SEQ ID NO: 2) of the humanized anti-HER2 antibody light chain is shown. [Figure 3]This figure shows the survival-prolonging effects of various drugs on mice subcutaneously transplanted with CT26.WT-hHER2 cells. The survival-prolonging effects were compared between monotherapy and combination therapy with antibody-drug conjugate (1) and anti-PD-1 antibody (clone RMP1-14). [Figure 4] This figure shows the survival-prolonging effects of various drugs on mice subcutaneously transplanted with CT26.WT-hHER2 cells. The survival-prolonging effects were compared between monotherapy and combination therapy with antibody-drug conjugate (1) and anti-PD-1 antibody (clone RMP1-14). [Figure 5] This figure shows the changes in tumor volume when CT26.WT-hHER2 cells or CT26.WT-mock cells were subcutaneously transplanted (re-transplanted) into antibody-drug conjugate (1) treated cured mice and control mice, respectively. [Figure 6] This figure shows the immune response (number of IFNγ-producing spleen cells) to antigens derived from CT26.WT-hHER2 cells when CT26.WT-hHER2 cells were subcutaneously transplanted (re-transplanted) into antibody-drug conjugate (1) treated and control mice, respectively. [Figure 7] This figure shows the immune response (number of IFNγ-producing spleen cells) to antigens derived from CT26.WT-mock cells when CT26.WT-hHER2 cells were subcutaneously transplanted (re-transplanted) into antibody-drug conjugate (1) treated and control mice, respectively. [Figure 8] This figure shows the immune response (number of IFNγ-producing spleen cells) to antigens derived from CT26.WT-hHER2 cells when CT26.WT-mock cells were subcutaneously transplanted (re-transplanted) into antibody-drug conjugate (1) treated and control mice, respectively. [Figure 9] This figure shows the immune response (number of IFNγ-producing spleen cells) to antigens derived from CT26.WT-mock cells when CT26.WT-mock cells were subcutaneously transplanted (re-transplanted) into antibody-drug conjugate (1) treated cured mice and control mice, respectively. [Figure 10] This figure shows the CD86 expression levels measured by flow cytometry in bone marrow-derived dendritic cells treated with compound (A) and DMSO. [Figure 11] This figure shows the MHC class II expression levels measured by flow cytometry in bone marrow-derived dendritic cells treated with compound (A) and DMSO. [Figure 12] This figure shows the number of dendritic cells in tumor lymphocytes measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in the antibody-drug conjugate (1) administration group and the control group. [Figure 13] This figure shows the number of CD86-positive cells in tumor dendritic cells measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in the antibody-drug conjugate (1) administration group and the control group. [Figure 14] This figure shows the expression levels of CD86 on tumor dendritic cells measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in both the antibody-drug conjugate (1) administration group and the control group, and is displayed as MFI. [Figure 15] This figure shows the MHC class I expression levels on cancer cells (human HER2-positive cells) measured by flow cytometry and displayed as MFI in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in both the antibody-drug conjugate (1) administration group and the control group. [Figure 16] This figure shows the expression levels of PD-L1 on cancer cells (human HER2-positive cells) measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in both the antibody-drug conjugate (1) administration group and the control group, and is displayed as MFI. [Figure 17] This figure shows the MHC class I expression levels measured by flow cytometry after treating cancer cells with compound (A) and DMSO. [Figure 18]This figure shows the changes in tumor volume in a mouse model in which CT26.WT-hHER2 cells were subcutaneously transplanted into nude mice, comparing the antibody-drug conjugate (1) administration group and the control group. [Figure 19] This figure shows the changes in tumor volume in mice subcutaneously transplanted with CT26.WT-hHER2 cells, specifically in the antibody-drug conjugate (1) administration group, the control antibody-drug conjugate administration group, and the control group. [Figure 20] This figure shows the changes in tumor volume in mice subcutaneously transplanted with EMT6-hHER2 cells, comparing monotherapy and combination therapy with antibody-drug conjugate (1) and anti-PD-1 antibody (clone RMP1-14). [Figure 21] This figure shows the survival-prolonging effects of various drugs on mice subcutaneously transplanted with CT26.WT-hHER2 cells. The survival-prolonging effects were compared between monotherapy and combination therapy with antibody-drug conjugate (1) and anti-PD-L1 antibody (clone 10F.9G2). [Figure 22] This figure shows the survival-prolonging effects of various drugs on mice subcutaneously transplanted with EMT6-hHER2 cells. The survival-prolonging effects were compared between monotherapy and combination therapy with antibody-drug conjugate (1) and anti-PD-L1 antibody (clone 10F.9G2). [Figure 23] This figure shows the changes in tumor volume in mice subcutaneously transplanted with CT26.WT-hHER2 cells, comparing the antibody-drug conjugate (1) and anti-CD4 antibody monotherapy and combination therapy. [Figure 24] This figure shows the changes in tumor volume in mice subcutaneously transplanted with CT26.WT-hHER2 cells, comparing the antibody-drug conjugate (1) and anti-CD8 antibody monotherapy and combination therapy. [Figure 25] This figure shows the percentage of CD8-positive T cells in the tumor biomass measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in the antibody-drug conjugate (1) administration group and the control group. [Figure 26] This figure shows the percentage of Granzyme B-positive cells among CD8-positive T cells in tumors measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in the antibody-drug conjugate (1) administration group and the control group. [Figure 27] This figure shows the percentage of Granzyme B-positive CD8-positive T cells in the tumor biomass measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in the antibody-drug conjugate (1) administration group and the control group. [Figure 28] This figure shows the percentage of CD4-positive T cells in the tumor biomass measured by flow cytometry in mice subcutaneously transplanted with CT26.WT-hHER2 cells, in the antibody-drug conjugate (1) administration group and the control group. [Figure 29] This figure shows images of excised tumors stained with anti-CD8 antibody from mice that received subcutaneous transplantation of CT26.WT-hHER2 cells, in both the antibody-drug conjugate (1) administration group and the control group. [Figure 30] This figure shows the number of CD8-positive cells per unit area within tumors measured by analyzing images of excised tumors stained with anti-CD8 antibody in mice that received subcutaneous transplantation of CT26.WT-hHER2 cells, in both the antibody-drug conjugate (1) administration group and the control group. [Figure 31] This figure shows the MHC class I expression levels measured by flow cytometry after treating cancer cells with compound (A), DM1-SMe, DM4-SMe, MMAE, and DMSO. [Figure 32] This figure shows the changes in tumor volume in mice subcutaneously transplanted with EMT6-hHER2 cells, comparing monotherapy and combination therapy with antibody-drug conjugate (1) and anti-CTLA-4 antibody (clone 9H10). [Modes for carrying out the invention]

[0038] Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. The embodiments described below are merely examples of typical embodiments of the present invention, and this should not be interpreted as narrowing the scope of the invention.

[0039] [Antibody-drug conjugates] The antibody-drug conjugate used in the present invention is,

[0040] [ka]

[0041] (In the formula, A indicates the binding site with the antibody.) This is an antibody-drug conjugate in which a drug linker, indicated by [the symbol], and an antibody are linked by a thioether bond.

[0042] In this invention, a substructure 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 thiol groups (in other words, sulfur atoms of cysteine ​​residues) formed at disulfide bond sites between antibody chains (two heavy chain-to-heavy chain bonds and two heavy chain-to-light chain bonds).

[0043] The drug linker of the present invention is composed of 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]indolidino[1,2-b]quinoline-10,13-dione, (also expressed as 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]indolidino[1,2-b]quinoline-10,13(9H,15H)-dione), a topoisomerase I inhibitor.

[0044] [Chemical formula]

[0045] It is a camptothecin derivative having an antitumor effect, represented by

[0046] The antibody-drug conjugate used in the present invention can also be represented by the following formula.

[0047] [Chemical formula]

[0048] Here, the drug linker is bound to the antibody by a thioether bond. Also, n is synonymous with the so-called average drug conjugation number (DAR; Drug-to-Antibody Ratio), and represents the average conjugation number of the drug linker per antibody.

[0049] The antibody-drug conjugate used in the present invention has an action of activating antitumor immunity.

[0050] In the present invention, "activating antitumor immunity" means promoting the exertion of an antitumor effect by activating at least one selected from the group consisting of T cells and B cells (Bracci L. et al., Cell Death Differ. (2014) 21, 15 - 25, Chen DS. Et al., Immunity (2013) 39, 1 - 10, Andersen MH. et al., Journal of Investigative Dermatology (2006) 126, 32 - 41).

[0051] The fact that the antibody-drug conjugate used in the present invention promotes the exertion of antitumor effects by activating at least one selected from the group consisting of T cells and B cells can be confirmed by comparing the antitumor effects of the antibody-drug conjugate used in the present invention in mice with normal immune function with those in mice in which the immune function of T cells and B cells is impaired (nude mice).

[0052] The antibody-drug conjugate used in the present invention is (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, It has at least one action selected from the group consisting of the following.

[0053] The "effect of promoting the increase of CD8-positive T cells in tumors" of the antibody-drug conjugate used in the present invention can be confirmed, for example, by measuring the percentage of CD45, CD3, and CD8-positive cells (CD8-positive T cells) among viable cells in tumor-bearing mice administered with the antibody-drug conjugate and in the control group using flow cytometry and examining the rate of increase. Alternatively, it can be confirmed by analyzing images of excised tumors stained with anti-CD8 antibody in tumor-bearing mice administered with the antibody-drug conjugate and in the control group, measuring the number of CD8-positive cells per unit area within the tumor, and examining the rate of increase.

[0054] The "activation effect of tumor-derived CD8-positive T cells" of the antibody-drug conjugate used in this invention can be confirmed, for example, by measuring the percentage of Granzyme B-positive cells among CD8-positive T cells in tumor-bearing mice administered with the antibody-drug conjugate and in a control group using flow cytometry and examining the rate of increase. It can also be confirmed by measuring the percentage of Granzyme B-positive cells among viable cells using flow cytometry and examining the rate of increase.

[0055] The antibody-drug conjugate used in this invention has the effect of promoting the formation of immunological memory against tumors. This effect contributes to the "effect of activating anti-tumor immunity" described above.

[0056] T cells are activated when they receive tumor-derived antigens from dendritic cells or cancer cells, thereby initiating an immune response and exerting an antitumor effect.

[0057] In this invention, "immunological memory formation against tumors" refers to the formation of a memory of the immune response to a tumor-derived antigen by generating memory T cells from T cells upon presentation of that antigen. This allows for a sustained antitumor effect against tumors possessing that antigen, and also enables the re-examination of the antitumor effect when the tumor possessing that antigen recurs.

[0058] The "effect of promoting the formation of immune memory against tumors" of the antibody-drug conjugate used in the present invention can be confirmed, for example, by administering the antibody-drug conjugate to tumor-bearing mice, transplanting the tumor back into mice whose tumors have completely regressed, and measuring the degree of suppression of tumor growth (recurrence). It can also be confirmed by removing the spleen from the above mice, adding tumor-derived antigens, and measuring the rate of increase in the immune response (e.g., the number of IFNγ-producing spleen cells).

[0059] Furthermore, the antibody-drug conjugate used in the present invention has the effect of promoting immunological memory formation not only in tumors that express the antigen for the antibody in the antibody-drug conjugate, but also in tumors within the same individual that do not express the antigen for the antibody in the antibody-drug conjugate.

[0060] For example, if the antibody in an antibody-drug conjugate is an anti-HER2 antibody, it not only promotes the formation of immunological memory against tumors that express HER2, but also promotes the formation of immunological memory against tumors that do not express HER2 within the same individual.

[0061] In addition, the antibody-drug conjugate used in the present invention has at least one action selected from the group consisting of (1) an action of promoting an increase in the number of dendritic cells in a tumor, (2) an action of activating dendritic cells, and (3) an action of promoting an increase in the expression level of MHC class I on cancer cells. These actions contribute to the above-mentioned "action of promoting the formation of immune memory against tumors", and ultimately contribute to the above-mentioned "action of activating anti-tumor immunity".

[0062] The "action of promoting an increase in the number of dendritic cells in a tumor" possessed by the antibody-drug conjugate used in the present invention can be confirmed, for example, by measuring the ratio of CD11c, MHC class II, and CD45-positive cells (dendritic cells, DC) occupying CD45-positive cells (lymphocytes) in the antibody-drug conjugate administration group and the control group in tumor-bearing mice using flow cytometry and examining the increase rate.

[0063] The "action of activating dendritic cells" possessed by the antibody-drug conjugate used in the present invention can be confirmed, for example, by measuring the ratio of dendritic cells expressing CD86 (activation marker) in the antibody-drug conjugate administration group and the control group in tumor-bearing mice using flow cytometry and examining the increase rate. Also, in tumor-bearing mice, for the antibody-drug conjugate administration group and the control group, the expression level (MFI (mean fluorescence intensity)) of CD86 on dendritic cells can be measured by flow cytometry and the increase rate can be examined to confirm it as well.

[0064] The "effect of promoting an increase in MHC class I expression on cancer cells" of the antibody-drug conjugate used in the present invention can be confirmed, for example, by measuring the amount of MHC class I expression (MFI) on cancer cells in tumor-bearing mice administered with the antibody-drug conjugate and in a control group using flow cytometry and examining the rate of increase.

[0065] The antibody-drug conjugate used in this invention may also have the effect of promoting an increase in PD-L1 expression on cancer cells. The immunosuppressive signal resulting from this can be released by an immune checkpoint inhibitor, allowing the antibody-drug conjugate to exhibit a stronger antitumor effect. Therefore, a stronger antitumor effect can be expected when the antibody-drug conjugate used in this invention is used in combination with an immune checkpoint inhibitor.

[0066] The "effect of promoting an increase in PD-L1 expression on cancer cells" of the antibody-drug conjugate used in the present invention can be confirmed, for example, by measuring the amount of PD-L1 expression (MFI) on cancer cells in tumor-bearing mice administered with the antibody-drug conjugate and in a control group using flow cytometry and examining the rate of increase.

[0067] The antibody-drug conjugate used in this invention undergoes cleavage of the linker portion after migrated into cancer cells.

[0068] formula

[0069] [ka]

[0070] It releases a compound represented by (hereinafter referred to as "compound (A)").

[0071] Compound (A) is considered to be the main component 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] Compound (A) has the effect of activating dendritic cells and promoting the increase in MHC class I expression on cancer cells.

[0073] The "dendritic cell activating effect" of compound (A) can be confirmed, for example, by measuring the CD86 expression level by flow cytometry after treating bone marrow-derived dendritic cells with compound (A) and DMSO, and examining the rate of increase.

[0074] The "effect of compound (A) that promotes an increase in MHC class I expression on cancer cells" can be confirmed, for example, by measuring the amount of MHC class I expression in cancer cells treated with compound (A) and DMSO using flow cytometry and examining the rate of increase.

[0075] The "dendritic cell activation effect" and the "effect of promoting increased MHC class I expression on cancer cells" of compound (A) are the same effects as those of the antibody-drug conjugate used in the present invention. Furthermore, as mentioned above, compound (A) is a compound released from the antibody-drug conjugate used in the present invention after the antibody-drug conjugate has migrated to cancer cells.

[0076] Therefore, a pharmaceutical composition that releases compound (A) within a tumor is similar to the antibody-drug conjugate used in the present invention. (1) An effect that promotes an increase in the number of dendritic cells in the tumor. (2) The effect of activating dendritic cells, and, (3) Action to promote the increase in MHC class I expression on cancer cells, It is expected to have at least one action selected from the group consisting of the following:

[0077] Furthermore, a pharmaceutical composition that releases compound (A) within a tumor is expected to have an effect of promoting the formation of immunological memory against the tumor, similar to the antibody-drug conjugate used in the present invention.

[0078] Furthermore, as mentioned above, compound (A) is a compound generated from the antibody-drug conjugate used in the present invention after the antibody-drug conjugate has migrated to cancer cells.

[0079] Therefore, a pharmaceutical composition that releases compound (A) within a tumor is similar to the antibody-drug conjugate used in the present invention. (1) The effect of promoting the increase of CD8-positive T cells in tumors, and, (2) The effect of activating CD8-positive T cells in tumors, It is expected to have at least one action selected from the group consisting of, Ultimately, it is expected to have the effect of "activating anti-tumor immunity."

[0080] Furthermore, the antibody-drug conjugate used in this invention is also known to have a bystander effect (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). This bystander effect is exerted when the antibody-drug conjugate used in the present invention, after being internalized in target-expressing cancer cells, releases compound (A), which then exerts an antitumor effect on nearby cancer cells that do not express the target. The bystander effect of the antibody-drug conjugate used in this invention is also demonstrated as an excellent antitumor effect when used in combination with an immune checkpoint inhibitor. [Antibodies used in the manufacture of antibody-drug conjugates] The antibodies used in the production of antibody-drug conjugates according to the present invention may be derived from any species, but preferably from humans, rats, mice, and rabbits. If the antibodies are derived from a species other than humans, it is preferable to chimerize or humanize them using well-known techniques. The antibodies of the present invention may be polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are preferred.

[0081] The antibody used in the production of the antibody-drug conjugate according to the present invention preferably has properties that can target cancer cells, and is preferably one that has the ability to recognize cancer cells, the ability to bind to cancer cells, the ability to be taken up and internalized into cancer cells, and / or cytotoxic activity against cancer cells.

[0082] The binding affinity of antibodies to cancer cells can be confirmed using flow cytometry. Antibody uptake into cancer cells can be confirmed using (1) an assay that visualizes antibodies taken up into cells using a fluorescence microscope with a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay that measures the amount of fluorescence when taken up into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay (Bio Techniques 28:162-165, January 2000), which uses an immunotoxin that binds to the therapeutic antibody and suppresses cell proliferation by releasing a toxin upon uptake into cells. Recombinant complex proteins of the catalytic domain of diphtheria toxin and protein G can also be used as immunotoxins.

[0083] The antitumor activity of an antibody can be confirmed in vitro by measuring its inhibitory activity on cell proliferation. For example, cancer cell lines 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 on focus formation, colony formation, and spheroid proliferation. In vivo, for example, antitumor activity can be confirmed by administering the antibody to nude mice transplanted with cancer cell lines highly expressing the target protein and measuring the changes in the cancer cells.

[0084] While it is desirable for the antibody itself to have antitumor effects, the antitumor effect of the antibody-drug conjugate is not essential, as it involves attaching a compound that exerts antitumor effects. For the purpose of specifically and selectively exerting the cytotoxic effects of the antitumor compound in cancer cells, it is important and desirable that the antibody has the property of internalizing and migrating into cancer cells.

[0085] The antibodies used in the production of antibody-drug conjugates according to the present invention can be obtained by known means. For example, they can be obtained by immunizing animals with antigenic polypeptides using methods commonly practiced in this art, and then collecting and purifying the antibodies produced in vivo. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from non-human animals such as mice and rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity between the obtained antibodies that bind to the heterologous antigens and human antigens.

[0086] Furthermore, monoclonal antibodies can also be obtained by establishing hybridomas and fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells, according to known methods (for example, Kohler and Milstein, Nature (1975) 256, p.495-497; Kennet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)).

[0087] Antigens can be obtained by genetically modifying host cells to produce the gene encoding the antigen protein. Specifically, a vector capable of expressing the antigen gene is created, introduced into host cells to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing animals with antigen-expressing cells or cell lines expressing the antigen, which have been genetically modified as described above.

[0088] The antibodies used in the production of antibody-drug conjugates according to the present invention are preferably genetically modified recombinant antibodies, such as chimeric antibodies or humanized antibodies, which are artificially modified for purposes such as reducing heterologous antigenicity against humans, or antibodies that have only the gene sequence of a human-derived antibody, i.e., human antibodies. These antibodies can be produced using known methods.

[0089] Chimeric antibodies are antibodies in which the variable region and constant region are heterogeneous, for example, chimeric antibodies in which the variable region of a mouse or rat-derived antibody is conjugated to the constant region of a human-derived antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0090] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody is incorporated into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies in which, in addition to the CDR sequence of a heterologous antibody, amino acid residues of a portion of the framework of the heterologous antibody are also transplanted into a human antibody using the CDR transplantation method (International Publication No. 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Patent No. 5821337).

[0091] Examples of human antibodies include antibodies produced using human antibody-producing mice that possess human chromosome fragments containing the genes for the heavy and light chains of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) 16, p.133-143; Kuroiwa, Y. et. al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et. al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.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, p.722-727, etc.). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be cited (see Wormstone, IM et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p.2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2), p.189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109(3), p.427-431, etc.).

[0092] The antibodies used in the production of antibody-drug conjugates according to the present invention also include modified antibodies. Such modified antibodies are those that have undergone chemical or biological modifications. Chemically modified antibodies include those having chemical moieties attached to an amino acid backbone, or chemical moieties attached to N- or O-linked carbohydrate chains. Biologically modified antibodies include those that have undergone post-translational modifications (e.g., addition of N- or O-linked glycans, N-terminus or C-terminus processing, deamidation, aspartic acid isomerization, methionine oxidation, etc.), or those that have a methionine residue added to the N-terminus by expression using prokaryotic host cells. Furthermore, modified antibodies that have been labeled to enable detection or isolation of the antibody or antigen according to the present invention, such as enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies, are also included in the meaning of such modified antibodies. Such modified antibodies according to the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and for detection or isolation of antibodies or antigens.

[0093] Furthermore, antibody-dependent cytotoxic activity can be enhanced by regulating the glycosylation (glycosylation, defucoseation, etc.) of the antibody bound to the antibody according to the present invention. Known techniques for regulating antibody glycosylation include, but are not limited to, International Publication No. 99 / 54342, International Publication No. 00 / 61739, and International Publication No. 02 / 31140. The antibody according to the present invention also includes antibodies in which such glycosylation has been regulated.

[0094] It is known that antibodies produced in mammalian cultured cells have a deletion of the lysine residue at the carboxyl terminus of their heavy chain (Journal of Chromatography A, 705: 129-134 (1995)), and also that two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain are deleted, and a proline residue located 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 function (such as complement activation or antibody-dependent cell-mediated cytotoxicity) of the antibody. Therefore, the antibodies according to 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 (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody according to the present invention are not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be one of the full-length heavy chains and heavy chains selected from the group consisting of the above-mentioned deletions, or a combination of either two types. The quantity ratio of each deletion may be affected by the type of mammalian cultured cell that produces the antibody according to the present invention and the culture conditions, but the antibody according to the present invention preferably has a deletion of one amino acid residue at the carboxyl terminal in both of the two heavy chains.

[0095] Examples of antibody isotypes according to the present invention include IgG (IgG1, IgG2, IgG3, IgG4), but IgG1 or IgG2 are preferred.

[0096] The antibodies that can be used in the production of the antibody-drug conjugate according to the present invention are not particularly limited, but examples include anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 antibody, anti-FOLR1 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD70 antibody, anti-PSMA antibody, anti-CEA antibody, and anti-Mesothelin antibody. Preferably, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, and anti-B7-H3 antibody can be used, and more preferably, anti-HER2 antibody can be used.

[0097] In the present invention, "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 to be internalized in HER2-expressing cells by binding to HER2.

[0098] Examples of anti-HER2 antibodies include trastuzumab (U.S. Patent No. 5821337) and pertuzumab (International Publication No. 01 / 00245), with trastuzumab being preferred.

[0099] In the present invention, "trastuzumab" may also be called HERCEPTIN®, huMAb4D5-8, or rhuMAb4D5-8, and is a humanized anti-HER2 antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (Figure 1) with amino acid numbers 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (Figure 2) with amino acid numbers 1 to 214.

[0100] A suitable anti-HER2 antibody used in the production of antibody-drug conjugates according to the present invention is: (1) An antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214, or (2) An antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[0101] In the present invention, "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 to be internalized in HER3-expressing cells by binding to HER3.

[0102] Examples of anti-HER3 antibodies include patritumab (U3-1287), U1-59 (International Publication No. 2007 / 077028), MM-121 (Seribantumab), the anti-ERBB3 antibody described in International Publication No. 2008 / 100624, RG-7116 (Lumretuzumab), and LJM-716 (Elgemtumab), with patritumab and U1-59 being preferred.

[0103] In the present invention, "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 to be internalized in TROP2-expressing cells by binding to TROP2.

[0104] An example of an anti-TROP2 antibody is hTINA1-H1L1 (International Publication No. 2015 / 098099).

[0105] In the present invention, "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 and, preferably, has the activity to be internalized in B7-H3-expressing cells by binding to B7-H3.

[0106] An example of an anti-B7-H3 antibody is M30-H1-L4 (International Publication No. 2014 / 057687). [Drug linker intermediate used in the manufacture of antibody-drug conjugates] The drug linker intermediate used in the production of the antibody-drug conjugate according to the present invention is represented by the following formula.

[0107] [ka]

[0108] The above drug linker intermediate is N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-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] It can be represented by the chemical name Pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide, and can be manufactured by referring to descriptions in International Publication Nos. 2014 / 057687, 2015 / 098099, 2015 / 115091, 2015 / 155998, etc.

[0109] [Conjugation of antibodies and drug linker intermediates] 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 known as a sulfhydryl group).

[0110] Antibodies containing 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, by using a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in an amount of 0.3 to 3 molar equivalents per interchain disulfide of the antibody, and reacting the antibody in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), an antibody containing sulfhydryl groups can be obtained in which the interchain disulfides of the antibody are partially or completely reduced.

[0111] Furthermore, by using 2 to 20 molar equivalents of a drug linker intermediate per antibody having a sulfhydryl group, an antibody-drug conjugate can be produced in which 2 to 8 drugs are bound to each antibody.

[0112] The average number of drug-bound molecules per antibody in the manufactured antibody-drug conjugate can be calculated, for example, by measuring the UV absorbance of the antibody-drug conjugate and its conjugation precursor at two wavelengths, 280 nm and 370 nm (UV method), or by treating the antibody-drug conjugate with a reducing agent and quantifying each resulting fragment by HPLC measurement (HPLC method).

[0113] The conjugation of antibodies and drug linker intermediates, and the calculation of the average number of drug conjugates per antibody molecule in antibody-drug conjugates, can be performed by referring to the descriptions in International Publication Nos. 2014 / 057687, 2015 / 098099, 2015 / 115091, 2015 / 155998, and others.

[0114] In the present invention, "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-HER2 antibody.

[0115] In the present invention, 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] The anti-HER2 antibody-drug conjugate used in this invention can be manufactured by referring to the description in International Publication No. 2015 / 115091, etc.

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

[0118] In the present invention, 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.

[0119] The anti-HER3 antibody-drug conjugate used in this invention can be manufactured by referring to the description in International Publication No. 2015 / 155998, etc.

[0120] In the present invention, "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-TROP2 antibody.

[0121] In the present invention, 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.

[0122] The anti-TROP2 antibody-drug conjugate used in this invention can be manufactured by referring to the description in International Publication No. 2015 / 098099, etc.

[0123] In the present invention, "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody is an anti-B7-H3 antibody.

[0124] In the present invention, 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.

[0125] The anti-B7-H3 antibody-drug conjugate used in this invention can be manufactured by referring to the description in International Publication No. 2014 / 057687, etc.

[0126] [Immune checkpoint inhibitors] In this invention, "immune checkpoint inhibitor" refers to a drug that inhibits the immunosuppressive system and activates tumor immunity.

[0127] The immune checkpoint inhibitors used in the present invention are not particularly limited, but preferably include anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and more preferably include anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0128] In the present invention, "anti-PD-1 antibody" refers to an antibody that specifically binds to PD-1 (Programmed cell death-1; CD279; PDCD1), thereby reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between PD-1 and its binding partners, PD-L1 and PD-L2. While there are no particular limitations on the anti-PD-1 antibody used in the present invention as long as its clinical efficacy and safety have been confirmed, preferred examples include nivolumab (International Publication No. 2006 / 121168, etc.) and pembrolizumab (International Publication No. 2008 / 156712, etc.). Furthermore, commercially available research-grade anti-PD-1 antibodies (e.g., clone RMP1-14) can be used in preclinical studies to confirm the combined effect with the antibody-drug conjugate used in the present invention.

[0129] In the present invention, "anti-PD-L1 antibody" refers to an antibody that specifically binds to PD-L1 (Programmed cell death ligand 1; CD274; B7-H1), thereby reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between PD-L1 and its binding partners, PD-1 and B7.1 (CD80). While there are no particular limitations on the anti-PD-L1 antibody used in the present invention, as long as its clinical efficacy and safety have been confirmed, preferred examples include atezolizumab (International Publication No. 2010 / 077634, etc.), durvalumab (International Publication No. 2011 / 066389, etc.), and avelumab (International Publication No. 2013 / 079174, etc.). Furthermore, to confirm the combined effect with the antibody-drug conjugate used in the present invention in preclinical studies, commercially available research-grade anti-PD-L1 antibodies (e.g., clone 10F.9G2) can also be used.

[0130] In the present invention, "anti-CTLA-4 antibody" refers to an antibody that specifically binds to CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4; CD152), thereby reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between CTLA-4 and its binding partners, B7.1 (CD80) and B7.2 (CD86). While there are no particular limitations on the anti-CTLA-4 antibody used in the present invention as long as its clinical efficacy and safety have been confirmed, preferred examples include ipilimumab (International Publication No. 2001 / 014424, etc.) and tremelimumab (International Publication No. 2000 / 037504, etc.). Furthermore, commercially available research-grade anti-CTLA-4 antibodies (e.g., clone 9H10) can also be used in preclinical studies to confirm the combined effect with the antibody-drug conjugate used in the present invention.

[0131] [Medical] The following describes a pharmaceutical composition and treatment method characterized by the administration of an antibody-drug conjugate and an immune checkpoint inhibitor in combination according to the present invention, as well as a pharmaceutical composition and treatment method characterized by the use of an antibody-drug conjugate according to the present invention for the treatment of diseases that are improved by activating antitumor immunity.

[0132] The pharmaceutical composition and therapeutic method of the present invention may be characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are each contained as active ingredients in separate formulations and administered simultaneously or at different times, or it may be characterized in that the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in a single formulation and administered. Furthermore, for the treatment of diseases that are improved by activating anti-tumor immunity, the antibody-drug conjugate according to the present invention may be contained as an active ingredient in a single formulation and administered.

[0133] The pharmaceutical compositions and therapeutic methods of the present invention can be used for the treatment of cancer, and preferably for the treatment of at least one disease selected from the group consisting of lung cancer (including non-small cell lung cancer), urothelial carcinoma, colorectal cancer (sometimes called colorectal cancer, including colon cancer and rectal cancer), prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer (sometimes called gastric adenocarcinoma), gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

[0134] The pharmaceutical composition and therapeutic method of the present invention can be selectively used as an agent for drug therapy, which is a major treatment for cancer, and as a result, it can slow the growth of cancer cells, suppress their proliferation, and even destroy them. Through these actions, cancer patients can be relieved from cancer-related symptoms and their quality of life can be improved, and the therapeutic effect can be achieved while preserving the life of the cancer patient. Even if the destruction of cancer cells is not achieved, the suppression and control of cancer cell proliferation can lead to a higher quality of life and longer survival for cancer patients.

[0135] In addition to being used alone in such drug therapies, the pharmaceutical composition and treatment method of the present invention can also be used as a drug in combination with other therapies in adjuvant therapy, and can be combined with surgical procedures, radiation therapy, hormone therapy, etc. Furthermore, it can also be used as a drug in neoadjuvant therapy.

[0136] In addition to the therapeutic uses described above, the pharmaceutical composition and therapeutic method of the present invention can also be expected to have preventive effects, such as suppressing and even destroying the proliferation of minute metastatic cancer cells. For example, it can be expected to have an effect of suppressing and destroying cancer cells in bodily fluids during the metastatic process, or suppressing and destroying minute cancer cells immediately after implantation in any tissue. Therefore, it can be expected to have an inhibitory and preventive effect on cancer metastasis, especially after surgical removal of cancer.

[0137] The pharmaceutical composition and therapeutic method of the present invention can be applied to patients as systemic therapy, as well as applied locally to cancerous tissue to expect therapeutic effects.

[0138] The pharmaceutical composition and therapeutic method of the present invention can be suitably used in mammals, and more preferably in humans.

[0139] The pharmaceutical composition of the present invention may be administered as a pharmaceutical composition containing one or more pharmaceutically compatible components. The substances used in the pharmaceutical composition of the present invention can be appropriately selected from pharmaceutical additives and others commonly used in the art, depending on the dosage and concentration. For example, the pharmaceutical composition typically includes one or more pharmaceutical carriers (e.g., sterile liquids). Here, the liquid includes, for example, water and oil (petroleum, animal, plant, or synthetic oils). The oil may be, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients can be appropriately selected from those known in the art. The composition may also optionally contain trace amounts of wetting agents or emulsifiers, or pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The prescription corresponds to the manner of administration.

[0140] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. Routes of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration may be by infusion or bolus injection, for example. In certain preferred embodiments, the antibody-drug conjugates and immune checkpoint inhibitors used in the present invention are administered by infusion. Parenteral administration is a preferred route of administration.

[0141] In typical embodiments, the pharmaceutical composition is formulated according to a standard procedure as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may also contain a solubilizer and a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the components are supplied either separately, or mixed together in a unit dosage form, as a dry lyophilized powder or an anhydrous concentrate in a sealed container, such as an ampoule or sachet indicating the amount of the activator. If the pharmaceutical composition is administered by infusion, it may be administered, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the pharmaceutical composition is administered by injection, an ampoule of sterile water or saline for injection may be provided, for example, so that the components can be mixed before administration.

[0142] The pharmaceutical composition and therapeutic method of the present invention may contain cancer therapeutic agents other than the antibody-drug conjugate and immune checkpoint inhibitor according to the present invention. The pharmaceutical composition and therapeutic method of the present invention can also be administered in combination with other cancer therapeutic agents, thereby enhancing the antitumor effect. Other cancer therapeutic agents used for such purposes may be administered to the individual simultaneously with the pharmaceutical composition of the present invention, separately, or consecutively, or with varying administration intervals. Examples of such cancer therapeutic agents include 5-fluorouracil (5-FU), pertuzumab, trastuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), and docetaxel (Docet). axel), pemetrexed, sorafenib, vinblastin, vinorelbine, everolims, tanespimycin, bevacizumab, oxaliplatin, lapatinib, trastuzumab emtansine Examples include emtansine (T-DM1) or drugs listed in International Publication No. 2003 / 038043, as well as LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), but the term is not limited to drugs with antitumor activity.

[0143] Such pharmaceutical compositions can be formulated as lyophilized or liquid formulations having the selected composition and required purity. When formulated as a lyophilized formulation, it may contain appropriate pharmaceutical additives used in this field. Similarly, liquid formulations can also be formulated as liquid formulations containing various pharmaceutical additives used in this field.

[0144] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-drug conjugate and immune checkpoint inhibitor contained in the pharmaceutical composition of the present invention can exert their therapeutic effect even at small doses if their affinity for the antigen is high (i.e., the dissociation constant (Kd value) for the antigen is low). Therefore, the dosage of the antibody-drug conjugate and immune checkpoint inhibitor can also be set based on the affinity for the antigen. When administering the antibody-drug conjugate and immune checkpoint inhibitor according to the present invention to humans, for example, approximately 0.001 to 100 mg / kg can be administered once or multiple times at intervals of 1 to 180 days.

[0145] In the case of the antibody-drug conjugate according to the present invention, for example, one method of administration is to administer 0.8 mg / kg to 8 mg / kg once every three weeks. Examples of dosages include 0.8 mg / kg, 1.6 mg / kg, 3.2 mg / kg, 5.4 mg / kg, 6.4 mg / kg, 7.4 mg / kg, and 8 mg / kg. Furthermore, while administration may be once every three weeks (q3w), it may also be once every week (q1w), once every two weeks (q2w), or once every four weeks (q4w). [Examples]

[0146] The present invention will be specifically illustrated by the following examples, but the present invention is not limited to these examples. Furthermore, these examples are not intended to be interpreted restrictively in any sense.

[0147] [Manufacturing Example 1: Preparation of Antibody-Drug Conjugates] Using a humanized anti-HER2 antibody (trastuzumab) according to the manufacturing method described in International Publication No. 2015 / 115091,

[0148] [ka]

[0149] (In the formula, A indicates the binding site with the antibody.) An antibody-drug conjugate (hereinafter referred to as "antibody-drug conjugate (1)") was manufactured in which a drug linker shown and an anti-HER2 antibody were linked by a thioether bond.

[0150] [Manufacturing Example 2: Preparation of Compound (A)] According to the manufacturing method described in International Publication No. 2014 / 057687, formula

[0151] [ka]

[0152] The compound (compound (A)) shown was produced.

[0153] [Evaluation Example 1: Life Sustaining Test] Mice: Six-week-old female BALB / c mice (BALB / c AnNCrlCrlj) (Charles River Co., Ltd. Japan) were used in the experiment. Measurement and calculation formula: The longest and shortest diameters of the tumor were measured twice a week using an electronic digital caliper (CD15-CX, Mitutoyo Corporation), and the tumor volume (mm³) was calculated. 3 The calculation was performed. The formula is as follows: Tumor volume (mm 3 ) = 0.5 × major axis (mm) × minor axis (mm) 2 Tumor volume is 3000 mm 3 Individuals exceeding a certain threshold were euthanized from the perspective of animal experimentation ethics.

[0154] The antibody-drug conjugate (1) (Drug-to-Antibody Ratio: 7.6) was diluted with a dedicated solvent (10 mM Histidine, 10% Trehalose, 0.02% Polysorbate 20, pH 5.5) before use. The anti-PD-1 antibody (clone RMP1-14) was purchased from Bio X Cell and diluted with DPBS (SIGMA-ALDRICH) before use. A dose of 10 mL / kg was administered intravenously via tail vein.

[0155] We used CT26.WT-hHER2 cells, which were created by introducing the human HER2 gene into the mouse colorectal cancer cell line CT26.WT (CRL2638), purchased from American Type Culture Collection, using a retroviral vector. These cells express the human HER2 protein on their cell membrane. The CT26.WT-hHER2 cells were suspended in physiological saline and 5.0 × 10⁶ cells were used. 6 Cells were subcutaneously transplanted into the right axilla of BALB / c mice, and randomization was performed after 6 days (Day 0). The antibody-drug conjugate (1) was administered intravenously in the tail vein twice at a dose of 10 mg / kg on Days 0 and 7. The anti-PD-1 antibody was administered intravenously in the tail vein five times at a dose of 2.5 mg / kg on Days 0, 3, 7, 10, and 14. In addition, a group receiving a combination of the antibody-drug conjugate (1) and the anti-PD-1 antibody, and a control group receiving only the specialized solvent for the antibody-drug conjugate (1) were established. Each group consisted of 6 mice, and tumor volume was measured until Day 43.

[0156] The results are shown in Figure 3. Tumor volume: 3000 mm³ 3The Kaplan-Meier curves are shown, with the endpoint being the point at which a certain threshold was exceeded. The vertical axis represents survival rate (%), and the horizontal axis represents the number of days from the first administration. In the control group, the number of mice was reduced from Day 17, and all mice were euthanized by Day 24. In contrast, in the antibody-drug conjugate (1) group, the number of mice was reduced from Day 28, and 3 mice survived by Day 43. In the anti-PD-1 antibody group, the number of mice was reduced from Day 21, and 2 mice survived by Day 43. Furthermore, all mice in the combination therapy group survived by Day 43. In addition, no weight loss was observed in any of the groups in this study. From the above, the antitumor effect of monotherapy with both drugs was confirmed, and it was confirmed that the effect was dramatically enhanced by the combination therapy of the two drugs.

[0157] [Evaluation Example 2: Life Sustaining Test] The study was conducted in the same manner as in Evaluation Example 1. The anti-PD-1 antibody was administered intravenously via tail vein four times at a dose of 5 mg / kg on days 0, 3, 7, and 10. Each group consisted of 20 mice, and tumor volume was measured up to day 38. Comparisons of efficacy between the control group, the antibody-drug conjugate (1) group, and the anti-PD-1 antibody group, as well as comparisons of efficacy between the antibody-drug conjugate (1) and anti-PD-1 antibody groups and the combined drug group, were performed using the Kaplan-Meier log-rank test (multi-group comparison). The estimated tumor volume was 3000 mm². 3 The day after the specified threshold (the day of euthanasia) was defined as the event date (date of death). Multiple-adjusted p-values ​​are presented to four decimal places, and p<0.05 (two-tailed test) was considered statistically significant. The results are shown in Figure 4. Compared to the control group, the antibody-drug conjugate (1) group showed significantly superior antitumor effects (P=0.0001). Also, compared to the control group, the anti-PD-1 antibody group showed significantly superior antitumor effects (P=0.0010). Furthermore, compared to the antibody-drug conjugate (1) group, the combination therapy group showed significantly superior antitumor effects (P=0.0006). Also, compared to the anti-PD-1 antibody group, the combination therapy group showed significantly superior antitumor effects (P<0.0001).

[0158] [Evaluation Example 3: Re-implantation Trial] Similar to Evaluation Example 1, antibody-drug conjugate (1) was administered to mice subcutaneously implanted with CT26.WT-hHER2 cells. Random grouping was performed on the 5th day after transplantation. Among these mice, mice with completely disappeared tumors were selected (hereinafter referred to as "antibody-drug conjugate (1)-treated cured mice"). In addition, untreated mice were used as controls (hereinafter referred to as "control mice"). Next, 5.0×10 6 cells of CT26.WT-hHER2 cells or CT26.WT-mock cells were subcutaneously implanted into the left axilla of each of the antibody-drug conjugate (1)-treated cured mice and control mice (re-implantation, Day 0), and the tumor volume was measured until Day 17. The number of mice in each group was 9. The results are shown in Figure 5. The vertical axis represents the tumor volume (mm 3 ), and the horizontal axis represents the number of days after re-implantation. When CT26.WT-hHER2 cells or CT26.WT-mock cells were re-implanted into control mice, tumor growth was observed respectively. On the other hand, when CT26.WT-hHER2 cells or CT26.WT-mock cells were re-implanted into antibody-drug conjugate (1)-treated cured mice, almost no tumor growth was observed, and rejection was confirmed. From the above, it was confirmed that immune memory against tumors was formed by administration of antibody-drug conjugate (1).

[0159] [Evaluation Example 4: ELISPOT Analysis] It was carried out using Murine IFNγ Single-Color Enzymatic ELISPOT Assay. The spleen was removed from the mice used in Evaluation Example 3, and spleen cells were prepared at 1.0 x 10 6 cells / mL using CTL test medium. In addition, each of CT26.WT-hHER2 cells and CT26.WT-mock cells was treated with 10 μg / mL of mitomycin C for 2 hours, washed, and the cells were collected and 1.0 x 10 6The antigen was prepared in cells / mL. Splenocytes and the antigen were added to PVDF-membrane plates coated with anti-IFNγ antibody at 100 μL / well each and co-cultured at 37°C for 24 hours, after which the number of IFNγ-producing splenocytes was measured. Comparison between the control group and the antibody-drug conjugate (1) group was performed using the Wilcoxon rank-sum test, with p-values ​​reported to four decimal places, and p<0.05 (two-tailed test) considered statistically significant. The results are shown in Figures 6 to 9. Compared to spleen cells of control mice treated with antibody-drug conjugates (1) and re-transplanted with CT26.WT-hHER2 cells, a significantly higher number of IFNγ-producing spleen cells was observed when treated with antigens derived from CT26.WT-hHER2 cells (P=0.0012, Figure 6). A significantly higher number of IFNγ-producing spleen cells was also observed when treated with antigens derived from CT26.WT-mock cells (P=0.0008, Figure 7). Furthermore, even after re-transplantation of CT26.WT-mock cells, the spleen cells of antibody-drug conjugate (1) treated mice showed a significantly higher number of IFNγ-producing spleen cells compared to the spleen cells of control mice when treated with antigens derived from CT26.WT-hHER2 cells (P=0.0116, Figure 8). A significantly higher number of IFNγ-producing spleen cells was also observed when treated with antigens derived from CT26.WT-mock cells (P=0.0052, Figure 9). These results suggest that antibody-drug conjugate (1) treatment healed mice induce T cells that recognize antigens derived from CT26.WT cells other than human HER2.

[0160] The results from evaluation examples 3 and 4 demonstrated that the antibody-drug conjugate (1) has the effect of promoting the formation of immunological memory against tumors. Furthermore, this effect was observed not only in tumors expressing HER2, but also in tumors of the same origin that do not express HER2.

[0161] Therefore, it has been shown that the antibody-drug conjugate used in the present invention has the effect of promoting immunological memory formation not only in tumors that express the antigen for the antibody in the antibody-drug conjugate, but also in tumors within the same individual that do not express the antigen for the antibody in the antibody-drug conjugate.

[0162] [Evaluation Example 5: Evaluation of effects on in vitro dendritic cells] After euthanasia of BALB / c mice, bone marrow cells were isolated from the femur and cultured for 11 days in 10% FBS, 55 μM 2-mercaptoethanol, 100 U / mL penicillin, 100 U / mL streptomycin, 1 mM sodium pyruvate, 1 × non-essential amino acid, 2 mM L-glutamine, 10 ng / mL mouse GM-CSF-containing RPMI 1640 medium to induce bone marrow-derived dendritic cells. Compound (A) was added to the culture medium of the induced dendritic cells at concentrations of 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM. DMSO was added as a control at the same volume as compound (A). After 24 hours, cells were stained with Pacific Blue labeled anti-mouse CD45 Antibody (103126, BioLegend), PE labeled anti-mouse CD86 (B7-2) (553692, Becton Dickinson), APC labeled anti-mouse CD11c (550261, Becton Dickinson), and FITC labeled anti-mouse MHC Class II (IA / IE) (11-5321-85, Thermo Fisher Scientific), and analyzed using FACS Canto II. Dead cells were stained with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit purchased from Thermo Fisher Scientific and excluded from the analysis.

[0163] Figures 10 and 11 show the results of flow cytometry measurements of CD86 and MHC class II expression levels in CD11c-positive cells. Compared to the control (DMSO), treatment with compound (A) was found to increase the expression levels of both CD86 and MHC class II, which are markers for dendritic cell maturation and activation.

[0164] The results of Evaluation Example 5 showed that compound (A) has the effect of activating dendritic cells.

[0165] [Evaluation Example 6: Analysis of Dendritic Cells within Tumors] Similar to Evaluation Example 1, mice were transplanted with CT26.WT-hHER2 and randomly assigned to groups 8 days later (Day 0). The antibody-drug conjugate (1) was administered intravenously at a dose of 10 mg / kg on Day 0. A control group was established, receiving a dedicated solvent for the antibody-drug conjugate (1). Each group consisted of 7 mice. On Day 8, the mice were euthanized and the tumors were excised. Using the Tumor Dissociation Kit, mouse purchased from Miltenyi Biotec, single-cell suspensions were prepared from the tumors and stained and analyzed in the same manner as in Evaluation Example 5. Comparison between the control group and the antibody-drug conjugate (1) group was performed using Student's t-test, with p-values ​​reported to 4 decimal places, and p<0.05 (two-tailed test) considered statistical significance.

[0166] The results are shown in Figures 12 to 14. It was confirmed that the proportion of CD11c, MHC class II, and CD45-positive cells (dendritic cells, DCs) among CD45-positive cells (lymphocytes) within the tumor significantly increased with the administration of an antibody-drug conjugate (1) (Figure 12). Furthermore, it was confirmed that dendritic cells expressing CD86 (an activation marker) significantly increased upon administration of the antibody-drug conjugate (1) (Figure 13). Furthermore, the expression level of CD86 on dendritic cells, as measured by MFI (mean fluorescence intensity), was also confirmed to be significantly increased by administration of the antibody-drug conjugate (1) (Figure 14). Based on these results, administration of the antibody-drug conjugate (1) to tumor-bearing mice resulted in an increase in the number of dendritic cells among tumor lymphocytes, an increase in CD86-positive cells among tumor dendritic cells, and an increase in CD86 expression on dendritic cells.

[0167] Furthermore, the results of Evaluation Example 5 indicate that compound (A), which is a drug released from antibody-drug conjugate (1), has the effect of activating dendritic cells. The "dendritic cell activating effect" of compound (A) is the same effect as the "dendritic cell activating effect" of the antibody-drug conjugate used in the present invention. In addition, compound (A) is a compound generated from the antibody-drug conjugate used in the present invention after it has migrated to cancer cells. Therefore, it is considered that a similar effect would be obtained even if the antibody portion of the antibody-drug conjugate was not an anti-HER2 antibody.

[0168] [Evaluation Example 7: Analysis of Intratumor Cancer Cells] Cell suspensions were prepared in the same manner as in Evaluation Example 6. Staining was performed with PE-labeled anti-human Her2 / neu (340552, Becton Dickinson), APC-labeled anti-mouse CD274 (B7-H1, PD-L1) (124312, BioLegend), and FITC-labeled anti-mouse H-2Dd (110606, BioLegend). The expression levels of MHC class I and PD-L1 on cancer cells were measured by flow cytometry. Dead cells were stained with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit purchased from Thermo Fisher Scientific and excluded from the analysis. Comparison between the control group and the antibody-drug conjugate (1) group was performed using Student's t-test, with P-values ​​rounded to four decimal places, and P<0.05 (two-tailed test) considered statistically significant.

[0169] The results are shown in Figures 15 and 16. The expression level of MHC class I on cancer cells (human HER2-positive cells) was significantly increased by administration of the antibody-drug conjugate (1) (Figure 15). MHC class I is a molecule necessary for T cells to recognize cancer cells. Therefore, it was suggested that the antibody-drug conjugate (1) activates anti-tumor immunity by promoting the increase in MHC class I expression on cancer cells. Furthermore, it was confirmed that the expression level of PD-L1 on cancer cells was significantly increased by the antibody-drug conjugate (1) (Figure 16). PD-L1 is known to act on PD-1 on T cells to impart immunosuppressive signals. Therefore, it is suggested that the antibody-drug conjugate (1) suppresses antitumor immunity by promoting the increase in PD-L1 expression on cancer cells, and that when used in combination with a PD-1 antibody, this suppressive signal is released, resulting in a stronger antitumor effect.

[0170] [Evaluation Example 8: In vitro cancer cell analysis] Compound (A) was added to the culture medium of CT26.WT-hHER2 cells at concentrations of 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM. DMSO was added as a control at the same volume as compound (A). After 24 hours, the cells were stained with PE-labeled anti-human Her2 / neu (340552, Becton Dickinson) and FITC-labeled anti-mouse H-2Dd (110606, BioLegend), and the expression level of MHC class I on cancer cells was measured by flow cytometry. Dead cells were stained with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit purchased from Thermo Fisher Scientific and excluded from the analysis. The mean fluorescence intensity (MFI) of MHC class I was calculated, and the adjusted MFI was defined as the MFI of the stained cells minus the MFI of the cells treated with the isotype control. Comparisons between the control group and compound (A) group were performed using Dunnett's test, with p-values ​​reported to four decimal places, and p<0.05 (two-tailed test) considered statistically significant. The results are shown in Figure 17. The expression level of MHC class I on CT26.WT-hHER2 cells was significantly increased by compound (A) (Figure 17). Therefore, it was suggested that compound (A) activates anti-tumor immunity by promoting the increase in MHC class I expression on cancer cells.

[0171] [Evaluation Example 9: Antitumor Test Using Nude Mice] Mice: Six-week-old female BALB / c-nu mice (CAnN.Cg-Foxn1[nu] / CrlCrlj〔Foxn1nu / Foxn1nu〕) (Charles River Co., Ltd. Japan) were used in the experiment. Measurement and calculation formula: The longest and shortest diameters of the tumor were measured twice a week using an electronic digital caliper (CD15-CX, Mitutoyo Corporation), and the tumor volume (mm³) was calculated. 3 The calculation was performed. The formula is as follows: Tumor volume (mm 3 ) = 0.5 × major axis (mm) × minor axis (mm) 2 Tumor volume is 3000 mm 3 Individuals exceeding a certain threshold were euthanized from the perspective of animal experimentation ethics.

[0172] The antibody-drug conjugate (1) was administered intravenously at a dose of 10 mL / kg. CT26.WT-hHER2 cells were suspended in physiological saline and 5.0 × 10⁶ cells were prepared. 6 Cells were subcutaneously transplanted into the right axilla of BALB / c-nu mice, and randomization was performed 3 days later (Day 0). The antibody-drug conjugate (1) was administered intravenously twice at a dose of 10 mg / kg on Days 0 and 7. A control group was established, receiving only the solvent for the antibody-drug conjugate (1). Each group consisted of 12 mice, and tumor volume was measured until Day 13.

[0173] The results are shown in Figure 18. The vertical axis represents tumor volume (mm³). 3 The horizontal axis shows the number of days from the first administration date. The antitumor effect observed in BALB / c mice with antibody-drug conjugate (1) administration was not observed in BALB / c-nu mice. In BALB / c-nu mice, the number of T cells and B cells was reduced, and their function was impaired, suggesting that these cells play an important role in the antitumor effect of antibody-drug conjugate (1).

[0174] [Evaluation Example 10: Antitumor Trial] Tumor volume changes were measured in mice subcutaneously transplanted with CT26.WT-hHER2 cells using the same method as in Evaluation Example 1 for the antibody-drug conjugate (1) administration group, the control antibody-drug conjugate administration group, and the control group. A control antibody-drug conjugate (Drug-to-Antibody Ratio: 7.8) using a human IgG1 antibody that binds to molecules other than those derived from mice and humans was used after being diluted with a dedicated solvent. Group assignment was performed 5 days after transplantation (Day 0). The control antibody-drug conjugate and antibody-drug conjugate (1) were administered intravenously in the tail vein twice at a dose of 10 mg / kg on Days 0 and 7. Each group consisted of 10 mice, and tumor volume was measured until Day 10. The efficacy of the control antibody-drug conjugate group and the antibody-drug conjugate (1) group was compared using the Wilcoxon rank-sum test. P values ​​were recorded to four decimal places, and P < 0.05 (two-tailed test) was considered statistically significant.

[0175] The results are shown in Figure 19. The vertical axis represents tumor volume (mm³). 3 The horizontal axis of the graph shows the number of days from the first administration date. At Day 10, the antibody-drug conjugate (1) group showed significantly superior antitumor efficacy compared to the control antibody-drug conjugate group (P=0.0003). Therefore, it was confirmed that the antitumor effect of antibody-drug conjugate (1) is target-dependent.

[0176] [Evaluation Example 11: Antitumor Trial] Tumor volume changes were measured in mice subcutaneously transplanted with EMT6-hHER2 cells, using the same method as in Evaluation Example 1, for monotherapy and combination therapy with antibody-drug conjugate (1) and anti-PD-1 antibody (clone RMP1-14). EMT6-hHER2 cells were created by introducing the human HER2 gene into the mouse mammary cancer cell line EMT6 (CRL-2755), purchased from American Type Culture Collection, using a lentiviral vector. These cells express the human HER2 protein on their cell membrane. EMT6-hHER2 cells were suspended in physiological saline and 1.0 × 10⁶ cells were used. 6Cells were subcutaneously transplanted into the right axilla of 5-week-old BALB / c mice, and randomization was performed 4 days after transplantation (Day 0). The antibody-drug conjugate (1) was administered once via tail vein at a dose of 10 mg / kg on Day 0. The anti-PD-1 antibody (clone RMP1-14) was prepared in D-PBS(-)(WAKO) and administered via tail vein a total of four times at a dose of 5.0 mg / kg on Days 0, 3, 7, and 10. In addition, a group receiving combination therapy with the antibody-drug conjugate (1) and the anti-PD-1 antibody, and a control group receiving only the specialized solvent for the antibody-drug conjugate (1) were established. Each group consisted of 11 mice, and tumor volume was measured until Day 17. The efficacy of the control group, the antibody-drug conjugate (1) group, and the anti-PD-1 antibody group was compared, as was the efficacy of the antibody-drug conjugate (1) and anti-PD-1 antibody groups compared with the group using both drugs in combination. Dunnett's test (multi-group comparison) was used to compare these efficacy levels. Multiplex-adjusted p-values ​​are presented to four decimal places, and a p-value of < 0.05 (two-tailed test) was considered statistically significant.

[0177] The results are shown in Figure 20. The vertical axis represents tumor volume (mm³). 3 The horizontal axis of the graph shows the number of days from the first administration date. At Day 17, the antibody-drug conjugate (1) group showed significantly superior antitumor effects compared to the control group (P<0.0001). The anti-PD-1 antibody group also showed significantly superior antitumor effects compared to the control group (P<0.0001). Furthermore, the combination therapy group showed significantly superior antitumor effects compared to the antibody-drug conjugate (1) group (P=0.0136). The combination therapy group also showed significantly superior antitumor effects compared to the anti-PD-1 antibody group (P=0.0372). In addition, no weight loss was observed in mice in any of the groups in this study. From the above, the antitumor effects of monotherapy with both drugs were confirmed, and it was confirmed that the effect was dramatically enhanced by the combination therapy of the two drugs.

[0178] [Evaluation Example 12: Life Sustaining Test] The survival-prolonging effect on mice subcutaneously transplanted with CT26.WT-hHER2 cells was measured in monotherapy and combination therapy groups of antibody-drug conjugate (1) and anti-PD-L1 antibody, using the same method as in Evaluation Example 1. The anti-PD-L1 antibody (clone 10F.9G2) was purchased from Bio X Cell and diluted with InVivoPure pH6.5 Dilution Buffer (Bio X Cell). Routing was performed 6 days after transplantation (Day 0). Antibody-drug conjugate (1) was administered intravenously at a dose of 10 mg / kg twice, on Day 0 and Day 7. The anti-PD-L1 antibody was administered intravenously at a dose of 5 mg / kg twice, on Day 0 and Day 3. In addition, a group receiving combination therapy with antibody-drug conjugate (1) and anti-PD-L1 antibody, and a control group receiving the dedicated solvent for antibody-drug conjugate (1) were established. Each group consisted of 15 mice, and tumor volume was measured until Day 38. The estimated tumor volume was 3000 mm³. 3 The day exceeding the threshold (the day of euthanasia) was defined as the event date (date of death). Survival times were compared between the control group, the antibody-drug conjugate (1) group, and the anti-PD-L1 antibody group, and between the antibody-drug conjugate (1) group and the anti-PD-L1 antibody group and the combined drug group. These comparisons were performed using the Kaplan-Meier log-rank test (multi-group comparison). Multiplex-adjusted p-values ​​are presented to four decimal places, and P<0.05 (two-tailed test) was considered statistically significant.

[0179] The results are shown in Figure 21. Compared to the control group, the antibody-drug conjugate (1) group showed significantly superior antitumor effects (P=0.0069). Also, compared to the control group, the anti-PD-L1 antibody group showed significantly superior antitumor effects (P=0.0037). Furthermore, compared to the antibody-drug conjugate (1) group, the combination therapy group showed significantly superior antitumor effects (P=0.0059). Also, compared to the anti-PD-L1 antibody group, the combination therapy group showed significantly superior antitumor effects (P=0.0091). In addition, no weight loss was observed in mice in any of the groups in this study. From the above, the antitumor effects of monotherapy with both drugs were confirmed, and it was confirmed that the effect was enhanced by the combination therapy of the two drugs.

[0180] [Evaluation Example 13: Life Sustaining Test] EMT6-hHER2 cells were subcutaneously transplanted into mice using the same method as in Evaluation Example 11, and the survival effect was measured in groups receiving either antibody-drug conjugate (1) or anti-PD-L1 antibody alone or in combination, as in Evaluation Example 12. Grouping was randomized 5 days after transplantation (Day 0). Antibody-drug conjugate (1) was administered once via tail vein at a dose of 10 mg / kg on Day 0. Anti-PD-L1 antibody was administered twice via tail vein at a dose of 5 mg / kg on Days 0 and 3. Additionally, a group receiving combination therapy with antibody-drug conjugate (1) and anti-PD-L1 antibody, and a control group receiving only the specialized solvent for antibody-drug conjugate (1) were established. Each group consisted of 6 mice, and tumor volume was measured until Day 60. Survival times were compared between the control group, the antibody-drug conjugate (1) group, and the anti-PD-L1 antibody group, as well as between the antibody-drug conjugate (1) group, the anti-PD-L1 antibody group, and the combined antibody-drug group using the Kaplan-Meier log-rank test (multi-group comparison). Multiplex-adjusted p-values ​​are presented to four decimal places, and P<0.05 (two-tailed test) was considered statistically significant.

[0181] The results are shown in Figure 22. Compared to the control group, the antibody-drug conjugate (1) group showed significantly superior antitumor effects (P=0.0006). Also, compared to the control group, the anti-PD-L1 antibody group showed significantly superior antitumor effects (P=0.0227). Furthermore, compared to the antibody-drug conjugate (1) group, the combination therapy group showed significantly superior antitumor effects (P=0.0039). In addition, no weight loss was observed in mice in any of the groups in this study. From the above, the antitumor effects of monotherapy with both drugs were confirmed, and it was confirmed that the effect was enhanced by the combination therapy of the two drugs.

[0182] [Evaluation Example 14: In vivo CD4 / 8 depletion study] Tumor volume changes were measured in mice subcutaneously transplanted with CT26.WT-hHER2 cells using the same method as in Evaluation Example 1, for both monotherapy and combination therapy with antibody-drug conjugate (1) and anti-CD4 antibody, as well as for monotherapy and combination therapy with antibody-drug conjugate (1) and anti-CD8 antibody. Antibody-drug conjugate (1) was administered at 10 mg / kg, while the depletion antibodies, anti-CD4 antibody (Bio X Cell, clone GK1.5) and anti-CD8 antibody (Bio X Cell, clone 53.6.7), were prepared to 1 mg / mL using D-PBS(-) immediately before administration. Each was administered intravenously to the tail vein at a dose of 200 μg / head to mice on days 0 and 7. A control group was also established, receiving only the specialized solvent for antibody-drug conjugate (1). Group assignment was performed on day 5 after transplantation (Day 0), and tumor volume was measured until Day 11.

[0183] The results are shown in Figures 23 and 24. The tumor volume in the antibody-drug conjugate (1) group on Day 11 was 651 mm². 3 The group using the antibody-drug conjugate (1) and anti-CD4 antibody combination was 561 mm. 3 Therefore, it was considered that CD4-positive cells did not contribute to the antitumor effect of the antibody-drug conjugate (1) (Figure 23). The tumor volume of the antibody-drug conjugate (1) group on Day 11 was 651 mm². 3 The group using the antibody-drug conjugate (1) and anti-CD8 antibody combination was 2247 mm. 3 Therefore, it was considered that CD8-positive cells contribute to the antitumor effect of the antibody-drug conjugate (1) (Figure 24). Furthermore, in the anti-CD4 antibody group and the anti-CD8 antibody group, some individuals had a tumor volume of 3000 mm³. 3 Because the growth rate exceeded a certain threshold, euthanasia was performed midway through the experiment. As a result, the tumor growth curve is interrupted at the point where euthanasia was performed. In the tumor model using immunodeficient nude mice in evaluation example 9, it was shown that T cells or B cells are involved in part of the drug effect of antibody-drug conjugate (1). Furthermore, based on these results, it is suggested that CD8-positive T cells contribute to the antitumor effect of antibody-drug conjugate (1).

[0184] [Evaluation Example 15: Tumor T Cell Analysis] In mice subcutaneously transplanted with CT26.WT-hHER2 cells, the proportion of CD8-positive T cells among living tumor cells, the proportion of Granzyme B-positive cells among CD8-positive T cells, the proportion of Granzyme B-positive CD8-positive T cells among living tumor cells, and the proportion of CD4-positive T cells among living tumor cells were measured by flow cytometry. Cell suspensions were prepared using the same method as in Evaluation Example 6. They were then stained with Pacific Blue labeled anti-mouse CD45 antibody (103126, BioLegend), PE labeled anti-mouse CD3e antibody (553064, Becton Dickinson), PerCP / Cy5.5 labeled anti-mouse CD4 antibody (100434, BioLegend), PE-Cy 7 labeled anti-mouse CD8a antibody (552877, Becton Dickinson), and Alexa FluorR 647 labeled anti-human / mouse Granzyme B antibody (515405, BioLegend), and measured by flow cytometry. Dead cells were stained with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit purchased from Thermo Fisher Scientific and excluded from the analysis. Comparisons between the control group and the antibody-drug conjugate (1) group were performed using Student's t-test, with p-values ​​reported to four decimal places, and p<0.05 (two-tailed test) considered statistically significant.

[0185] The results are shown in Figures 25 to 28. The proportion of CD45, CD3, and CD8-positive cells (CD8-positive T cells) among living cells was confirmed to increase significantly with the administration of antibody-drug conjugate (1) (Figure 25). The proportion of Granzyme B-positive cells among CD8-positive T cells was confirmed to increase significantly with the administration of antibody-drug conjugate (1) (Figure 26). The proportion of Granzyme B-positive CD8-positive T cells among living cells was confirmed to increase significantly with the administration of antibody-drug conjugate (1) (Figure 27). The proportion of CD45, CD3, and CD4-positive cells (CD4-positive T cells) among living cells showed an increasing trend with the administration of antibody-drug conjugate (1), but the difference was not statistically significant (Figure 28). Therefore, it was suggested that the antibody-drug conjugate (1) activates anti-tumor immunity by increasing the number of CD8-positive T cells in tumors and promoting their activation.

[0186] [Evaluation example 16: CD8 IHC analysis] In mice subcutaneously transplanted with CT26.WT-hHER2 cells, the number of CD8-positive cells per unit area in the tumor was measured by IHC after administration of an antibody-drug conjugate (1). Using the same method as in Evaluation Example 6, control groups and the antibody-drug conjugate (1) were administered, and the mice were euthanized 8 days after administration. Each group consisted of 5 mice, and tumors from a median of 3 mice were excised, infiltrated into 4% paraformaldehyde-phosphate buffer, and paraffin blocks were prepared. The tissues were stained with anti-CD8 antibody (clone: ​​4SM16), and sample images were captured using NanoZoomer 2.0-HT (Hamamatsu Photonics). The entire tissue region was analyzed using the image analysis software Tissue Studio 3.0 (Definiens).

[0187] The results are shown in Figures 29 and 30. It was confirmed that the antibody-drug conjugate (1) tended to increase the number of CD8-positive cells per unit area within the tumor. [Evaluation Example 17: In vitro cancer cell analysis] The expression levels of MHC class I were measured when cancer cells were treated with various compounds. Using the same method as in Evaluation Example 8, compound (A), DM1-SMe, DM4-SMe (J. Med. Chem. (2014), 57, 16, 6949-6964), and MMAE (Molecular Cancer Therapeutics (2011), 10, 9, 1728-1739) were added at concentrations of 20 nM, 100 nM, and 500 nM, and the expression levels of MHC class I on cancer cells were measured by flow cytometry. The experiment was performed using a triplicate. Comparisons between the control group and each drug concentration group were performed using Dunnett's test, with p-values ​​reported to four decimal places, and P<0.05 (two-tailed test) considered statistical significance (***: P<0.001, **: P<0.01).

[0188] The results are shown in Figure 31. Among the concentrations examined (20 nM, 100 nM, 500 nM), all evaluated drugs significantly increased MHC class I expression on CT26.WT-hHER2 cells compared to the control group. Of these, compound (A) showed the greatest increase in MHC class I expression on CT26.WT-hHER2 cells.

[0189] [Evaluation Example 18: Antitumor Trial] Tumor volume changes were measured in mice subcutaneously transplanted with EMT6-hHER2 cells, using the same method as in Evaluation Example 11, for monotherapy and combination therapy with antibody-drug conjugate (1) and anti-CTLA-4 antibody. Anti-CTLA-4 antibody (clone 9H10) was purchased from Bio X Cell and used after dilution with D-PBS(-). Group assignment was performed on day 5 post-transplant (Day 0). Antibody-drug conjugate (1) was administered once via tail vein at a dose of 10 mg / kg on Day 0. Anti-CTLA-4 antibody was administered three times via tail vein at a dose of 5.0 mg / kg on Days 0, 3, and 7. In addition, a group receiving combination therapy with antibody-drug conjugate (1) and anti-CTLA-4 antibody, and a control group receiving only the dedicated solvent for antibody-drug conjugate (1) were established. Each group consisted of 10 mice, and tumor volume was measured until Day 14. The efficacy of the control group, the antibody-drug conjugate (1) group, and the anti-CTLA-4 antibody group was compared, as was the efficacy of the antibody-drug conjugate (1) and anti-CTLA-4 antibody groups compared with the group using both drugs in combination. Dunnett's test (multi-group comparison) was used to compare these efficacy levels. Multiplex-adjusted p-values ​​are presented to four decimal places, and a p-value of < 0.05 (two-tailed test) was considered statistically significant.

[0190] The results are shown in Figure 32. The vertical axis represents tumor volume (mm³). 3 The horizontal axis of the graph shows the number of days from the first administration date. At Day 14, the antibody-drug conjugate (1) group showed significantly superior antitumor effects compared to the control group (P=0.0011). The anti-CTLA-4 antibody group also showed significantly superior antitumor effects compared to the control group (P=0.0006). Furthermore, the combination therapy group showed significantly superior antitumor effects compared to the antibody-drug conjugate (1) group (P=0.0115). The combination therapy group also showed significantly superior antitumor effects compared to the anti-CTLA-4 antibody group (P=0.0309). In summary, the antitumor effects of monotherapy with both drugs were confirmed, and it was confirmed that the effect was dramatically enhanced by the combination therapy of the two drugs.

[0191] From the experimental results described above, it was found that the antibody-drug conjugate according to the present invention exhibits dramatically superior antitumor effects when administered in combination with an immune checkpoint inhibitor. Furthermore, it was shown that the antibody-drug conjugate according to the present invention has the effect of activating antitumor immunity. This makes it possible to provide a pharmaceutical composition and treatment method that are superior in terms of antitumor effect and safety. [Sequence Listing Free Text]

[0192] Sequence ID 1: Amino acid sequence of the humanized anti-HER2 antibody heavy chain Sequence ID 2: Amino acid sequence of the light chain of a humanized anti-HER2 antibody

Claims

1. A combination drug comprising an antibody-drug conjugate and an immune checkpoint inhibitor, An antibody-drug conjugate and an immune checkpoint inhibitor are administered in combination. The antibody-drug conjugate is, 【Chemistry 1】 (In the formula, A indicates the binding site with the antibody.) This is an antibody-drug conjugate in which a drug linker, shown as indicated, and an antibody are linked by a thioether bond. The antibody in the antibody-drug conjugate is an anti-HER2 antibody. Anti-HER2 antibodies, An antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449, and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214, or An antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2, Immune checkpoint inhibitors An anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, and clone RMP1-14. An anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, avelumab, and clone 10F.9G2, or This is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and clone 9H10. Combination medicine.

2. A pharmaceutical composition comprising an antibody-drug conjugate for use in combination with an immune checkpoint inhibitor, The antibody-drug conjugate is, 【Chemistry 2】 (In the formula, A indicates the binding site with the antibody.) This is an antibody-drug conjugate in which a drug linker, shown as indicated, and an antibody are linked by a thioether bond. The antibody in the antibody-drug conjugate is an anti-HER2 antibody. Anti-HER2 antibodies, An antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449, and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214, or An antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2, Immune checkpoint inhibitors An anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, and clone RMP1-14. An anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, avelumab, and clone 10F.9G2, or This is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and clone 9H10. Pharmaceutical composition.

3. The combination pharmaceutical or pharmaceutical composition according to claim 1 or 2, wherein the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 to 449 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 to 214.

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

2.

5. The combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 4, wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

6. The combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 4, 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 combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 4, wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 7.5 to 8.

8. The combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 7, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, and clone RMP1-14.

9. The combination pharmaceutical or pharmaceutical composition according to claim 8, wherein the immune checkpoint inhibitor is nivolumab.

10. The combination pharmaceutical or pharmaceutical composition according to claim 8, wherein the immune checkpoint inhibitor is pembrolizumab.

11. The combination pharmaceutical or pharmaceutical composition according to claim 8, wherein the immune checkpoint inhibitor is clone RMP1-14.

12. The combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 7, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, avelumab, and clone 10F.9G2.

13. The combination pharmaceutical or pharmaceutical composition according to claim 12, wherein the immune checkpoint inhibitor is atezolizumab.

14. The combination pharmaceutical or pharmaceutical composition according to claim 12, wherein the immune checkpoint inhibitor is durvalumab.

15. The combination pharmaceutical or pharmaceutical composition according to claim 12, wherein the immune checkpoint inhibitor is avelumab.

16. The combination pharmaceutical or pharmaceutical composition according to claim 12, wherein the immune checkpoint inhibitor is clone 10F.9G2.

17. The combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 7, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, and clone 9H10.

18. A combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 17, characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are each contained as active ingredients in separate formulations and administered simultaneously or at different times.

19. A combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 17, characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are contained as active ingredients in a single formulation and administered.

20. A combination pharmaceutical or pharmaceutical composition according to any one of claims 1 to 19, which is used for the treatment of cancer.

21. The combination pharmaceutical or pharmaceutical composition according to claim 20, wherein the cancer is at least one selected from the group consisting of lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastroesophageal junction adenocarcinoma, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer, mesothelioma, Paget's disease, and sarcoma.

22. The combination pharmaceutical or pharmaceutical composition according to claim 20, wherein the cancer is colorectal cancer.

23. The combination pharmaceutical or pharmaceutical composition according to claim 20, wherein the cancer is breast cancer.

24. The combination pharmaceutical or pharmaceutical composition according to claim 20, wherein the cancer is lung cancer.

25. The combination pharmaceutical or pharmaceutical composition according to claim 20, wherein the cancer is bladder cancer.

26. The combination pharmaceutical or pharmaceutical composition according to claim 20, wherein the cancer is gastric cancer.

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