Combination therapy with anti-CCR8 antibodies and chemotherapy agents

Abstract

The combined use of an anti-CCR8 antibody and a chemotherapeutic agent was found to be useful in the treatment or prevention of cancer.

Description

[Technical Field]

[0001] This invention relates to the combined use of an anti-CCR8 antibody and a chemotherapeutic agent. [Background technology]

[0002] The powerful negative regulatory mechanisms, including immunosuppression mediated by regulatory T cells (Treg cells) within the tumor microenvironment, pose a major obstacle to tumor treatment (Non-Patent Literature 1). For example, CD4-positive Treg cells infiltrating tumors may strongly inhibit the anti-tumor immune response, which could be a major obstacle to effective cancer treatment. Tumor immunosuppression mediated by CD4-positive FoxP3-positive Treg cells has been well-established in animal tumor models, and antitumor effects can be obtained by systemic removal of Treg cells, including those within the tumor. However, it has been reported that removal of about 50% of Treg cells that have infiltrated the tumor does not produce any effect (Non-Patent Literature 2).

[0003] In humans, an increase in the ratio of CD4-positive CD25-positive Treg cells (the cell population including Treg cells) within the overall CD4-positive T cell population has been detected in tumors of various cancer patients, including lung, breast, and ovarian tumors, and a negative correlation has been reported between the abundance of Treg cells and patient survival rates (Non-patent documents 3-8).

[0004] CCR8, also known as CY6, CKR-L1, or TER1, is a G protein-coupled, seven-transmembrane CC chemokine receptor protein expressed in organs such as the thymus and spleen. Its gene is located in chromosome 3p21. Human CCR8 consists of 355 amino acids (Non-Patent Literature 9). CCL1 is known as an endogenous ligand for CCR8 (Non-Patent Literature 10). Human CCR8 cDNA is composed of the nucleotide sequence shown in Genbank ACC No. NM_005201.3, and mouse CCR8 cDNA is composed of the nucleotide sequence shown in Genbank ACC No. NM_007720.2.

[0005] CCR8 is specifically expressed in tumor-infiltrating Treg cells, and studies have shown that when mammary cancer cells were transplanted into CCR8-deficient mice and wild-type mice, the proliferation and metastasis of mammary cancer in CCR8-deficient mice were suppressed compared to wild-type mice (Patent Document 1 and Non-Patent Document 11). Furthermore, it has been disclosed that administering anti-CCR8 antibodies to cancer model animals showed antitumor effects (Patent Documents 2, 3, and 9).

[0006] Regarding the combined use of anti-CCR8 antibodies with other drugs, the following reports have been made: Patent documents 2 and 3 show the antitumor effect of the combined use of anti-CCR8 antibodies and anti-PD-1 antibodies. Patent document 4 shows the antitumor effect of the combined use of anti-CCR8 antibodies and Listeria cancer vaccine. Patent document 5 discloses a biantibody against CCR8 and CTLA-4. However, all of these relate to the combined use of anti-CCR8 antibodies with immunotherapy agents, and the combined use of anti-CCR8 antibodies with chemotherapeutic agents is not shown at all.

[0007] Regarding the combination of immunotherapeutic agents other than anti-CCR8 antibodies with chemotherapeutic agents, the following reports have been made: Patent Document 6 shows the antitumor effect of combining an anti-PD-1 antibody with axitinib, a VEGF receptor inhibitor. Patent Document 7 shows the antitumor effect of combining an anti-PD-1 antibody with dinacyclib. Patent Document 8 shows the antitumor effect of combining an anti-CTLA-4 antibody with various chemotherapeutic agents such as etoposide. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 10087259 [Patent Document 2] International Publication No. 2018 / 181425 [Patent Document 3] International Publication No. 2018 / 112032 [Patent Document 4] International Publication No. 2019 / 157098 [Patent Document 5] China Patent Publication No. 110835374 [License 6] Patent No. 6591428 [License 7] Patent No. 6586087 [License 8] Patent No. 5589077 [License 9] International Publication No. 2020 / 138489 [Non-licensed literature]

[0009] [Non-licensed Document 1] Nat. Rev. Immunol., 2006, Vol. 6, No. 4, pp. 295-307 [Non-licensed Document 2] Eur. J. Immunol., 2010, Vol. 40, pp. 3325-3335 [Non-licensed Document 3] J. Clin. Oncol., 2006, Vol. 24, pp. 5373-5380 [Non-licensed Document 4] Nat. Med., 2004, Volume 10, p.942-949 [Non-licensed Document 5] J. Clin. Oncol., 2007, Vol. 25, pp. 2586-2593 [Non-licensed Document 6] Cancer, 2006, Volume 107, p.2866-2872 [Non-licensed Document 7] Eur. J. Cancer, 2008, Volume 44, p.1875-1882 [Non-licensed Document 8] Cell. Mol. Immunol. 2011, Volume 8, p.59-66 [Non-licensed Document 9] J. Immunol., 1996, Vol. 157, No. 7, pp. 2759-63 [Non-licensed Document 10] J. Biol. Chem., 1997, Volume 272, No. 28, p.17251-4 [Non-Patent Document 11] Cancer Res., 2016, Vol. 76, No. 4, Supp. 1, P4-04-11 [Overview of the project] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a combination of an anti-CCR8 antibody and a chemotherapeutic agent. Furthermore, it is to provide a combination of an anti-CCR8 antibody and a chemotherapeutic agent that is useful for cancer treatment. [Means for solving the problem]

[0011] As a result of diligent research, the inventors have found that administering an anti-CCR8 antibody and a chemotherapeutic agent exhibits a combined effect. Furthermore, they have found that the combined use of the anti-CCR8 antibody and chemotherapeutic agent of the present invention is useful for the treatment or prevention of cancer.

[0012] This invention relates to the following: (1) A pharmaceutical composition containing an anti-CCR8 antibody, to be used together with a chemotherapeutic agent. (2) A pharmaceutical composition containing a chemotherapeutic agent, to be used together with an anti-CCR8 antibody. (3) A pharmaceutical composition according to (1) or (2) for the treatment of cancer. (4) The pharmaceutical composition according to (3), for the treatment of cancer in which treatment with anti-PD-1 antibody or anti-PD-L1 antibody is ineffective. (5) A pharmaceutical composition according to any one of (1) to (4), wherein the cancer is breast cancer, lung cancer, colorectal cancer, kidney cancer, sarcoma, liver cancer, bladder cancer, or ovarian cancer. (6) The pharmaceutical composition according to (5), wherein the cancer is breast cancer, lung cancer, bladder cancer, kidney cancer, or colorectal cancer. (6') The pharmaceutical composition according to (5), wherein the cancer is breast cancer, lung cancer, bladder cancer, or colorectal cancer. (7) The pharmaceutical composition according to (5), wherein the cancer is breast cancer, lung cancer, or colorectal cancer. (8) A pharmaceutical composition according to any one of (1) to (7), wherein the chemotherapeutic agent is a platinum complex, a taxane, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide, or doxorubicin. (8') A pharmaceutical composition according to any one of (1) to (7), wherein the chemotherapeutic agent is a platinum complex, a taxane, gemcitabine, or fluorouracil. (9) The pharmaceutical composition according to (8), wherein the chemotherapeutic agent is gemcitabine. (10) The pharmaceutical composition according to (8), wherein the chemotherapeutic agent is a platinum complex, and the platinum complex is carboplatin, cisplatin, or oxaliplatin. (11) The pharmaceutical composition according to (10), wherein the platinum complex is carboplatin. (12) The pharmaceutical composition according to (8), wherein the chemotherapeutic agent is a taxane, and the taxane is paclitaxel or docetaxel. (13) The pharmaceutical composition according to (8), wherein the chemotherapeutic agent is fluorouracil. (14) Anti-CCR8 antibody, 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 Heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7 (Here, it is possible to have one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine. C) The fourth asparagine molecule in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine. D) The fifth leucine in the amino acid sequence of SEQ ID NO: 3 is replaced with isoleucine. E) The fourth leucine in the amino acid sequence of SEQ ID NO: 4 is replaced with isoleucine. F) The 6th tyrosine in the amino acid sequence of SEQ ID NO: 4 is replaced with phenylalanine. G) The 16th serine in the amino acid sequence of SEQ ID NO: 6 is replaced with threonine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO: 6 is replaced with glutamic acid. 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7; 3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 10, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 11; (Here, it is possible to have one or more of the following substitutions: A) The second serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. B) The third serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. C) The fifth serine in the amino acid sequence of SEQ ID NO: 2 is replaced with threonine. D) The second glutamine in the amino acid sequence of Sequence ID No. 10 is replaced with asparagine. E) The first threonine in the amino acid sequence of sequence number 8 is replaced with serine. F) The 14th alanine in the amino acid sequence of sequence number 6 is replaced with valine. G) The 18th lysine in the amino acid sequence of sequence number 6 is replaced with arginine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO. 6 is replaced with glutamic acid. I) The fifth asparagine in the amino acid sequence of SEQ ID NO: 11 is replaced with glutamine. J) The 12th tyrosine in the amino acid sequence of sequence number 11 is replaced with phenylalanine. 4) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 10, and CDR1, consisting of the amino acid sequence of SEQ ID NO: 12, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 13; 5) CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 and the amino acid sequence of SEQ ID NO: 15 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 16, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 17, CDR2 and the amino acid sequence of SEQ ID NO: 18 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 19; or 6) CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 22, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 Heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 25 A pharmaceutical composition according to any one of (1) to (13), which is a monoclonal antibody or antibody fragment containing the above. (15) Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 It includes a heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7, It has one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine; D) The fifth leucine in the amino acid sequence of SEQ ID NO: 3 is replaced with isoleucine; E) The fourth leucine in the amino acid sequence of SEQ ID NO: 4 is replaced with isoleucine. F) The 6th tyrosine in the amino acid sequence of SEQ ID NO: 4 is replaced with phenylalanine. G) The 16th serine in the amino acid sequence of SEQ ID NO: 6 is replaced with threonine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO. 6 is replaced with glutamic acid. The pharmaceutical composition according to (14), which is a monoclonal antibody or antibody fragment containing the above. (16) The anti-CCR8 antibody is a monoclonal antibody or antibody fragment having one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with leucine or arginine; C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine. (15) The pharmaceutical composition described above. (17) The anti-CCR8 antibody has the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 replaced with glutamine. Furthermore, there is one of the following substitutions: It is a monoclonal antibody or antibody fragment that may be present. A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with leucine or arginine. The pharmaceutical composition described in (15) or (16). (18) The pharmaceutical composition according to (17), wherein the anti-CCR8 antibody is a monoclonal antibody or antibody fragment in which the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine, and the eleventh glycine in the amino acid sequence of SEQ ID NO: 2 is further replaced with arginine. (18') Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 10, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 It contains a heavy chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 11, The pharmaceutical composition according to (14), wherein the monoclonal antibody or antibody fragment is a monoclonal antibody or antibody fragment in which the 18th lysine of the amino acid sequence of SEQ ID NO: 6 is substituted with arginine. (18'') Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 22, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 25; The pharmaceutical composition according to (14), which is a monoclonal antibody or antibody fragment containing the above. (19) The pharmaceutical composition according to any one of (14) to (18), wherein the anti-CCR8 antibody is a humanized monoclonal antibody or an antibody fragment thereof. (20) A humanized monoclonal antibody or a fragment thereof 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 3) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 4) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 5) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 6) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 7) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 8) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 9) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 10) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 11) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; or 12) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 Includes, It may have one or more of the following substitutions: A) The 33rd asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine; D) The 59th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; E) The 97th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; F) The 99th tyrosine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with phenylalanine; G) The 65th serine in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with threonine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with glutamic acid. (19) The pharmaceutical composition described above. (21) A humanized monoclonal antibody or an antibody fragment thereof has one or more of the following substitutions: A) The 33rd asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine; D) The 59th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; E) The 97th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; F) The 99th tyrosine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with phenylalanine; G) The 65th serine in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with threonine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with glutamic acid. (20) The pharmaceutical composition described above. (22) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, It has one or more of the following substitutions: A) Substitution of asparagine at position 33 of the amino acid sequence of SEQ ID NO: 40 or 42 with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) Substitution of asparagine at position 58 of the amino acid sequence of SEQ ID NO: 40 or 42 with glutamine; D) The 59th leucine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with isoleucine; E) The 97th leucine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with isoleucine; F) The 99th tyrosine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with phenylalanine; G) The 65th serine molecule in the amino acid sequence of SEQ ID NO: 41 is replaced with threonine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 is replaced with glutamic acid. (21) The pharmaceutical composition described above. (23) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, It has one or more of the following substitutions: A) Substitution of asparagine at position 33 of the amino acid sequence of SEQ ID NO: 40 or 42 with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine. The pharmaceutical composition described in (21) or (22). (24) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, In the amino acid sequence of SEQ ID NO: 40 or 42, the 58th asparagine is substituted with glutamine. Furthermore, it may have one of the following substitutions: A) Substitution of asparagine at position 33 of the amino acid sequence of SEQ ID NO: 40 or 42 with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine. A pharmaceutical composition as described in any of (21) to (23). (25) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, In the amino acid sequence of SEQ ID NO: 42, the 58th asparagine molecule is replaced with glutamine, and the 34th glycine molecule is replaced with arginine. (24) The pharmaceutical composition described above. (26) The pharmaceutical composition according to (24), wherein the humanized monoclonal antibody or antibody fragment thereof has a light chain variable region having the amino acid sequence of SEQ ID NO: 59 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41. (26') The pharmaceutical composition according to (19), wherein the humanized monoclonal antibody or antibody fragment thereof has a light chain variable region having the amino acid sequence of SEQ ID NO: 56 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 60. (27) A humanized monoclonal antibody or a fragment thereof Furthermore, a light chain constant region having the amino acid sequence of SEQ ID NO: 52, and It has a heavy chain constant region having the amino acid sequence of SEQ ID NO: 53, A pharmaceutical composition according to any one of (19) to (26), wherein lysine may or may not be added to the C-terminus of sequence number 53. (28) The pharmaceutical composition according to any one of (1) to (27), wherein the anti-CCR8 antibody is a neutralizing antibody. (29) The pharmaceutical composition according to any one of (1) to (27), wherein the anti-CCR8 antibody has ADCC activity. (30) The pharmaceutical composition according to any one of (1) to (27), wherein the anti-CCR8 antibody is an IgG antibody. (31) A method for treating cancer, characterized by administering an anti-CCR8 antibody for use in conjunction with a chemotherapeutic agent. (32) A method for treating cancer, characterized by administering a chemotherapeutic agent for use in conjunction with an anti-CCR8 antibody. (33) An anti-CCR8 antibody used in conjunction with chemotherapy agents to treat cancer. (34) A chemotherapeutic agent used in conjunction with an anti-CCR8 antibody to treat cancer. (35) The use of an anti-CCR8 antibody in conjunction with a chemotherapeutic agent to manufacture a cancer treatment drug. (36) Use of chemotherapeutic agents in combination with anti-CCR8 antibodies for the manufacture of cancer treatment drugs.

[0013] (1-2) A pharmaceutical product comprising an anti-CCR8 antibody and a chemotherapeutic agent. (3-2) A pharmaceutical product described in (1-2) for the treatment of cancer. (4-2) The pharmaceutical product described in (3-2) for the treatment of cancer in which treatment with anti-PD-1 antibody or anti-PD-L1 antibody is ineffective. (5-2) A pharmaceutical product as described in any of (1-2) to (4-2), wherein the cancer is breast cancer, lung cancer, colorectal cancer, kidney cancer, sarcoma, liver cancer, bladder cancer, or ovarian cancer. (6-2) The pharmaceutical product described in (5-2), wherein the cancer is breast cancer, lung cancer, bladder cancer, kidney cancer, or colorectal cancer. (6'-2) The pharmaceutical product described in (5-2), wherein the cancer is breast cancer, lung cancer, bladder cancer, or colorectal cancer. (7-2) The pharmaceutical product described in (5-2), wherein the cancer is breast cancer, lung cancer, or colorectal cancer. (8-2) A pharmaceutical product according to any of (1-2) to (7-2), wherein the chemotherapeutic agent is a platinum complex, taxane, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide, or doxorubicin. (8'-2) A pharmaceutical product according to any one of (1-2) to (7-2), wherein the chemotherapeutic agent is a platinum complex, a taxane, gemcitabine, or fluorouracil. (9-2) The pharmaceutical product described in (8-2), wherein the chemotherapeutic agent is gemcitabine. (10-2) The pharmaceutical product according to (8-2), wherein the chemotherapeutic agent is a platinum complex, and the platinum complex is carboplatin, cisplatin, or oxaliplatin. (11-2) The pharmaceutical product described in (10-2), wherein the platinum complex is carboplatin. (12-2) The pharmaceutical product described in (8-2), wherein the chemotherapeutic agent is a taxane, and the taxane is paclitaxel or docetaxel. (13-2) The pharmaceutical product described in (8-2), wherein the chemotherapeutic agent is fluorouracil. (14-2) Anti-CCR8 antibody, 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 Heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7 (Here, it is possible to have one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine. C) The fourth asparagine molecule in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine. D) The fifth leucine in the amino acid sequence of SEQ ID NO: 3 is replaced with isoleucine. E) The fourth leucine in the amino acid sequence of SEQ ID NO: 4 is replaced with isoleucine. F) The 6th tyrosine in the amino acid sequence of SEQ ID NO: 4 is replaced with phenylalanine. G) The 16th serine in the amino acid sequence of SEQ ID NO: 6 is replaced with threonine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO: 6 is replaced with glutamic acid. 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7; 3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 10, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 11; (Here, it is possible to have one or more of the following substitutions: A) The second serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. B) The third serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. C) The fifth serine in the amino acid sequence of SEQ ID NO: 2 is replaced with threonine. D) The second glutamine in the amino acid sequence of Sequence ID No. 10 is replaced with asparagine. E) The first threonine in the amino acid sequence of sequence number 8 is replaced with serine. F) The 14th alanine in the amino acid sequence of sequence number 6 is replaced with valine. G) The 18th lysine in the amino acid sequence of sequence number 6 is replaced with arginine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO. 6 is replaced with glutamic acid. I) The fifth asparagine in the amino acid sequence of SEQ ID NO: 11 is replaced with glutamine. J) The 12th tyrosine in the amino acid sequence of sequence number 11 is replaced with phenylalanine. 4) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 10, and CDR1, consisting of the amino acid sequence of SEQ ID NO: 12, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 13; 5) CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 and the amino acid sequence of SEQ ID NO: 15 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 16, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 17, CDR2 and the amino acid sequence of SEQ ID NO: 18 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 19; or 6) CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 22, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 Heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 25 A pharmaceutical product according to any of (1-2) to (13-2), which is a monoclonal antibody or antibody fragment containing [the specified substance]. (15-2) Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 The light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 It includes a heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7, It has one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine; D) The fifth leucine in the amino acid sequence of SEQ ID NO: 3 is replaced with isoleucine; E) The fourth leucine in the amino acid sequence of SEQ ID NO: 4 is replaced with isoleucine. F) The 6th tyrosine in the amino acid sequence of SEQ ID NO: 4 is replaced with phenylalanine. G) The 16th serine in the amino acid sequence of SEQ ID NO: 6 is replaced with threonine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO. 6 is replaced with glutamic acid. The pharmaceutical product according to (14-2), which is a monoclonal antibody or antibody fragment containing a monoclonal antibody. (16-2) The anti-CCR8 antibody is a monoclonal antibody or antibody fragment having one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with leucine or arginine; C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine. The pharmaceutical product described in (15-2). (17-2) The anti-CCR8 antibody has a variant in which the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine. Furthermore, the monoclonal antibody or antibody fragment may have one of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with leucine or arginine. The pharmaceutical product described in (15-2) or (16-2). (18-2) The pharmaceutical product according to (17-2), wherein the anti-CCR8 antibody is a monoclonal antibody or antibody fragment in which the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamine, and the eleventh glycine in the amino acid sequence of SEQ ID NO: 2 is substituted with arginine. (18'-2) Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 10, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 It contains a heavy chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 11, The pharmaceutical product according to (14-2), which is a monoclonal antibody or antibody fragment in which the 18th lysine of the amino acid sequence of SEQ ID NO: 6 is substituted with arginine. (18''-2) Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 The light chain variable region including CDR3, which consists of the amino acid sequence of SEQ ID NO: 22, and CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 25; The pharmaceutical product according to (14-2), which is a monoclonal antibody or antibody fragment containing a monoclonal antibody. (19-2) A pharmaceutical product according to any of (14-2) to (18-2), wherein the anti-CCR8 antibody is a humanized monoclonal antibody or an antibody fragment thereof. (20-2) A humanized monoclonal antibody or a fragment thereof 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 3) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 4) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 5) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 6) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 7) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 8) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 9) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 10) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 11) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; or 12) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 Includes, It may have one or more of the following substitutions: A) The 33rd asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine; D) The 59th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; E) The 97th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; F) The 99th tyrosine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with phenylalanine; G) The 65th serine in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with threonine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with glutamic acid. The pharmaceutical product described in (19-2). (21-2) A humanized monoclonal antibody or an antibody fragment thereof has one or more of the following substitutions: A) The 33rd asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine; D) The 59th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; E) The 97th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine; F) The 99th tyrosine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with phenylalanine; G) The 65th serine in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with threonine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with glutamic acid. The pharmaceutical product described in (20-2). (22-2) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, It has one or more of the following substitutions: A) Substitution of asparagine at position 33 of the amino acid sequence of SEQ ID NO: 40 or 42 with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine; C) Substitution of asparagine at position 58 of the amino acid sequence of SEQ ID NO: 40 or 42 with glutamine; D) The 59th leucine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with isoleucine; E) The 97th leucine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with isoleucine; F) The 99th tyrosine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with phenylalanine; G) The 65th serine molecule in the amino acid sequence of SEQ ID NO: 41 is replaced with threonine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 is replaced with glutamic acid. The pharmaceutical product described in (21-2). (23-2) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, It has one or more of the following substitutions: A) Substitution of asparagine at position 33 of the amino acid sequence of SEQ ID NO: 40 or 42 with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine. The pharmaceutical product described in (21-2) or (22-2). (24-2) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, In the amino acid sequence of SEQ ID NO: 40 or 42, the 58th asparagine is substituted with glutamine. Furthermore, it may have one of the following substitutions: A) Substitution of asparagine at position 33 of the amino acid sequence of SEQ ID NO: 40 or 42 with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine. A medicine described in any of (21-2) to (23-2). (25-2) A humanized monoclonal antibody or a fragment thereof A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and It contains a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, In the amino acid sequence of SEQ ID NO: 42, the 58th asparagine molecule is replaced with glutamine, and the 34th glycine molecule is replaced with arginine. The pharmaceutical product described in (24-2). (26-2) The pharmaceutical product according to (24-2), wherein the humanized monoclonal antibody or antibody fragment thereof has a light chain variable region having the amino acid sequence of SEQ ID NO: 59 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41. (26'-2) The pharmaceutical product according to (19-2), wherein the humanized monoclonal antibody or antibody fragment thereof has a light chain variable region having the amino acid sequence of SEQ ID NO: 56 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 60. (27-2) A humanized monoclonal antibody or a fragment thereof Furthermore, a light chain constant region having the amino acid sequence of SEQ ID NO: 52, and It has a heavy chain constant region having the amino acid sequence of SEQ ID NO: 53, A pharmaceutical product as described in any of (19-2) to (26-2), with or without lysine added to the C-terminus of sequence number 53. (28-2) A pharmaceutical product as described in any of (1-2) to (27-2), wherein the anti-CCR8 antibody is a neutralizing antibody. (29-2) A pharmaceutical product according to any of (1-2) to (27-2), wherein the anti-CCR8 antibody has ADCC activity. (30-2) A pharmaceutical product as described in any of (1-2) to (27-2), wherein the anti-CCR8 antibody is an IgG antibody. (31-2) A method for treating cancer, characterized by administering an anti-CCR8 antibody and a chemotherapeutic agent. [Effects of the Invention]

[0014] The combination of the anti-CCR8 antibody and chemotherapeutic agent of the present invention exhibits a significantly greater effect when used in combination compared to the effects of the anti-CCR8 antibody alone or the chemotherapeutic agent alone. Therefore, the combination of the anti-CCR8 antibody and chemotherapeutic agent of the present invention is extremely useful as a pharmaceutical product, particularly as a pharmaceutical product for the treatment or prevention of cancer. [Brief explanation of the drawing]

[0015] [Figure 1] A comparison of the amino acid sequences of human CCR8, human CCR4, and chimeric organisms in which the N-terminal region, loop1 region, loop2 region, or loop3 region corresponding to the extracellular domain of human CCR8 is replaced with the corresponding N-terminal region, loop1 region, loop2 region, or loop3 region of human CCR4, respectively. [Figure 2] Kabat numbering and alignment results for the light chain variable regions of 10A11, 27G1, 1H4, 19D7, 8F7, and 2C7. [Figure 3] Kabat numbering and alignment results for the heavy-chain variable regions of 10A11, 27G1, 1H4, 19D7, 8F7, and 2C7. [Figure 4] Kabat numbering and alignment results for the light chain variable region of 10A11, IGKV4-1, IGKV3-20, IGKV1-39, IGKV2-40, IGKV2-28, and IGKV1-16. [Figure 5] Kabat numbering and alignment results for the heavy-chain variable region of 10A11, IGHV3-15 T94R, and IGHV3-73. [Figure 6] Kabat numbering and alignment results for the light chain variable region of 19D7, IGKV3-15, IGKV2-18, and IGKV3-20. [Figure 7] Kabat numbering and alignment results for the heavy-chain variable region of 19D7, IGHV3-15 T94R, and IGHV3-73. [Figure 8]In human CCR8 knock-in mice transplanted with colorectal cancer-derived CT26 cells, the antitumor activity of anti-human CCR8 antibodies was evaluated by measuring tumor volume after transplantation. ** indicates a significance level of p<0.01 by Welch's test, and *** indicates p<0.001. [Figure 9] In mice transplanted with breast cancer-derived 4T1 cells, the antitumor activity of a combination of anti-mouse CCR8 antibody and carboplatin (CBDCA) was evaluated. [Figure 10] In mice transplanted with breast cancer-derived 4T1 cells, the antitumor activity of a combination of anti-mouse CCR8 antibody and gemcitabine (GEM) was evaluated. [Figure 11] In human CCR8 knock-in mice transplanted with breast cancer-derived 4T1 cells, the antitumor activity of a combination of anti-human CCR8 antibody and cisplatin (CDDP) was evaluated. [Figure 12] In mice transplanted with bladder cancer-derived MB49 cells, the antitumor activity of a combination of anti-mouse CCR8 antibody and carboplatin (CBDCA) was evaluated. [Figure 13] In mice transplanted with bladder cancer-derived MB49 cells, the antitumor activity of a combination of anti-mouse CCR8 antibody and cisplatin (CDDP) was evaluated. [Figure 14] In mice transplanted with Colon26 cells derived from colorectal cancer, the antitumor activity of a combination of anti-mouse CCR8 antibody and oxaliplatin was evaluated. [Figure 15] In mice transplanted with Colon26 cells derived from colorectal cancer, the antitumor activity of a combination of anti-mouse CCR8 antibody and fluorouracil (5-FU) was evaluated. [Figure 16] In human CCR8 knock-in mice transplanted with Colon26 cells derived from colorectal cancer, the antitumor activity of a combination of anti-human CCR8 antibody and oxaliplatin was evaluated. [Figure 17] In human CCR8 knock-in mice transplanted with lung cancer-derived ASB-XIV cells, the antitumor activity of a combination of anti-human CCR8 antibody and cisplatin (CDDP) was evaluated. [Figure 18]In human CCR8 knock-in mice transplanted with lung cancer-derived ASB-XIV cells, the antitumor activity of a combination of anti-human CCR8 antibody and paclitaxel (PTX) was evaluated. [Figure 19] In human CCR8 knock-in mice transplanted with breast cancer-derived 4T1 cells, the antitumor activity of a combination of anti-human CCR8 antibody and docetaxel (DTX) was evaluated. [Figure 20] In mice transplanted with breast cancer-derived 4T1 cells, the antitumor activity of a combination of anti-mouse CCR8 antibody and docetaxel (DTX) was evaluated. [Figure 21] In human CCR8 knock-in mice transplanted with Colon26 cells derived from colorectal cancer, the antitumor activity of a combination of anti-human CCR8 antibody and oxaliplatin was evaluated. [Figure 22] In human CCR8 knock-in mice transplanted with Colon26 cells derived from colorectal cancer, the antitumor activity of a combination of anti-human CCR8 antibody and oxaliplatin was evaluated. [Figure 23] In human CCR8 knock-in mice transplanted with colorectal cancer-derived CT26 cells, the antitumor activity of a combination of anti-human CCR8 antibody and fluorouracil (5-FU) was evaluated. [Modes for carrying out the invention]

[0016] Unless otherwise specified, terms used herein shall be used in the sense commonly used in the art.

[0017] In the present invention, antibody production methods known in the art can be used. For example, methods described in Immunochemistry in Practice (Blackwell Scientific Publications) can be used. Furthermore, known genetic manipulation techniques in this field are available. Examples include the methods described in Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012) and Current Protocols Essential Laboratory Techniques, Current Protocols (2012).

[0018] The hybridoma that produces anti-CCR8 antibodies according to the present invention can be prepared using CCR8 protein, a gene encoding the full length of CCR8, CCR8-expressing cells, etc., as immunogens. For example, when using a gene encoding the full length of human CCR8 as an immunogen, a hybridoma that produces anti-CCR8 antibodies can be obtained by fusing spleen cells of a mouse that has been DNA-immunized with the gene as an antigen with mouse myeloma cells.

[0019] The amino acid sequence of human CCR8 is shown in UniProtKB / Swiss-Prot:P51685 (SEQ ID NO: 1).

[0020] One embodiment of the monoclonal antibody or antibody fragment according to the present invention is a monoclonal antibody or antibody fragment having a CDR or heavy chain variable region / light chain variable region as described herein. The antibody or antibody fragment may be derived from any class (e.g., IgG, IgE, IgM, IgD, or IgA; preferably IgG) or subclass of an immunoglobulin molecule, and may be obtained from any species, for example, including mouse, rat, shark, rabbit, pig, hamster, camel, llama, goat, or human. The antibody or antibody fragment is preferably a humanized monoclonal antibody or an antibody fragment of a humanized monoclonal antibody.

[0021] The CDR sequence of the monoclonal antibody according to the present invention preferably has the following sequence. (1) Light chain CDR1: Sequence IDs 2, 14, or 20. (2) Light chain CDR2: Sequence IDs 3, 9, 15, or 21. (3) Light chain CDR3: Sequence IDs 4, 10, 16, or 22. (4) Heavy chain CDR1: Sequence IDs 5, 8, 12, 17, or 23. (5) Heavy chain CDR2: Sequence IDs 6, 18, or 24. (6) Heavy chain CDR3: SEQ ID NOs. 7, 11, 13, 19, or 25. More preferably, the following arrangement is present. (1) Light chain CDR1: Sequence ID 2. (2) Light chain CDR2: Sequence ID 3 or 9. (3) Light chain CDR3: Sequence ID No. 4 or 10. (4) Heavy chain CDR1: Sequence IDs 5, 8, or 12. (5) Heavy chain CDR2: Sequence ID 6. (6) Heavy chain CDR3: SEQ ID NOs: 7, 11, or 13. Most preferably, it has the following arrangement: (1) Light chain CDR1: Sequence ID 2. (2) Light chain CDR2: Sequence ID 3. (3) Light chain CDR3: Sequence ID 4. (4) Heavy chain CDR1: Sequence ID No. 5. (5) Heavy chain CDR2: Sequence ID 6. (6) Heavy chain CDR3: Sequence ID 7. Furthermore, in each of the above amino acid sequences (SEQ ID NOs. 2 to 25), one or two amino acids may be deleted, substituted, inserted, and / or added. Here, in the monoclonal antibody of the present invention, asparagine in the CDR sequence may be substituted with glutamine or lysine, aspartic acid with glutamic acid, leucine with isoleucine, tyrosine with phenylalanine, serine with threonine, and glycine with glutamine, threonine, alanine, lysine, leucine, or arginine. Preferably, it may have one or more of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine. C) The fourth asparagine molecule in the amino acid sequence of Sequence ID No. 3 is replaced with glutamine. D) The fifth leucine in the amino acid sequence of SEQ ID NO: 3 is replaced with isoleucine. E) The fourth leucine in the amino acid sequence of Sequence ID No. 4 is replaced with isoleucine. F) The 6th tyrosine in the amino acid sequence of SEQ ID NO: 4 is replaced with phenylalanine. G) The 16th serine molecule in the amino acid sequence of Sequence ID No. 6 is replaced with threonine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO. 6 is replaced with glutamic acid. More preferably, the following substitutions may be present: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with leucine or arginine. C) The fourth asparagine molecule in the amino acid sequence of Sequence ID No. 3 is replaced with glutamine. More preferably, the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamine. Furthermore, it may have one of the following substitutions: A) The 10th asparagine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with lysine; B) The 11th glycine in the amino acid sequence of Sequence ID No. 2 is replaced with leucine or arginine. Furthermore, if the monoclonal antibody of the present invention has the following sequence, (1) Light chain CDR1: Sequence ID 2. (2) Light chain CDR2: Sequence ID 9. (3) Light chain CDR3: Sequence ID 10. (4) Heavy chain CDR1: Sequence ID 8. (5) Heavy chain CDR2: Sequence ID 6. (6) Heavy chain CDR3: Sequence ID 11. It may have one or more of the following substitutions: A) The second serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. B) The third serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. C) The fifth serine molecule in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. D) The second glutamine in the amino acid sequence of SEQ ID NO. 10 is replaced with asparagine. E) The first threonine in the amino acid sequence of sequence number 8 is replaced with serine. F) The 14th alanine in the amino acid sequence of sequence number 6 is replaced with valine. G) The 18th lysine in the amino acid sequence of Sequence ID No. 6 is replaced with arginine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO. 6 is replaced with glutamic acid. I) The fifth asparagine molecule in the amino acid sequence of SEQ ID NO. 11 is replaced with glutamine. J) The 12th tyrosine in the amino acid sequence of SEQ ID NO. 11 is replaced with phenylalanine.

[0022] "Antibody fragment of a monoclonal antibody" means a part of the monoclonal antibody according to the present invention that specifically binds to CCR8 and selectively inhibits CCR8, similar to the monoclonal antibody.

[0023] Specifically, examples include Fab (fragment of antigen binding) that specifically binds to CCR8, Fab', F(ab')2, single-chain antibodies (single-chain Fv; hereafter referred to as scFv), disulfide-stabilized antibodies (disulfide-stabilized Fv; hereafter referred to as dsFv), dimerized V region fragments (hereafter referred to as Diabody), and peptides containing CDRs (Expert Opinion on Therapeutic Patents, Vol. 6, No. 5, pp. 441-456, 1996).

[0024] Fab is an antibody fragment with antigen-binding activity, consisting of approximately half of the N-terminal side of the H chain and the entire L chain. It is obtained by degrading the peptide portion above the two disulfide bonds (SS bonds) that cross-link the two H chains in the hinge region of IgG with the enzyme papain. The Fab used in this invention can be obtained by treating the monoclonal antibody according to the present invention with papain. Alternatively, Fab can be produced by inserting the DNA encoding the Fab of the monoclonal antibody according to the present invention into a cell expression vector and expressing the vector in cells.

[0025] Fab' is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, obtained by cleaving the disulfide bond between the hinges of F(ab')2. The Fab' used in this invention can be obtained by treating the monoclonal antibody F(ab')2 according to the present invention with the reducing agent dithiothreitol. Alternatively, Fab' can also be produced by inserting the DNA encoding the monoclonal antibody Fab' according to the present invention into a cell expression vector and expressing the vector in E. coli, yeast, or animal cells.

[0026] F(ab')2 is an antibody fragment with antigen-binding activity and a molecular weight of approximately 100,000, obtained by degrading the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin. It consists of two Fab' regions joined at the hinge portion. The F(ab')2 used in this invention can be obtained by pepsin treatment of the monoclonal antibody according to the present invention. Alternatively, F(ab')2 can also be produced by inserting the DNA encoding F(ab')2 of the monoclonal antibody of the present invention into a cell expression vector and expressing the vector in E. coli, yeast, or animal cells.

[0027] scFv is a VH-P-VL or VL-P-VH polypeptide, formed by linking one VH molecule and one VL molecule using a suitable peptide linker (hereinafter referred to as P), and is an antibody fragment having antigen-binding activity. The VH and VL molecules contained in the scFv used in this invention may be those of the monoclonal antibody according to the present invention. The scFv used in this invention can be produced by constructing an scFv expression vector using the cDNA encoding the VH and VL molecules of the monoclonal antibody according to the present invention, and then introducing it into E. coli, yeast, or animal cells to induce expression.

[0028] dsFv refers to a polypeptide in which one amino acid residue in VH and VL is replaced with a cysteine ​​residue, and these polypeptides are linked via a disulfide bond. The amino acid residue to be replaced with the cysteine ​​residue can be selected based on the prediction of the antibody's three-dimensional structure according to the method shown by Reiter et al. (Protein Engineering, 7, 697 (1994)). The VH or VL contained in the dsFv used in this invention may be any of the monoclonal antibody according to the present invention. The dsFv used in this invention can be produced by constructing a dsFv expression vector by inserting the cDNA encoding the VH and VL of the monoclonal antibody according to the present invention into a suitable expression vector, and then introducing the expression vector into E. coli, yeast, or animal cells and expressing the dsFv.

[0029] A Diabody is an antibody fragment in which scFvs with the same or different antigen-binding specificities form a dimer, and is an antibody fragment having bivalent antigen-binding activity against the same antigen or two types of specific antigen-binding activity against different antigens. For example, a bivalent Diabody that specifically reacts with the monoclonal antibody according to the present invention can be produced by constructing DNA encoding an scFv having a peptide linker of 3 to 10 residues using cDNA encoding VH and VL of the monoclonal antibody according to the present invention, inserting the DNA into a cell expression vector, and expressing the Diabody by introducing the expression vector into E. coli, yeast, or animal cells.

[0030] A peptide containing a CDR is composed of at least one region of a VH or VL CDR. Multiple CDRs can be linked directly or via a suitable peptide linker. The peptide containing a CDR used in the present invention can be produced by constructing a DNA encoding the CDR using the cDNA encoding the VH and VL of the monoclonal antibody according to the present invention, inserting the DNA into an expression vector for animal cells, and expressing the vector by introducing it into E. coli, yeast, or animal cells. Alternatively, the peptide containing a CDR can also be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method).

[0031] The monoclonal antibody or antibody fragment thereof according to the present invention is characterized by its ability to bind to CCR8. In particular, those that bind specifically are preferred.

[0032] Specific binding is at least about 1 × 10⁻⁶ -6 The equilibrium dissociation constant can be M or less (for example, a smaller Kd indicates a tighter bond). The Kd value is preferably 1 × 10⁻⁶. -7 M or less, more preferably 1 × 10 -8 M or less, more preferably 1 × 10 -9 The value is M or less. Methods for measuring whether two molecules specifically bind are well known in the art, and include competitive ELISA, surface plasmon resonance, and similar methods.

[0033] The monoclonal antibody or antibody fragment thereof according to the present invention is characterized by inhibiting the binding of either CCR8 or a CCR8 ligand. The "CCR8 ligand" is not particularly limited to any substance that binds to CCR8, such as CCL1, CCL8, or CCL18, but is preferably CCL1 or CCL18, and particularly preferably CCL1. Regarding the ability to inhibit the binding of CCR8 to CCR8 ligands, for example, in the case of human CCL1, human CCR8-expressing 293 cells were used, and the addition of human CCL1 inhibited Ca 2+The influx can be measured, and the IC50 value can be calculated by setting the signal without human CCL1 addition as 100% inhibition and the signal with human CCL1 addition and without antibody addition as 0% inhibition. The binding inhibitory ability of other CCR8 ligands can also be determined in the same manner as in the case of human CCL1 described above.

[0034] Human CCL1 has the amino acid sequence shown in UniProtKB / Swiss-Prot No. P22362, etc. Human CCL8 has the amino acid sequence shown in GenBank No. AAI26243.1, etc. Human CCL18 has the amino acid sequence shown in GenBank No. EAW80102.1, etc.

[0035] The monoclonal antibody according to the present invention can be prepared by conventional methods in the art using the CDR or heavy chain variable region / light chain variable region described herein.

[0036] The monoclonal antibodies according to the present invention also include chimeric, humanized, fully human, antibody-small molecule conjugates (ADCs), and bispecific antibodies. Humanized monoclonal antibodies are useful for therapeutic purposes when administered to humans because their antigenicity in the human body is reduced. Humanized monoclonal antibodies are created by transplanting the complementarity determining region (CDR) of an antibody from a non-human mammal, such as a mouse antibody, into the framework region (FR) of a human antibody. Therefore, the FR of a humanized monoclonal antibody is of human origin. An appropriate FR can be selected by referring to the literature of Kabat EA et al. In this case, the FR should be one in which the CDR can form a good antigen-binding site. If necessary, amino acids in the FR of the variable region of the antibody may be substituted so that the CDR of the reconstituted humanized monoclonal antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. 1993, Vol. 53, p. 851). The proportion of amino acids in the FR to be substituted is 0-15% of the total FR region, preferably 0-5%.

[0037] The humanized monoclonal antibody according to the present invention is preferably, The amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 or Light chain variable regions consisting of amino acid sequences that are 95% or more identical to the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47, and The amino acid sequence of SEQ ID NO: 41 or 46 or It contains a heavy chain variable region consisting of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 41 or 46. more, 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 3) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 4) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 5) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 6) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 7) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 8) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 9) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 10) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 11) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; or 12) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 Includes, It may have one or more of the following substitutions. A) The 33rd asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with lysine. B) The 34th glycine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine. C) The 58th asparagine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with glutamine. D) The 59th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine. E) The 97th leucine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with isoleucine. F) The 99th tyrosine in the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 is replaced with phenylalanine. G) The 65th serine in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with threonine. H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 or 46 is replaced with glutamic acid. More preferably, 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; or 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 Includes, It may have one or more of the following substitutions: A) The 33rd asparagine molecule in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with lysine. B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine, threonine, alanine, lysine, leucine, or arginine. C) The 58th asparagine molecule in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine. D) The 59th leucine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with isoleucine. E) The 97th leucine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with isoleucine. F) The 99th tyrosine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with phenylalanine. G) The 65th serine molecule in the amino acid sequence of SEQ ID NO: 41 is replaced with threonine. H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 41 is replaced with glutamic acid. Particularly preferred, 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; or 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 Includes, It may have one or more of the following substitutions: A) The 33rd asparagine molecule in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with lysine. B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine. C) The 58th asparagine molecule in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine. Most preferably, 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; or 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 Includes, In the amino acid sequence of SEQ ID NO: 40 or 42, the 58th asparagine is substituted with glutamine. Furthermore, it may have one of the following substitutions: A) The 33rd asparagine molecule in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with lysine. B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine. Most preferred is a humanized monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 42 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41, wherein the 58th asparagine in the amino acid sequence of SEQ ID NO: 42 is substituted with glutamine and the 34th glycine in the amino acid sequence of SEQ ID NO: 42 is substituted with arginine, i.e., a humanized monoclonal antibody having a light chain variable region having the amino acid sequence of SEQ ID NO: 59 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 41.

[0038] Examples of sequences for the light chain variable region and heavy chain variable region in the humanized monoclonal antibody according to the present invention include the combinations shown in Table 1. [Table 1] Here, each symbol in the "Type of Substitution" column of Table 1 above represents one of the following substitutions: (1) Light chain variable region having the amino acid sequence of SEQ ID NO: 40 or 42 A) The 33rd asparagine molecule in the amino acid sequence is replaced with lysine. B1) The 34th glycine in the amino acid sequence is replaced with glutamine. B2) The 34th glycine in the amino acid sequence is replaced with threonine. B3) The 34th glycine in the amino acid sequence is replaced with alanine. B4) The 34th glycine in the amino acid sequence is replaced with lysine. B5) The 34th glycine in the amino acid sequence is replaced with leucine. B6) The 34th glycine molecule in the amino acid sequence is replaced with arginine. C) The 58th asparagine molecule in the amino acid sequence is replaced with glutamine. D) The 59th leucine in the amino acid sequence is replaced with isoleucine. E) The 97th leucine in the amino acid sequence is replaced with isoleucine. F) The 99th tyrosine in the amino acid sequence is replaced with phenylalanine. (2) Heavy chain variable region having the amino acid sequence of SEQ ID NO: 41 G) The 65th serine molecule in the amino acid sequence is replaced with threonine. H) The 68th aspartic acid in the amino acid sequence is replaced with glutamic acid.

[0039] Another embodiment of the humanized monoclonal antibody according to the present invention is: The amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47 or The light chain variable region consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NOs. 40, 42, 43, 44, 45, or 47, wherein the 26th amino acid position is serine, the 27th is lysine, the 30th is leucine, and the 98th is glutamic acid, and The amino acid sequence of SEQ ID NO: 41 or 46 or It consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 41 or 46, and includes a heavy chain variable region in which the 33rd amino acid is alanine, the 35th is tyrosine, the 52nd is arginine, the 56th is asparagine, the 62nd is tyrosine, the 102nd is arginine, the 103rd is phenylalanine, the 104th is tyrosine, the 109th is glycine, and the 113th is aspartic acid. more, The amino acid sequence of SEQ ID NO: 40 or 42 or The light chain variable region consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 40 or 42, wherein the 26th amino acid position is serine, the 27th is lysine, the 30th is leucine, and the 98th is glutamic acid, and The amino acid sequence of SEQ ID NO: 41 or 46 or It consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 41 or 46, and includes a heavy chain variable region in which the 33rd amino acid is alanine, the 35th is tyrosine, the 52nd is arginine, the 56th is asparagine, the 62nd is tyrosine, the 102nd is arginine, the 103rd is phenylalanine, the 104th is tyrosine, the 109th is glycine, and the 113th is aspartic acid. More preferably, The amino acid sequence of SEQ ID NO: 42 or The light chain variable region consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of Sequence ID No. 42, wherein the 26th amino acid position is serine, the 27th is lysine, the 30th is leucine, and the 98th is glutamic acid, and The amino acid sequence of sequence number 41 or 46 or It consists of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 41 or 46, and includes a heavy chain variable region in which the 33rd amino acid is alanine, the 35th is tyrosine, the 52nd is arginine, the 56th is asparagine, the 62nd is tyrosine, the 102nd is arginine, the 103rd is phenylalanine, the 104th is tyrosine, the 109th is glycine, and the 113th is aspartic acid.

[0040] The humanized monoclonal antibody according to the present invention is also preferably, The amino acid sequence of SEQ ID NOs. 54, 55, or 56 or A light chain variable region consisting of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NOs. 54, 55, or 56, and The amino acid sequence of SEQ ID NO: 57 or 58 or It contains a heavy chain variable region consisting of an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO: 57 or 58. more, 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 54, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 57; 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 55, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 57; 3) A light chain variable region having the amino acid sequence of SEQ ID NO: 56, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 57; 4) A light chain variable region having the amino acid sequence of SEQ ID NO: 54, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 58; 5) A light chain variable region having the amino acid sequence of SEQ ID NO: 55, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 58; 6) A light chain variable region having the amino acid sequence of SEQ ID NO: 56, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 58 It may include and have one or more of the following substitutions: A) The 25th serine in the amino acid sequence of SEQ ID NOs. 54, 55, or 56 is replaced with threonine; B) The 26th serine in the amino acid sequence of SEQ ID NOs. 54, 55, or 56 is replaced with threonine; C) The 28th serine in the amino acid sequence of SEQ ID NOs. 54, 55, or 56 is replaced with threonine; D) The 95th glutamine in the amino acid sequence of SEQ ID NOs. 54, 55, or 56 is replaced with asparagine; E) The 31st threonine in the amino acid sequence of SEQ ID NO: 57 or 58 is replaced with serine; F) The 63rd alanine in the amino acid sequence of sequence number 57 or 58 is replaced with valine; G) The 67th lysine in the amino acid sequence of SEQ ID NO. 57 or 58 is replaced with arginine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 57 or 58 is substituted with glutamic acid; I) The 99th asparagine in the amino acid sequence of SEQ ID NO: 57 or 58 is substituted with glutamine, J) The 105th tyrosine in the amino acid sequence of SEQ ID NO: 57 or 58 is substituted with phenylalanine. More preferably, It includes a light chain variable region having the amino acid sequence of SEQ ID NO: 56 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57, and may have one or more of the following substitutions: A) The 25th serine in the amino acid sequence of SEQ ID NO: 56 is substituted with threonine; B) The 26th serine in the amino acid sequence of SEQ ID NO: 56 is substituted with threonine; C) The 28th serine in the amino acid sequence of SEQ ID NO: 56 is substituted with threonine; D) The 95th glutamine in the amino acid sequence of SEQ ID NO: 56 is substituted with asparagine; E) The 31st threonine in the amino acid sequence of SEQ ID NO: 57 is substituted with serine; F) The 63rd alanine in the amino acid sequence of SEQ ID NO: 57 is substituted with valine; G) The 67th lysine in the amino acid sequence of SEQ ID NO: 57 is substituted with arginine; H) The 68th aspartic acid in the amino acid sequence of SEQ ID NO: 57 is substituted with glutamic acid; I) The 99th asparagine in the amino acid sequence of SEQ ID NO: 57 is substituted with glutamine, J) The 105th tyrosine in the amino acid sequence of SEQ ID NO: 57 is substituted with phenylalanine. A humanized monoclonal antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 56 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57, in which the 67th lysine in the amino acid sequence of SEQ ID NO: 57 is substituted with arginine, that is, a humanized monoclonal antibody having a light chain variable region having the amino acid sequence of SEQ ID NO: 56 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 60 is particularly preferred.

[0041] In the humanized monoclonal antibody according to the present invention, the constant region of a human antibody is used. Preferred constant regions of human antibodies include Cγ as the heavy chain, for example, Cγ1, Cγ2, Cγ3, Cγ4, and Cκ, Cλ as the light chain can be used. Further, in order to improve the stability of the antibody or its production, the C region of the human antibody may be modified. The human antibody used in humanization may be a human antibody of any isotype such as IgG, IgM, IgA, IgE, IgD, etc., but in the present invention, it is preferable to use IgG, and more preferably IgG1 or IgG4.

[0042] The humanized monoclonal antibody according to the present invention may or may not have a lysine added to the C-terminus of the heavy chain constant region. Preferably, it has a light chain constant region of the amino acid sequence of SEQ ID NO: 52 and a heavy chain constant region of the amino acid sequence of SEQ ID NO: 53, and may or may not have a lysine added to the C-terminus of SEQ ID NO: 53.

[0043] The humanized monoclonal antibody can be produced by a general production method (see, for example, WO95 / 14041, WO96 / 02576, etc.). Specifically, first, a DNA sequence encoding a variable region designed to link the CDR of a mouse antibody and the FR of a human antibody is synthesized by the PCR method from several oligonucleotides prepared to have an overlapping portion at the end (see WO98 / 13388). The obtained DNA is ligated to the DNA encoding the constant region of the human antibody and then incorporated into an expression vector. Alternatively, the DNA encoding the variable region of the antibody may be incorporated into an expression vector containing the DNA of the constant region of the antibody. To produce the antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region, for example, an enhancer / promoter. Next, the host cell can be transformed with this expression vector to express the antibody.

[0044] Examples of host cells for the above-mentioned transformants include vertebrate cells such as COS cells and CHO cells, prokaryotic cells, and yeast. The transformants can be cultured according to methods well known to those skilled in the art, and the monoclonal antibody of the present invention is produced either inside or outside the transformant cells during this culture. Various culture media commonly used can be appropriately selected depending on the host cells adopted. For example, in the case of COS cells, media such as RPMI-1640 medium or Dulbecco's Modified Eagle Minimum Essential Medium (DMEM) can be used, with serum components such as fetal bovine serum (FBS) added as needed. The culture temperature for culturing the transformants can be any temperature that does not significantly reduce the intracellular protein synthesis capacity, but it is preferably 32-42°C, and most preferably 37°C. If necessary, the cells can also be cultured in air containing 1-10% (v / v) carbon dioxide.

[0045] The fraction containing the monoclonal antibody according to the present invention, produced intracellularly or extracellularly in the transformant as described above, can be separated and purified by various known separation methods that utilize the physical and chemical properties of the protein. Specifically, such methods include treatment with conventional protein precipitants, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), dialysis, and combinations thereof. By this method, the monoclonal antibody according to the present invention can be easily produced in high yield and high purity.

[0046] The monoclonal antibody or antibody fragment thereof according to the present invention may be further modified with various molecules such as polyethylene glycol (PEG), radioactive materials, or toxins. Methods known in the art can be used for modifying the antibody.

[0047] Furthermore, the monoclonal antibody or antibody fragment thereof according to the present invention may have another protein fused to its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). The protein to be fused can be appropriately selected by those skilled in the art.

[0048] The monoclonal antibody or antibody fragment thereof according to the present invention includes an antibody in which an N-glycosidic linkage glycan is bound to the Fc region of the antibody. Note that fucose does not need to be bound to the N-acetylglucosamine at the reducing end of the N-glycosidic linkage glycan. An example of an antibody in which an N-glycosidic linkage glycan is bound to the Fc region of the antibody, but fucose is not bound to the N-acetylglucosamine at the reducing end of the N-glycosidic linkage glycan, is an antibody produced using CHO cells lacking the α1,6-fucosyltransferase gene (International Publication No. 2005 / 035586, International Publication No. 02 / 31140). The antibody of the present invention, in which an N-glycosidic linkage glycan is bound to the Fc region of the antibody, and fucose is not bound to the N-acetylglucosamine at the reducing end of the N-glycosidic linkage glycan, has high ADCC activity.

[0049] The anti-CCR8 antibody according to the present invention is preferably an antibody or antibody fragment thereof that has ADCC (Antibody-dependent cell-mediated cytotoxicity) activity against cells expressing CCR8, from the viewpoint of eliminating Treg cells or macrophage cells. ADCC activity refers to the activity in which an antibody bound to a cell surface antigen such as a target cell activates effector cells through the binding of the antibody's Fc region to Fc receptors present on the surface of effector cells, thereby damaging the target cells. Examples of effector cells include natural killer cells and activated macrophages. In addition, the present invention also includes cases in which an antibody bound to a cell surface antigen (CCR8) such as a Treg cell or macrophage cell activates effector cells through the binding of the antibody's Fc region to Fc receptors present on the surface of effector cells, damaging Treg cells or macrophage cells, and consequently damaging tumor cells.

[0050] The anti-CCR8 antibody according to the present invention is preferably a CCR8 neutralizing antibody or a neutralizing antibody fragment. A CCR8 neutralizing antibody or neutralizing antibody fragment means an antibody or antibody fragment that has neutralizing activity against CCR8. Whether or not it has neutralizing activity against CCR8 can be determined, for example, by measuring whether or not it suppresses the physiological effects of any of the CCR8 ligands (e.g., CCL1) on CCR8. Examples, though not limited to these, include the binding of CCL1 to CCR8, or the migration of CCR8-expressing cells or intracellular Ca by CCL1. 2+ This includes measuring changes in the expression of genes that are increased or sensitive to CCL1 stimulation. Alternatively, it can be measured using the methods described in the example tests below. The neutralizing activity of the anti-CCR8 antibody according to the present invention against the binding of CCR8 and CCL1 is determined beforehand by Ca 2+ The indicator can be incorporated into 293 human CCR8-expressing cells, and the antibody dilution diluted in culture medium can be added for measurement. The affinity between CCR8 and its ligand is strongest for CCL1, and antibodies with high inhibitory activity against CCL1 are useful. The neutralizing activity of the monoclonal antibody or antibody fragment according to the present invention is preferably such that the IC50 value is 10 nM or less. More preferably, the neutralizing activity is such that the IC50 value is 5 nM or less, even more preferably 2 nM or less, particularly preferably 1 nM or less, and most preferably 0.5 nM or less.

[0051] In the anti-CCR8 antibody according to the present invention, an antibody or antibody fragment that strongly recognizes CCR8 is preferred. When selecting an antibody or antibody fragment that strongly recognizes CCR8, the strength of neutralization activity can be used as an indicator to select an antibody or antibody fragment that more strongly recognizes CCR8.

[0052] The anti-CCR8 antibody according to the present invention is preferably one that has the effect of removing tumor-infiltrating Treg cells. Whether or not the monoclonal antibody according to the present invention has the effect of removing tumor-infiltrating Treg cells can be measured, for example, by the method described in the examples of Patent Document 2.

[0053] The anti-CCR8 antibody according to the present invention is preferably one that has the effect of removing tumor-infiltrating macrophage cells. Whether or not the antibody or antibody fragment of the present invention has the effect of removing tumor-infiltrating macrophage cells can be measured, for example, by the method described in the examples of Patent Document 2.

[0054] "Chemotherapy agents" refer to drugs that have the effect of suppressing the proliferation of tumor cells. An "immunotherapy agent" refers to a drug that damages tumor cells by activating the immune system. The anti-CCR8 antibody according to the present invention is classified as an immunotherapy agent because, in vivo, it binds to the cell surface antigen (CCR8) of immune cells such as macrophages, and activates effector cells through the binding of the antibody's Fc region to Fc receptors present on the surface of effector cells, thereby damaging Treg cells or macrophage cells, and consequently damaging tumor cells.

[0055] Examples of chemotherapeutic agents according to the present invention include alkylating agents, platinum complexes, antimetabolites, topoisomerase inhibitors, taxanes, and molecularly targeted drugs.

[0056] Alkylating agents are drugs that exert an inhibitory effect on tumor cell proliferation by binding alkyl groups to DNA. Examples include nitrogen mustards such as cyclophosphamide, ifosfamide, melphalan, busulfan, and thiotepa, and nitrosoureas such as nimustine, ranimustine, dacarbazine, procarbazine, temozolomide, carmustine, streptozotocin, and bendamustine.

[0057] Platinum complexes are drugs that contain platinum in their structure and induce apoptosis in tumor cells by binding to DNA. Examples include cisplatin, carboplatin, oxaliplatin, and nedaplatin. Preferably, cisplatin, carboplatin, or oxaliplatin, and more preferably, carboplatin.

[0058] Antimetabolites are drugs that inhibit the proliferation of tumor cells by inhibiting the uptake of purines and pyrimidines during DNA synthesis. Examples include folic acid antimetabolites such as pemetrexed, sulfadiazine, sulfamethoxazole, methotrexate, trimethoprim, and pyrimethamine; pyrimidine antimetabolites such as fluorouracil and flucytosine; purine antimetabolites such as 6-mercaptopurine, azathioprine, and pentostatin; urea derivatives such as hydroxyurea; purine analogs such as thioguanine, fludarabine, and cladribine; and pyrimidine analogs such as gemcitabine and cytarabine. Preferably, pemetrexed, fluorouracil, or gemcitabine are used. More preferably, fluorouracil or gemcitabine are used.

[0059] Topoisomerase inhibitors are drugs that exhibit an inhibitory effect on the growth of tumor cells by inhibiting topoisomerase, which changes the helical structure of DNA. Examples include type I topoisomerase inhibitors such as irinotecan and nogitecan, and type II topoisomerase inhibitors such as doxorubicin and etoposide. Preferably, it is irinotecan, doxorubicin or etoposide.

[0060] Taxanes are drugs that inhibit the depolymerization of microtubules, which play an important role in cell division, inhibit cell division, and exhibit an inhibitory effect on the growth of tumor cells. Examples include paclitaxel and docetaxel.

[0061] Molecular target drugs are drugs that inhibit the signal transduction of specific molecules involved in the growth of tumor cells, etc. Examples include tyrosine kinase inhibitors such as gefitinib, erlotinib, osimertinib, afatinib, dacomitinib, imatinib, dasatinib, bosutinib, vandetanib, sunitinib, axitinib, pazopanib, lenvatinib, lapatinib, nintedanib, nilotinib, ibrutinib, girentuximab, crizotinib, ceritinib, alectinib, lorlatinib, Raf kinase inhibitors such as sorafenib, vemurafenib, dabrafenib, MEK inhibitors such as trametinib, cyclin-dependent kinase inhibitors such as palbociclib, abemaciclib, PARP inhibitors such as olaparib, and monoclonal antibodies such as cetuximab, trastuzumab, bevacizumab, pertuzumab, panitumumab, ramucirumab.

[0062] The chemotherapeutic agent according to the present invention is preferably a platinum complex, taxane, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide or doxorubicin, more preferably a platinum complex, taxane, gemcitabine or fluorouracil, still more preferably carboplatin, cisplatin, oxaliplatin, fluorouracil or gemcitabine, still more preferably carboplatin, cisplatin, oxaliplatin or gemcitabine, and still more preferably carboplatin or gemcitabine.

[0063] The pharmaceutical composition or pharmaceutical of the present invention is extremely useful as a pharmaceutical for the treatment and / or prevention of CCR8-related diseases. In particular, it is extremely useful as a pharmaceutical for the treatment and / or prevention of cancers in which CCR8-expressing Treg cells invade the tumor. For example, it is extremely useful as a pharmaceutical agent for treating and / or preventing cancers such as breast cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric (gastric adenocarcinoma), non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial carcinoma, sarcoma, hematopoietic cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, and other cancers, preferably breast cancer, lung cancer, colorectal cancer, kidney cancer, sarcoma, liver cancer, bladder cancer, or ovarian cancer, more preferably breast cancer, lung cancer, bladder cancer, kidney cancer, or colorectal cancer, and even more preferably breast cancer, lung cancer, or colorectal cancer. Herein, the pharmaceutical composition or pharmaceutical of the present invention is also effective in treating and / or preventing cancers with low immunogenicity. For example, it is also effective in treating and / or preventing cancers with low tumor mutational burden (TMB) values. The TMB value is preferably 10 mutations / Megabase or less, more preferably 5 mutations / Megabase or less, even more preferably 2 mutations / Megabase or less, and particularly preferably 1 mutation / Megabase or less. Furthermore, in the breast cancer-derived 4T1 cells used in Examples 1 and 2 of this specification, treatment with anti-PD-1 antibodies is ineffective, as described in the literature by Grasselli et al. (Frontiers in Immunology, (2018), 9, Article 2100). In other words, the pharmaceutical composition or pharmaceutical of the present invention is also useful for treating cancers that are ineffective with anti-PD-1 antibodies or anti-PD-L1 antibodies. Ineffective treatment with anti-PD-1 antibodies or anti-PD-L1 antibodies means that cancer growth cannot be suppressed by the administration of anti-PD-1 antibodies or anti-PD-L1 antibodies.

[0064] In the "cancer treatment" aspect of the present invention, "cancer" includes all solid tumors and hematological cancers. Specifically, examples include breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, lung cancer, gastric (gastric adenocarcinoma), non-small cell lung cancer, pancreatic cancer, head and neck squamous cell carcinoma, esophageal cancer, bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkin lymphoma, urothelial carcinoma, sarcoma, hematological cancer (leukemia, lymphoma, etc.), bile duct cancer, gallbladder cancer, thyroid cancer, testicular cancer, thymic cancer, liver cancer, etc. Preferably, breast cancer, lung cancer, colorectal cancer, kidney cancer, sarcoma, liver cancer, bladder cancer, or ovarian cancer; more preferably, breast cancer, lung cancer, bladder cancer, kidney cancer, or colorectal cancer; even more preferably, breast cancer, lung cancer, bladder cancer, or colorectal cancer; even more preferably, breast cancer, lung cancer, or colorectal cancer; and even more preferably, breast cancer. Furthermore, in the "cancer treatment" aspect of the present invention, "cancer" preferably refers to cancer that expresses tumor-specific antigens.

[0065] In this specification, "cancer" refers not only to epithelial malignant tumors such as ovarian cancer and gastric cancer, but also to non-epithelial malignant tumors including hematopoietic cancers such as chronic lymphocytic leukemia and Hodgkin lymphoma. In this specification, terms such as "cancer," "carcinoma," "tumor," and "neoplasm" are not distinguished from each other and are interchangeable.

[0066] The "pharmaceutical composition for use with a chemotherapeutic agent containing an anti-CCR8 antibody" of the present invention may also use two or more chemotherapeutic agents according to the present invention together.

[0067] The "pharmaceutical composition containing a chemotherapeutic agent for use together with an anti-CCR8 antibody" or "pharmaceutical comprising an anti-CCR8 antibody and a chemotherapeutic agent" of the present invention may contain two or more chemotherapeutic agents according to the present invention.

[0068] The combination of the anti-CCR8 antibody and the chemotherapeutic agent of the present invention exhibits a significantly greater effect when used in combination compared to the effects of the anti-CCR8 antibody alone or the chemotherapeutic agent alone. Specifically, the antitumor effect of the combination of the anti-CCR8 antibody and the chemotherapeutic agent shows a synergistic or greater effect compared to the antitumor effect of the anti-CCR8 antibody alone or the antitumor effect of the chemotherapeutic agent alone.

[0069] The combination of the anti-CCR8 antibody and chemotherapeutic agent of the present invention exhibits a significantly greater effect when used in combination compared to the effects of the anti-CCR8 antibody alone or the chemotherapeutic agent alone. Therefore, the antitumor effect can be achieved at lower doses of the anti-CCR8 antibody and chemotherapeutic agent compared to the doses used when each is administered individually. Accordingly, the combination of the anti-CCR8 antibody and chemotherapeutic agent of the present invention can be expected to reduce the side effects caused by the anti-CCR8 antibody and / or chemotherapeutic agent.

[0070] The pharmaceutical composition or pharmaceutical of the present invention can be administered orally or parenterally, systemically or topically. Parenteral administration can be selected from, for example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, intranasal administration, inhalation, etc.

[0071] The "pharmaceutical product comprising an anti-CCR8 antibody and a chemotherapeutic agent" of the present invention may be a combination of a pharmaceutical composition containing an anti-CCR8 antibody and a pharmaceutical composition containing a chemotherapeutic agent, or it may be a combination of an anti-CCR8 antibody and a chemotherapeutic agent. A "combination drug" refers to a preparation containing two or more active ingredients. For example, a combination drug of an anti-CCR8 antibody and a chemotherapeutic agent contains both the anti-CCR8 antibody and the chemotherapeutic agent in a single preparation.

[0072] When the "pharmaceutical comprising an anti-CCR8 antibody and a chemotherapeutic agent" of the present invention is administered as a combination agent, with the pharmaceutical composition containing the anti-CCR8 antibody and the pharmaceutical composition containing the chemotherapeutic agent being administered as separate formulations, simultaneous administration and administration with a time difference are included. Furthermore, in the case of administration with a time difference, the pharmaceutical composition containing the chemotherapeutic agent may be administered first and the pharmaceutical composition containing the anti-CCR8 antibody may be administered later, or the pharmaceutical composition containing the anti-CCR8 antibody may be administered first and the pharmaceutical composition containing the chemotherapeutic agent may be administered later, and the respective administration methods may be the same or different. The ratio of anti-CCR8 antibody to chemotherapeutic agent in the combination agent can be any value. The weight ratio of anti-CCR8 antibody to chemotherapeutic agent in the combination agent is, for example, 1000:1 to 1:100, preferably 100:1 to 1:100, more preferably 10:1 to 1:10, and even more preferably 5:1 to 1:5.

[0073] When the "pharmaceutical comprising an anti-CCR8 antibody and a chemotherapeutic agent" of the present invention is a combination of an anti-CCR8 antibody and a chemotherapeutic agent, the ratio of the anti-CCR8 antibody to the chemotherapeutic agent in the combination can take any value. The ratio of the anti-CCR8 antibody to the chemotherapeutic agent in the combination is, for example, 1000:1 to 1:100, preferably 100:1 to 1:100, more preferably 10:1 to 1:10, and even more preferably 5:1 to 1:5.

[0074] The pharmaceutical composition or formulation according to the present invention may be mixed with various pharmaceutical additives such as excipients, binders, disintegrants, and lubricants as needed, depending on the dosage form.

[0075] The therapeutically effective dose of the pharmaceutical composition or formulation according to the present invention is the amount that suppresses the symptoms of the indicated disease compared to an individual that has not received the drug. The specific effective dose is not fixed and is appropriately determined depending on the form of administration, method of administration, purpose of use, and the age, weight, symptoms, etc. of the individual. [Examples]

[0076] Examples of the present invention are given below to explain the present invention in more detail, but the present invention is not limited thereto.

[0077] Test Example 1: Preparation of anti-human CCR8 antibody-producing mouse hybridoma A gene encoding the full length of human CCR8 (UniProtKB / Swiss-Prot: P51685, SEQ ID NO: 1) was used as an antigen to immunize A / J Jms Slc female mice by DNA immunization. DNA immunization was repeated two or three times at two-week intervals, and was boosted by intraperitoneal administration of human CCR8-expressing Expi293 cells one week after the final immunization. Three days later, the spleen was excised, and spleen cells and mouse myeloma cells (p3×6363-Ag8., Tokyo Institute of Oncology) were fused using the PEG method and selected in a medium containing hypoxanthine, aminopterin and thymidine. Anti-human CCR8 antibody and anti-human CCR8 neutralizing antibody were selected from this culture supernatant by the following method. Anti-human CCR8 antibody was reacted with culture supernatant with human CCR8-expressing Expi293 cells and human CCR4-expressing Expi293 cells, respectively, and clones specifically binding only to human CCR8 were selected by detecting with Alexa488-labeled anti-mouse IgG antibody (manufactured by Thermo Fisher Scientific). Anti-human CCR8 neutralizing antibody was added to human CCR8-expressing 293 cells pre-incorporated with an indicator, and after sufficient reaction of the culture supernatant, Ca 2+ influx was measured by FLIPR upon addition of 200 nM hCCL1 (manufactured by BioLegend), and clones inhibiting Ca 2+ influx by hCCL1 stimulation were selected. 2+ Clones showing particularly strong neutralizing activity (% inhibition>80%) and clones without neutralizing activity were each cloned to establish hybridomas. Clones showing particularly strong neutralizing activity (% inhibition>80%) and clones without neutralizing activity were each cloned to establish hybridomas.

[0078] Test Example 2: Preparation of anti-human CCR8 antibody-producing rat hybridoma Human CCR8-expressing Rat-1 cells as an immunogen were transfected into pQCXIP (Clontech) The expression vector containing the cloned human CCR8 gene was transfected into Rat-1 cells, followed by drug selection with puromycin (1 ug / ml) for one month. Human CCR8-expressing Rat-1 cells were immunized once in rats, and approximately two weeks later, lymph nodes were collected and hybridomas were constructed using a standard method. Anti-human CCR8 antibodies were selected by reacting hybridoma culture supernatants with human CCR8-expressing 293 cells and 293 cells, respectively, and detecting the antibodies with Alexa647-labeled anti-rat IgG antibody (Thermo Fisher Scientific). Clones capable of specifically binding to human CCR8 were then selected.

[0079] Test Example 3: Epitope Analysis of Anti-Human CCR8 Neutralizing and Non-Neutralizing Antibodies For the anti-human CCR8 antibody-producing hybridoma established in Test Example 1, purified antibodies from 40 clones were obtained from the serum-free culture supernatant by Protein G purification and gel filtration. Of the 40 clones obtained, 27 neutralizing antibodies and 13 non-neutralizing antibodies were used to perform antigen binding assays using the method described below, thereby identifying epitopes important for neutralizing activity. The neutralizing activity of each clone was determined by adding 100 nM hCCL1 (BioLegend) as described in Test Example 1. 2+ Inhibitory activity against influx was measured by FLIPR. Each neutralizing antibody was tested for Ca iontophoresis induced by 100 nM hCCL1 stimulation. 2+ The IC50 value for the influx inhibitory activity was 2 nM or less. The full-length genes encoding human CCR8 (UniProtKB / Swiss-Prot:P51685, SEQ ID NO: 1), human CCR4 (UniProtKB / Swiss-Prot:P51679, SEQ ID NO: 48), and mouse CCR8 (UniProtKB / Swiss-Prot:P56484, SEQ ID NO: 39) were cloned into pcDNA3.4 vectors. Expi293 cells expressing human CCR8, human CCR4, and mouse CCR8 transiently were generated by transfection. These cells were reacted with dilution series of purified antibodies from each clone, and their binding affinity was evaluated by detection with Alexa488-labeled anti-mouse IgG antibody (Thermo Fisher Scientific). The results showed that all antibodies bound to human CCR8, but not to human CCR4 or mouse CCR8. Therefore, we created chimeric bodies (Figure 1) in which the N-terminal region (amino acids 1-35 of SEQ ID NO: 1), loop1 region (amino acids 94-107 of SEQ ID NO: 1), loop2 region (amino acids 172-202 of SEQ ID NO: 1), and loop3 region (amino acids 264-280 of SEQ ID NO: 1), corresponding to the extracellular domain of human CCR8, were replaced with the corresponding N-terminal region (amino acids 1-39 of SEQ ID NO: 48), loop1 region (amino acids 98-111 of SEQ ID NO: 48), loop2 region (amino acids 176-206 of SEQ ID NO: 48), and loop3 region (amino acids 268-284 of SEQ ID NO: 48) of human CCR4. The binding affinity of each antibody was then evaluated using the same method as described above. Binding was evaluated at 5 ug / mL, 0.5 ug / mL, and 0.05 ug / mL. Compared to wild-type human CCR8 (hCCR8), organisms showing significantly reduced binding activity were marked with △, and those showing binding below the detection limit were marked with ×. A blank space indicates that the organism shows binding activity at a similar level to wild-type human CCR8 (hCCR8). As a result, as shown in Table 2, the binding activity of all antibodies exhibiting neutralizing activity was reduced to below the detection limit when the N-terminal region was substituted with human CCR4 (N-ter hCCR4-hCCR8), revealing that the N-terminal region is an important epitope for the exertion of neutralizing activity. Furthermore, binding activity was also reduced for loop1 human CCR4-substituted human CCR8 and loop2 human CCR4-substituted human CCR8. From these findings, it is thought that anti-CCR8 antibodies with strong neutralizing activity strongly recognize the N-terminal region and also perform conformational recognition that allows them to bind to loop1 and loop2. Furthermore, antibodies that do not recognize human CCR8, human CCR4, or mouse CCR8 were used as controls, but the control antibodies did not bind to any of the antigens. In addition, by tagging the N-terminus of each antigen and detecting it with an anti-tagged antibody, it was confirmed that the mutation did not reduce hCCR8 expression. Furthermore, to analyze the N-terminal region, which is a common epitope region for all clones exhibiting strong neutralization, in more detail, human CCR8 point mutants were created as shown in Table 2. Using the same method as described above, the binding activity of each clone to these mutants was evaluated. The results showed that all 27 neutralizing antibodies exhibited significantly reduced binding activity to the mutant in which Y at position 17 of human CCR8 was replaced with A (hCCR8(Y17A)), falling below the detection limit, suggesting that they recognize Y at position 17. In addition, although only clone No. 8F7 is shown in Table 2, there were also several neutralizing antibodies that showed reduced binding activity to the mutant in which I at position 20 of human CCR8 was replaced with A (hCCR8(I20A)), suggesting that some neutralizing antibodies also exhibit structural recognition that allows them to bind to isoleucine at position 20. On the other hand, the 13 non-neutralizing antibodies listed above did not show a decrease in binding activity to hCCR8(Y17A), suggesting that they recognize the same N-terminal region but do not recognize Y at position 17. This suggests that Y at position 17 is an extremely important amino acid for neutralizing activity. Table 2 shows the results for representative neutralizing antibodies (clone No. 10A11, 27G1, 1H4, 8F7, 2C7) and non-neutralizing antibodies (clone No. 5B5).

[0080] [Table 2]

[0081] Test Example 4: Determination of Antibody Sequence Of the established clones, the amino acid sequences of the light chain variable region and heavy chain variable region of the mouse antibodies shown in Table 3 and the rat antibodies shown in Table 4 were determined from hybridoma cells using a standard method.

[0082] [Table 3]

[0083] [Table 4]

[0084] Test Example 5: Alignment of Antibody Sequences The amino acid sequences of the light and heavy chains of the mouse hybridoma-derived anti-human CCR8 neutralizing antibody described in Test Example 4 were analyzed using Kabat numbering and alignment with the antibody sequence analysis software abYsis (Figures 2 and 3). As a result, the amino acid sequences of 10A11, 27G1, 1H4, 19D7, and 8F7 contained sequences similar to those of CDR, as described below. The light chain CDR1 was composed of the 16 amino acids shown in SEQ ID NO: 2. The light chain CDR2 consisted of seven amino acids of R-Xaa1-SNLAS (where Xaa1 is M or V: SEQ ID NO: 49) (SEQ ID NO: 3 or 9). The light chain CDR3 consisted of nine amino acids MQHLEYP-Xaa1-T (where Xaa1 is L or F: SEQ ID NO: 50) (SEQ ID NO: 4 or 10). The heavy chain CDR1 consisted of five amino acids Xaa1-YA-Xaa2-Y (where Xaa1 is T or P and Xaa2 is L or M: SEQ ID NO: 51) (SEQ ID NOs: 5, 8, or 12). The heavy chain CDR2 consisted of the 19 amino acids shown in SEQ ID NO: 6. The heavy chain CDR3 has a common sequence of 10A11, 27G1, and 1H4, and is composed of the 14 amino acids shown in SEQ ID NO: 7.

[0085] Test Example 6: Evaluation of Neutralizing Activity The established hybridomas were cultured in serum-free medium, and the culture supernatant was purified by Protein G affinity purification and gel filtration to obtain purified antibodies. The neutralizing activity of these purified antibodies was measured using the method described below. In advance Ca 2+ To 293 human CCR8-expressing cells that had incorporated an indicator, an antibody dilution diluted in culture medium was added, and Ca was measured by FLIPR with the addition of 200 nM hCCL1 (BioLegend). 2+ Intake was measured. The inhibition rate was calculated by setting the signal without hCCL1 addition as 100% inhibition and the signal with hCCL1 addition and without antibody addition as 0% inhibition. The antibody concentration showing a 50% inhibition rate was defined as IC50. At least three evaluations were performed, and the IC50 was expressed as Average ± SD. (Table 5)

[0086] [Table 5]

[0087] Test Example 7: Humanization of Antibodies (10A11, 2C7) Humanization was performed on 10A11 and 2C7 using the following method. Kabat numbering and CDR definitions were performed using the antibody sequence analysis software abYsis. Human germline acceptor sequences similar to the V gene region sequences of the heavy and light chains of mouse antibody amino acid sequences were searched for and selected using the sequence analysis software Absis. For the J chain region, sequences with high homology to the mouse antibody DNA sequence were searched for using IMGT (http: / / www.imgt.org / ) and designated as human framework sequences. Humanized monoclonal antibody sequences were designed by transplanting mouse antibody heavy chains CDR1, CDR2, and CDR3 and mouse antibody light chains CDR1, CDR2, and CDR3, as defined by Kabat numbering (Wu, TT and Kabat, EA, J Exp. Med. Aug1; 132(2): 211-50. (1970)) onto these human framework sequences (light chain; Figure 4, heavy chain; Figure 5). Neutralization activity was measured using the method shown in Test Example 6, and the humanized monoclonal antibodies shown in Table 6 showed affinity equal to or greater than that of the mouse antibodies.

[0088] [Table 6]

[0089] Test Example 7-2: Humanization of Antibodies (19D7) Humanization of 19D7 was performed using the same method as in Test Example 7 (light chain; Figure 6, heavy chain; Figure 7). Neutralization activity was measured using the method shown in Test Example 6, and the humanized monoclonal antibodies shown in Table 7 showed affinity equal to or greater than that of the mouse antibodies.

[0090] [Table 7]

[0091] Test Example 8: Identification of amino acids important for the activity of humanized 10A11 For the humanized 10A11 shown in Figures 4 and 5, mutants were created by introducing point mutations in the amino acids corresponding to the CDR, and the neutralizing activity of each mutant was calculated using the method shown in Test Example 6. The neutralizing activity was evaluated multiple times for each mutant, and its IC50 value was expressed as the ratio of the IC50 value of the WT to the IC50 value, expressed as the IC50 ratio (WT IC50 / mutant IC50). The results are shown in Tables 8 and 9. nd (=not detectable) indicates that the IC50 value of the mutant was above the detection limit of 10 nM, meaning its activity was reduced to below the detection limit. Neutralizing activity evaluation revealed that for the light chain, mutants S26T, K27R, L27cI, and E93D, which had mutations at the L26, L27, L27c, and L93 amino acid sites according to Kabat numbering, showed a more than 10-fold decrease in activity (Table 8). For the heavy chain, mutants A33V, Y35F, R52K, N53Q, Y59F, R96K, F97L, Y98F, G100cA, and D101E, which had mutations at the H33, H35, H52, H53, H59, H96, H97, H98, H100c, and H101 amino acid sites according to Kabat numbering, also showed a more than 10-fold decrease in activity (Table 9). These amino acids are extremely important for activity.

[0092] [Table 8] *The amino acid site is determined by Kabat's numbering system.

[0093] [Table 9] *The amino acid site is determined by Kabat's numbering system.

[0094] Test Example 8-2: Identification of amino acids important for the activity of humanized 19D7 For the humanized 19D7 shown in Figures 6 and 7, mutants were created by introducing point mutations in the amino acids corresponding to the CDR, and the neutralizing activity of each mutant was calculated using the method shown in Test Example 6. The neutralizing activity was evaluated multiple times for each mutant, and its IC50 value was expressed as the ratio of the IC50 value of the WT to the IC50 value, expressed as the IC50 ratio (WT IC50 / mutant IC50). The results are shown in Tables 10 and 11. nd (=not detectable) indicates that the IC50 value of the mutant is above the measurement system's limit of 10 nM, meaning its activity has decreased to below the detection limit.

[0095] [Table 10] *The amino acid site is determined by Kabat's numbering system.

[0096] [Table 11] *The amino acid site is determined by Kabat's numbering system.

[0097] Test Example 9: Improvement of Humanized 10A11 Activity Humanized 10A11 was optimized by combining the humanization framework found in Test Example 7 with the activity-enhancing mutations found in Test Example 8 and mutations in the N at the L28 amino acid site and the G at the L29 amino acid site, corresponding to deamidation risk sequences, as determined by Kabat numbering. As a result, the humanized 10A11 variants shown in Table 12 were found.

[0098] [Table 12] *The amino acid site is determined by Kabat's numbering system.

[0099] Test Example 9-2: Enhancement of Humanized 19D7 Activity Point mutations were introduced into the humanization framework identified in Test Example 7-2 to optimize humanized 19D7. As a result, the humanized 19D7 mutants shown in Table 13 were found.

[0100] [Table 13]

[0101] Test Example 10: Antitumor activity of anti-human CCR8 antibody in tumor-transplanted human CCR8 knock-in mice (hereinafter referred to as hCCR8-KI (KI / KI) mice) (1) Creation of hCCR8-KI(KI / KI) mice By removing the full length of the ORF of the gene encoding mouse CCR8 (SEQ ID NO: 39) and simultaneously inserting the full length of the ORF of the gene encoding human CCR8 (SEQ ID NO: 1), we created hCCR8-KI (KI / KI) mice in which the expressed CCR8 protein is completely humanized. DNA fragments used as homologous recombination arms were obtained by PCR amplification of approximately 2 kb (kilobases) of mouse genome sequence upstream and downstream of the ORF of the mouse CCR8 gene, and the mouse genome sequence was designed so that only the ORF was removed. A targeting vector was created by seamlessly connecting the homologous recombination arms before and after the full-length ORF sequence of the human CCR8 gene, and subjected to homologous recombination to create hCCR8-KI(KI / +)Balb / c mice. For the created hCCR8-KI(KI / +)Balb / c mice, it was confirmed by PCR and DNA base sequence analysis that the full-length ORF of the mouse CCR8 gene and the full-length ORF of the human CCR8 gene were correctly replaced, and that there were no extraneous sequence insertions or deletions. hCCR8-KI(KI / KI) mice were created by crossing hCCR8-KI(KI / +)Balb / c mice in a normal mating.

[0102] (2) Confirmation of human CCR8 expression in tumor-invading cells of the colorectal cancer CT26 cell line in hCCR8-KI (KI / KI) mice. hCCR8-KI(KI / KI) mice (6 weeks old, female) had 3.5 × 10⁴ cells in the dorsal dermis. 5 CT26 cells (50 μL) were transplanted, and tumors were collected from 5 individuals 17 days after transplantation (N=5). The tumor masses of CT26 cells were finely cut with scissors, and tumor-infiltrating cells were prepared using commercially available kits (Tumor Dissociation Kit, mouse, Miltenyi and The gentleMACS(TM) Dissociator, Miltenyi) according to the kit protocol. The prepared cells were passed through a 70 μm cell strainer and washed twice with 10 mM HEPES / HBSS / 2% FBS. They were then treated with erythrocyte lysate (BD Biosciences) for 5 minutes to remove red blood cells, and washed twice again with 2% FBS / 10 mM HEPES / HBSS buffer. Tumor-infiltrating cells were stained using the following methods and antibodies. Infiltrating cells were stained using the Zombie NIR Fixable Viability Kit (BioLegend) reagent in ice for 30 minutes. After washing once with 2% FBS / 10mM HEPES / HBSS, the cells were stained with Bv510-labeled anti-mouse CD45 (30-F11, BioLegend), FITC-labeled anti-mouse CD4 (RM4-4, BioLegend), PE / Cy7-labeled anti-mouse CD8 (53-6.7, BioLegend), PerCP / Cy5.5-labeled anti-mouse TCRβ (H57-597, BioLegend), PE-labeled anti-mouse CD25 (PC61, BioLegend), and BV421-labeled anti-human CCR8 antibody (433H, BD Biosciences) (or BV421-labeled isotype control antibody). Staining was performed in ice for 30 minutes. After washing twice with 2% FBS / HEPES / HBSS, the cells were analyzed using a flow cytometer. Human CCR8 expression in CD45+TCRβ+CD4+CD25+ T cells was analyzed. Negative cell regions were determined by staining with isotype control antibodies, and cells positive with anti-human CCR8 antibody were designated as human CCR8+ cells. Their frequency was calculated 17 days after transplantation. As a result, human CCR8 was detected in approximately 47% of CD45+TCRβ+CD4+CD25+ cells in mouse tumors.

[0103] (3) Evaluation of the antitumor effect of administering anti-human CCR8 antibody to mice transplanted with CT26 cells derived from colorectal cancer. hCCR8-KI(KI / KI) mouse (8 weeks old, female) had 4 × 10 cells in the dorsal dermis. 5 N=10 patients were transplanted with CT26 cells (50 μL) derived from colorectal cancer. Humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41)) was administered intravenously at doses of 100 μg (100 μL) or 200 μg (100 μL) at 4 and 11 days after tumor transplantation. Controls were administered 100 μL of a medium (phosphate-buffered saline) (N=10). Tumor volume was measured at 4, 7, 10, 11, 14, 16, 18, 21, and 24 days after tumor transplantation. Tumor volume (mm²) 3 The measurement was taken using the formula: major diameter (mm) × minor diameter (mm) × minor diameter (mm) / 2. As a result, tumor volume was significantly smaller in all doses of anti-human CCR8 antibody administered at 11, 14, 16, 18, 21, and 24 days after transplantation (Figure 8; significance level was determined using Welch's test, with **; p<0.01 at 10 days and ***; p<0.001 from 11 days onward for all doses). Furthermore, some individuals in the anti-human CCR8 antibody administration groups showed complete regression, with tumors almost completely disappearing in 5 out of 10 mice in the 100 μg anti-human CCR8 antibody group and 6 out of 10 mice in the 200 μg anti-human CCR8 antibody group at 24 days.

[0104] Example 1: Evaluation of the antitumor effect of combined administration of anti-mouse CCR8 antibody and carboplatin in breast cancer-derived 4T1 cell-transplanted mice. Balb / c mouse (6 weeks old, female) had 2 × 10⁶ cells in the dorsal dermis.5 One breast cancer-derived 4T1 cell (50 μL) was transplanted. In the anti-mouse CCR8 antibody monotherapy group, 50 μg (200 μL) of anti-mouse CCR8 antibody (clone SA214G2, BioLegend) was administered intravenously 5 and 8 days after tumor transplantation, and 11.4 mL / kg body weight of physiological saline was administered intravenously 5 days after tumor transplantation (N=10). In the carboplatin monotherapy group, 50 μg (200 μL) of isotype control antibody (clone LTF-2, Bio X Cell) was administered intravenously 5 and 8 days after tumor transplantation, and 114 mg / kg body weight of carboplatin (product name: Paraplatin, Bristol-Myers Squibb) was administered intravenously 5 days after tumor transplantation (N=10). In the group receiving carboplatin in combination with an anti-mouse CCR8 antibody, 50 μg of anti-mouse CCR8 antibody was administered intravenously 5 and 8 days after tumor transplantation, and 114 mg / kg body weight of carboplatin was administered intravenously 5 days after tumor transplantation (N=10). In the control group, 50 μg of isotype control antibody was administered intravenously 5 and 8 days after tumor transplantation, and physiological saline was administered intravenously 5 days after tumor transplantation (N=10). Tumor volume was measured every 3-4 days starting 5 days after tumor transplantation. Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) × minor diameter (mm) × minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 9. At 8, 12, 15, 18, and 22 days after tumor transplantation, there was no significant difference in mean tumor volume between the control group and the anti-mouse CCR8 antibody monotherapy group, nor between the control group and the carboplatin monotherapy group. On the other hand, in the group administered anti-mouse CCR8 antibody and carboplatin in combination, a significant reduction in tumor volume was observed from 8 days after tumor transplantation compared to the control group. Furthermore, the tumor volume was significantly smaller from 8 days after day compared to the anti-mouse CCR8 antibody monotherapy group and the carboplatin monotherapy group. Based on these results, it became clear that co-administration of carboplatin with an anti-mouse CCR8 antibody, which does not significantly affect tumor volume when administered alone, shows a synergistic or greater antitumor effect compared to each drug administered alone.

[0105] Example 2: Evaluation of the antitumor effect of combined administration of anti-mouse CCR8 antibody and gemcitabine in breast cancer-derived 4T1 cell-transplanted mice. Balb / c mouse (6 weeks old, female) had 2 × 10⁶ cells in the dorsal dermis. 5 One breast cancer-derived 4T1 cell (50 μL) was transplanted. In the anti-mouse CCR8 antibody monotherapy group, 50 μg (200 μL) of anti-mouse CCR8 antibody (clone SA214G2, BioLegend) was administered intravenously 5 and 8 days after tumor transplantation, and 10 mL / kg body weight of physiological saline was administered intravenously 5 days after tumor transplantation (N=10). In the gemcitabine monotherapy group, 50 μg (200 μL) of isotype control antibody (clone LTF-2, Bio X Cell) was administered intravenously 5 and 8 days after tumor transplantation, and 50 mg / kg body weight of gemcitabine (product name: Gemzar, Eli Lilly) was administered intravenously 5 days after tumor transplantation (N=10). In the gemcitabine and anti-mouse CCR8 antibody combination therapy group, 50 μg of anti-mouse CCR8 antibody was administered intravenously 5 and 8 days after tumor transplantation, and 50 mg / kg body weight of gemcitabine was administered intravenously 5 days after tumor transplantation (N=10). In the control group, 50 μg of isotype control antibody was administered intravenously 5 and 8 days after tumor transplantation, and physiological saline was administered intravenously 5 days after tumor transplantation (N=10). Tumor volume was measured every 3-4 days starting 5 days after tumor transplantation. Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) × minor diameter (mm) × minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 10. At 8, 12, 15, 18, and 22 days after tumor transplantation, the gemcitabine monotherapy group showed a significant reduction in tumor volume compared to the control group. No significant difference in tumor volume was observed between the anti-mouse CCR8 antibody monotherapy group and the control group. On the other hand, in the group receiving both anti-mouse CCR8 antibody and gemcitabine, a significant reduction in tumor volume was observed from 8 days after tumor transplantation compared to the control group or the anti-mouse CCR8 antibody monotherapy group, and from 12 days onward, the tumor volume was significantly smaller compared to the gemcitabine monotherapy group. Based on these results, it became clear that co-administration of the anti-mouse CCR8 antibody, which does not significantly affect tumor volume when administered alone, with gemcitabine resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0106] Example 3: Evaluation of the antitumor effect of combined administration of anti-human CCR8 antibody and cisplatin in hCCR8-KI (KI / KI) mice transplanted with breast cancer-derived 4T1 cells. In the dorsal skin of the hCCR8-KI(KI / KI) mice (14 weeks old, female) prepared in Test Example 10, 2 × 10 5 One breast cancer-derived 4T1 cell (50 μL) was transplanted. In the anti-human CCR8 antibody monotherapy group, 400 μg (200 μL) of anti-human CCR8 antibody (humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41))) was administered intravenously 5 and 8 days after tumor transplantation, and 16 mL / kg body weight of physiological saline was administered intravenously 5 days after tumor transplantation (N=10). In the cisplatin monotherapy group, 200 μL of control solution (buffer used for the antibody diluted with physiological saline according to the antibody dilution ratio) was administered intravenously 5 and 8 days after tumor transplantation, and 8 mg / kg body weight of cisplatin (product name: Randa, Nippon Kayaku) was administered intravenously 5 days after tumor transplantation (N=10). In the group receiving combination therapy with anti-human CCR8 antibody and cisplatin, 400 μg of anti-human CCR8 antibody was administered intravenously 5 and 8 days after tumor transplantation, and 8 mg / kg body weight of cisplatin was administered intravenously 5 days after tumor transplantation (N=10). In the control group, 200 μL of control solution was administered intravenously 5 and 8 days after tumor transplantation, and physiological saline was administered intravenously 5 days after tumor transplantation (N=10). Tumor volume was measured every 2-4 days starting 5 days after tumor transplantation. Tumor volume (mm 3 The weight was measured using the formula: major axis (mm) x minor axis (mm) x minor axis (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 11. At 8, 11, 13, 15, 18, 21, 25, and 29 days after tumor transplantation, the cisplatin monotherapy group showed a significant reduction in tumor volume compared to the control group. No significant reduction in tumor volume was observed in the anti-human CCR8 antibody monotherapy group. On the other hand, in the combination therapy group of anti-human CCR8 antibody and cisplatin, a significant reduction in tumor volume was observed from 8 days after tumor transplantation compared to the control group or the anti-human CCR8 antibody monotherapy group, and from 18 days onward, the tumor volume was significantly reduced compared to the cisplatin monotherapy group. Based on these results, it became clear that co-administration of the anti-human CCR8 antibody, which does not significantly affect tumor volume when administered alone, with cisplatin resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0107] Example 4 Evaluation of antitumor effects of combined administration of anti-mouse CCR8 antibody and carboplatin or cisplatin in bladder cancer-derived MB49 cell-transplanted mice. C57BL / 6 mouse (6 weeks old, male) had 2 × 10 cells in the dorsal skin. 5 One bladder cancer-derived MB49 (50 μL) was transplanted. In the group receiving anti-mouse CCR8 antibody monotherapy, 50 μg (200 μL) of anti-mouse CCR8 antibody (clone SA214G2, BioLegend) was administered intravenously 4 and 7 days after tumor transplantation, and physiological saline was administered intravenously at a volume of 10 mL / kg body weight 4 days after tumor transplantation (N=10). In the carboplatin monotherapy group, 50 μg (200 μL) of isotype control antibody (Clone LTF-2, Bio X Cell) was administered intravenously 4 and 7 days after tumor transplantation, and carboplatin (product name: Paraplatin, Bristol-Myers Squibb) 40 mg / kg body weight was administered intravenously at a volume of 10 mL / kg body weight 4 days after tumor transplantation (N=10). In the combination therapy group of anti-mouse CCR8 antibody and carboplatin, 50 μg of anti-mouse CCR8 antibody was administered intravenously 4 and 7 days after tumor transplantation, and carboplatin 40 mg / kg body weight was administered intravenously 4 days after tumor transplantation (N=10). In the cisplatin monotherapy group, 50 μg (200 μL) of isotype control antibody was administered intravenously 4 and 7 days after tumor transplantation, and 3 mg / kg body weight of cisplatin (product name: Randa, Nippon Kayaku) was administered intravenously at a volume of 10 mL / kg body weight 4 days after tumor transplantation (N=10). In the cisplatin and anti-mouse CCR8 antibody combination therapy group, 50 μg of anti-mouse CCR8 antibody was administered intravenously 4 and 7 days after tumor transplantation, and 3 mg / kg body weight of cisplatin was administered intravenously 4 days after tumor transplantation (N=10). The control group received intravenous administration of 50 μg of isotype control antibody 4 and 7 days after tumor transplantation, and intravenous administration of normal saline 4 days after tumor transplantation (N=10). Tumor volume was measured every 1 to 4 days starting 4 days after tumor transplantation. Tumor volume (mm 3The weight was measured using the formula: major axis (mm) x minor axis (mm) x minor axis (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 12 (carboplatin) and Figure 13 (cisplatin). At 7, 10, 14, 17, and 18 days after tumor transplantation, no significant difference in mean tumor volume was observed between the control group and the anti-mouse CCR8 antibody monotherapy group, the control group and the carboplatin monotherapy group, or the control group and the cisplatin monotherapy group. On the other hand, in the group administered with anti-mouse CCR8 antibody and carboplatin, a significant reduction in tumor volume was observed from 10 days after tumor transplantation compared to the control group. Furthermore, the tumor volume was significantly reduced from 10 days after tumor transplantation compared to the anti-mouse CCR8 antibody monotherapy group and the carboplatin monotherapy group. In the group administered with anti-mouse CCR8 antibody and cisplatin, a significant reduction in tumor volume was also observed from 14 days after tumor transplantation compared to the control group. Furthermore, the tumor volume was significantly reduced from 10 days after tumor transplantation compared to the anti-mouse CCR8 antibody monotherapy group and the cisplatin monotherapy group. Based on these results, it became clear that co-administration of an anti-mouse CCR8 antibody, which does not significantly affect tumor volume when administered alone, with carboplatin or cisplatin resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0108] Example 5: Evaluation of antitumor effects in mice transplanted with Colon26 cells derived from colorectal cancer by the combined administration of anti-mouse CCR8 antibody and oxaliplatin or fluorouracil (5-FU). A Balb / c mouse (5 weeks old, female) had a 3.5 × 10⁴ cavity inside its dorsal skin. 5 One colon cancer-derived Colon26 (50 μL) was transplanted. In the group receiving anti-mouse CCR8 antibody monotherapy, 50 μg (200 μL) of anti-mouse CCR8 antibody (clone SA214G2, BioLegend) was administered intravenously 5 days after tumor transplantation, and 1 hour after anti-mouse CCR8 antibody administration, physiological saline was administered intravenously at a volume of 10 mg / kg body weight (N=10). In the oxaliplatin monotherapy group, 50 μg (200 μL) of isotype control antibody was administered intravenously 5 days after tumor transplantation, and oxaliplatin (product name: Elplat, Yakult Honsha) 5 mg / kg body weight was administered intravenously at a volume of 10 mL / kg body weight 1 hour after administration of the isotype control antibody (N=10). In the combination therapy group of anti-mouse CCR8 antibody and oxaliplatin, 50 μg of anti-mouse CCR8 antibody was administered intravenously 5 days after tumor transplantation, and oxaliplatin 5 mg / kg body weight was administered intravenously 1 hour after administration of the anti-mouse CCR8 antibody (N=10). In the 5-FU monotherapy group, 50 μg (200 μL) of isotype control antibody was administered intravenously 5 days after tumor transplantation, and 5-FU (product name: 5-FU Injection 250 Kyowa, Kyowa Hakko Kirin) was administered intravenously at a dose of 30 mg / kg body weight and a volume of 10 mL / kg (N=10). In the combination therapy group of anti-mouse CCR8 antibody and 5-FU, 50 μg of anti-mouse CCR8 antibody was administered intravenously 5 days after tumor transplantation, and 5-FU was administered intravenously at a dose of 30 mg / kg body weight 1 hour after anti-mouse CCR8 antibody administration (N=10). The control group received 50 μg of isotype control antibody intravenously 5 days after tumor transplantation, and physiological saline was administered intravenously 1 hour after the administration of the isotype control antibody (N=10). Tumor volume was measured every 2-3 days starting 4 days after tumor transplantation (the day before antibody administration). Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 14 (oxaliplatin) and Figure 15 (5-FU). At 4, 7, 10, 12, 14, and 17 days after tumor transplantation, no significant reduction in tumor volume was observed in the oxaliplatin monotherapy group or the anti-mouse CCR8 antibody monotherapy group compared to the control group. On the other hand, in the group administered with anti-mouse CCR8 antibody and oxaliplatin in combination, a significant reduction in tumor volume was observed from 10 days after tumor transplantation compared to the control group, from 10 days after tumor transplantation compared to the oxaliplatin monotherapy group, and from 12 days after tumor transplantation compared to the anti-mouse CCR8 antibody monotherapy group. In the 5-FU monotherapy group, a significant reduction in tumor volume was observed only at 7 days after tumor transplantation compared to the control group. On the other hand, in the group administered with anti-mouse CCR8 antibody and 5-FU in combination, a significant reduction in tumor volume was observed from 10 days after tumor transplantation compared to the control group, from 12 days after tumor transplantation compared to the 5-FU monotherapy group, and from 14 days after tumor transplantation compared to the anti-mouse CCR8 antibody monotherapy group. Based on these results, it became clear that co-administration of an anti-mouse CCR8 antibody, which does not significantly affect tumor volume when administered alone, with oxaliplatin or 5-FU resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0109] Example 6: Evaluation of the antitumor effect of combined administration of anti-human CCR8 antibody and oxaliplatin in hCCR8-KI (KI / KI) mice transplanted with colon cancer-derived Colon26 cells. In the dorsal skin of the hCCR8-KI(KI / KI) mice (12 weeks old, female) prepared in Test Example 10, 3.5 × 10 5 One colon cancer-derived Colon26 (50 μL) was transplanted. In the group receiving anti-human CCR8 antibody monotherapy, 15 mg / kg body weight of anti-human CCR8 antibody (humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41))) was administered intravenously 5 days after tumor transplantation, and physiological saline was administered intravenously 3 hours after the administration of anti-human CCR8 antibody (N=10). In the group receiving oxaliplatin monotherapy, physiological saline was administered intravenously 5 days after tumor transplantation, and oxaliplatin (product name: Elplat, Yakult Honsha) 5 mg / kg body weight was administered intravenously 3 hours after the administration of physiological saline (N=10). In the group receiving combination therapy with anti-human CCR8 antibody and oxaliplatin, 15 mg / kg body weight of anti-human CCR8 antibody was administered intravenously 5 days after tumor transplantation, followed by 5 mg / kg body weight of oxaliplatin intravenously 3 hours after anti-human CCR8 antibody administration (N=10). In the control group, saline was administered intravenously 5 days after tumor transplantation, followed by another intravenous administration of saline 3 hours after saline administration (N=10). In all samples, tail vein administration was performed at a volume of 10 mL / kg body weight. Tumor volume was measured every 1 to 3 days starting 4 days after tumor transplantation (the day before antibody administration). Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 16. At 4, 7, 10, 12, 14, 17, and 18 days after tumor transplantation, no significant reduction in tumor volume was observed in the oxaliplatin monotherapy group or the anti-human CCR8 antibody monotherapy group compared to the control group. On the other hand, in the group receiving combination therapy with anti-human CCR8 antibody and oxaliplatin, a significant reduction in tumor volume was observed from 7 days after tumor transplantation compared to the control group, from 10 days after tumor transplantation compared to the anti-human CCR8 antibody monotherapy group, and from 12 days after tumor transplantation compared to the oxaliplatin monotherapy group. Based on these results, anti-human CCR8 antibodies, when administered alone, do not significantly affect tumor volume. Oxaliplatin It was revealed that the combined administration of these drugs showed a synergistic or greater antitumor effect compared to each drug administered alone.

[0110] Example 7 Evaluation of the antitumor effect of combined administration of anti-human CCR8 antibody and cisplatin in lung cancer-derived ASB-XIV cell-transplanted hCCR8-KI (KI / KI) mice. In the dorsal skin of the hCCR8-KI (KI / KI) mice (8 weeks old, female) prepared in Test Example 10, 5 × 10 5 One lung cancer-derived ASB-XIV cell (50 μL) was transplanted. In the anti-human CCR8 antibody monotherapy group, anti-human CCR8 antibody was administered intravenously at a dose of 1 mg / 10 mL / kg body weight (humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41))) and 10 mL / kg body weight of physiological saline 5 days after tumor transplantation (N=10). In the cisplatin monotherapy group, a control solution was administered intravenously at a dose of 10 mL / kg body weight and cisplatin (product name: Randa, Nippon Kayaku) at a dose of 3 mg / 10 mL / kg body weight 5 days after tumor transplantation (N=10). In the anti-human CCR8 antibody and cisplatin combination therapy group, anti-human CCR8 antibody was administered intravenously at a dose of 1 mg / 10 mL / kg body weight and cisplatin at a dose of 3 mg / 10 mL / kg body weight 5 days after tumor transplantation (N=10). The control group received intravenous administration of control solution at 10 mL / kg body weight and physiological saline at 10 mL / kg body weight 5 days after tumor transplantation (N=10). Tumor volume was measured every 2-5 days starting 5 days after tumor transplantation. Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 17. At 14, 17, and 19 days after tumor transplantation, no significant reduction in tumor volume was observed in the anti-human CCR8 antibody monotherapy group or the cisplatin monotherapy group. On the other hand, the group receiving combination therapy with anti-human CCR8 antibody and cisplatin showed a significant reduction in tumor volume compared to the control group, and at 14 days, the tumor volume was significantly reduced compared to the cisplatin monotherapy group or the anti-human CCR8 antibody monotherapy group. Based on these results, it has become clear that co-administration of anti-human CCR8 antibody and cisplatin, which do not significantly affect tumor volume when administered alone, shows a significant antitumor effect.

[0111] Example 8 Evaluation of the antitumor effect of combined administration of anti-human CCR8 antibody and paclitaxel in lung cancer-derived ASB-XIV cell-transplanted hCCR8-KI (KI / KI) mice. In the dorsal skin of the hCCR8-KI(KI / KI) mice (9 weeks old, female) prepared in Test Example 10, 5 × 10 5 One lung cancer-derived ASB-XIV cell (50 μL) was transplanted. In the group receiving anti-human CCR8 antibody monotherapy, 0.5 mg / 10 mL / kg body weight of anti-human CCR8 antibody (humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41))) and 10 mL / kg body weight of physiological saline were administered intravenously 5 days after tumor transplantation (N=10). In the group receiving paclitaxel monotherapy, 10 mL / kg body weight of physiological saline and 8 mg / 10 mL / kg body weight of paclitaxel (product name: Taxol, Bristol-Myers Squib) were administered intravenously 5 days after tumor transplantation (N=10). In the group receiving combination therapy with anti-human CCR8 antibody and paclitaxel, anti-human CCR8 antibody was administered intravenously at a dose of 0.5 mg / 10 mL / kg body weight and paclitaxel at a dose of 8 mg / 10 mL / kg body weight five days after tumor transplantation (N=10). The control group received intravenous administration of physiological saline at a dose of 20 mL / kg body weight five days after tumor transplantation (N=10). Tumor volume was measured every 2-5 days starting five days after tumor transplantation. Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Student's T: one-sided test method (significance level: P<0.05). The results are shown in Figure 18. At 10, 12, 14, and 18 days after tumor transplantation, no significant reduction in tumor volume was observed in the anti-human CCR8 antibody monotherapy group. In the paclitaxel monotherapy group, a significant reduction in tumor volume was observed at 10 and 12 days after tumor transplantation, but the significant difference disappeared at 14 and 17 days. On the other hand, in the group receiving combination therapy with anti-human CCR8 antibody and paclitaxel, a significant reduction in tumor volume was observed from 10 days after transplantation compared to the control group, and at 18 days, the tumor volume was significantly reduced compared to the paclitaxel monotherapy group or the anti-human CCR8 antibody monotherapy group. Based on these results, it became clear that co-administration of an anti-human CCR8 antibody, which does not significantly affect tumor volume when administered alone, with paclitaxel resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0112] Example 9 Evaluation of antitumor effect of combined administration of anti-human CCR8 antibody and docetaxel in hCCR8-KI (KI / KI) mice transplanted with breast cancer-derived 4T1 cells. In the dorsal skin of the hCCR8-KI(KI / KI) mice (9 weeks old, female) prepared in Test Example 10, 2 × 10 5 One breast cancer-derived 4T1 cell (50 μL) was transplanted. In the group receiving anti-human CCR8 antibody monotherapy, 15 mg / 10 mL / kg body weight of anti-human CCR8 antibody (humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41))) and 10 mL / kg body weight of physiological saline were administered intravenously 5 days after tumor transplantation (N=10). In the group receiving docetaxel monotherapy, 10 mL / kg body weight of physiological saline and 30 mg / 10 mL / kg body weight of docetaxel (product name: OneTaxotere, Sanofi) were administered intravenously 5 days after tumor transplantation (N=10). In the group receiving combination therapy with anti-human CCR8 antibody and docetaxel, anti-human CCR8 antibody was administered intravenously at a dose of 15 mg / 10 mL / kg body weight and docetaxel at a dose of 30 mg / 10 mL / kg body weight five days after tumor transplantation (N=10). The control group received intravenous administration of 20 mL / kg body weight of normal saline five days after tumor transplantation (N=10). Tumor volume was measured every 3-4 days starting five days after tumor transplantation. Tumor volume (mm3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 19. At 8, 12, 15, 18, and 21 days after tumor transplantation, no significant reduction in tumor volume was observed in the group administered anti-human CCR8 antibody alone. On the other hand, in the group administered anti-human CCR8 antibody in combination with docetaxel, a significant reduction in tumor volume was observed compared to the control group or the group administered anti-human CCR8 antibody alone, and from day 12 onwards, the tumor volume was significantly reduced compared to the group administered docetaxel alone. Based on these results, it became clear that co-administration of an anti-human CCR8 antibody, which does not significantly affect tumor volume when administered alone, with docetaxel resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0113] Example 10 Evaluation of antitumor effect of combined administration of anti-mouse CCR8 antibody and docetaxel in breast cancer-derived 4T1 cell-transplanted mice. Balb / c mouse (6 weeks old, female) had 2 × 10⁶ cells in the dorsal dermis. 5 One breast cancer-derived 4T1 cell (50 μL) was transplanted. In the anti-mouse CCR8 antibody monotherapy group, anti-human CCR8 antibody was administered intravenously at 2.5 mg / 10 mL / kg body weight (clone SA214G2, BioLegend) 5 and 8 days after tumor transplantation, and physiological saline was administered intravenously at 10 mL / kg body weight 5 days after tumor transplantation (N=10). In the docetaxel monotherapy group, isotype control antibody was administered intravenously at 2.5 mg / 10 mL / kg body weight (clone LTF-2, Bio X Cell) 5 and 8 days after tumor transplantation, and docetaxel (product name: OneTaxotere, Sanofi) was administered intravenously at 30 mg / 10 mL / kg body weight 5 days after tumor transplantation (N=10). In the group receiving combination therapy with anti-mouse CCR8 antibody and docetaxel, anti-mouse CCR8 antibody was administered intravenously at a dose of 2.5 mg / 10 mL / kg body weight 5 and 8 days after tumor transplantation, and docetaxel was administered intravenously at a dose of 30 mg / 10 mL / kg body weight 5 days after tumor transplantation (N=10). In the control group, isotype control antibody was administered intravenously at a dose of 2.5 mg / 10 mL / kg body weight and physiological saline at a dose of 10 mL / kg body weight 5 days after tumor transplantation (N=10). Tumor volume was measured every 3-4 days starting 5 days after tumor transplantation. Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 20. At 8, 11, 15, 18, and 21 days after tumor transplantation, no significant reduction in tumor volume was observed in the anti-mouse CCR8 antibody monotherapy group. On the other hand, in the group receiving anti-mouse CCR8 antibody in combination with docetaxel, a significant reduction in tumor volume was observed compared to the control group or the anti-mouse CCR8 antibody monotherapy group, and from day 15 onwards, the tumor volume was significantly reduced compared to the docetaxel monotherapy group. Based on these results, it became clear that co-administration of an anti-mouse CCR8 antibody, which does not significantly affect tumor volume when administered alone, with docetaxel resulted in a synergistic or greater antitumor effect compared to each monotherapy group.

[0114] Example 11: Evaluation of antitumor effects of combined administration of anti-human CCR8 antibody and oxaliplatin in hCCR8-KI (KI / KI) mice transplanted with colon cancer-derived Colon26 cells (Part 2) In the same manner as in Example 6, only the anti-human CCR8 antibody was evaluated using two different antibodies (2-7B antibody (light chain variable region: SEQ ID NO: 37 / heavy chain variable region: SEQ ID NO: 38), humanized 19D7 antibody (light chain variable region: IGKV3-20 (SEQ ID NO: 56) / heavy chain variable region: IGHV3-15 K64R+T94R (SEQ ID NO: 60))). Significant difference testing was performed using the Student's T: one-sided test method (significance level: P<0.05). The results are shown in Figure 21 (2-7B antibody) and Figure 22 (humanized 19D7 antibody). 1)2-7B antibody At 10, 14, and 17 days after tumor transplantation, no significant reduction in tumor volume was observed in the oxaliplatin monotherapy group compared to the control group. Similarly, at 14 and 17 days after tumor transplantation, no significant reduction in tumor volume was observed in the anti-human CCR8 antibody monotherapy group compared to the control group. On the other hand, in the group receiving combination therapy with anti-human CCR8 antibody and oxaliplatin, a significant reduction in tumor volume was observed from 10 days after tumor transplantation compared to the control group and the anti-human CCR8 antibody monotherapy group, and from 14 days after tumor transplantation compared to the oxaliplatin monotherapy group. 2) Humanized 19D7 antibody At 10 and 14 days after tumor transplantation, no significant reduction in tumor volume was observed in the oxaliplatin monotherapy group compared to the control group. Similarly, at 14 days after tumor transplantation, no significant reduction in tumor volume was observed in the anti-human CCR8 antibody monotherapy group compared to the control group. On the other hand, in the group receiving combination therapy with anti-human CCR8 antibody and oxaliplatin, a significant reduction in tumor volume was observed from 10 days after tumor transplantation compared to the control group and the anti-human CCR8 antibody monotherapy group, and from 14 days after tumor transplantation compared to the oxaliplatin monotherapy group. Based on these results, anti-human CCR8 antibodies, when administered alone, do not significantly affect tumor volume. Oxaliplatin It was revealed that the combined administration of these drugs showed a synergistic or greater antitumor effect compared to each drug administered alone.

[0115] Example 12: Evaluation of the antitumor effect of combined administration of anti-human CCR8 antibody and fluorouracil (5-FU) in hCCR8-KI (KI / KI) mice transplanted with CT26 cells derived from colorectal cancer. In the dorsal skin of the hCCR8-KI(KI / KI) mouse (14 weeks old, female) prepared in Test Example 10, 3.5 × 10 5 One CT26 (50 μL) derived from colorectal cancer was transplanted. In the group receiving anti-human CCR8 antibody monotherapy, 0.1 mg / kg body weight of anti-human CCR8 antibody (humanized 10A11 antibody (light chain variable region: IGKV4-1 N53Q+G29R (SEQ ID NO: 59) / heavy chain variable region: IGHV3-15 T94R (SEQ ID NO: 41))) was administered intravenously 5 days after tumor transplantation, and physiological saline was administered intravenously 3 hours after anti-human CCR8 antibody administration (N=8). In the group receiving 5-FU monotherapy, physiological saline was administered intravenously 5 days after tumor transplantation, and 5-FU (product name: 5-FU Injection 250 Kyowa, Kyowa Hakko Kirin) was administered intravenously at 30 mg / kg body weight 3 hours after physiological saline administration (N=8). In the group receiving combination therapy with anti-human CCR8 antibody and 5-FU, anti-human CCR8 antibody was administered intravenously at a dose of 0.1 mg / kg body weight five days after tumor transplantation, and 5-FU was administered intravenously at a dose of 30 mg / kg body weight three hours after anti-human CCR8 antibody administration (N=8). In the control group, physiological saline was administered intravenously five days after tumor transplantation, and physiological saline was administered intravenously three hours after physiological saline administration (N=8). For all samples, tail vein administration was performed at a volume of 10 mL / kg body weight. Tumor volume was measured every 3-4 days starting five days after tumor transplantation (the day before antibody administration). Tumor volume (mm 3 The weight was measured using the formula: major diameter (mm) x minor diameter (mm) x minor diameter (mm) / 2, and a significance test was performed using the Mann-Whitney U method (significance level: P<0.05). The results are shown in Figure 23. At 8, 12, and 15 days after tumor transplantation, the 5-FU monotherapy group and the anti-human CCR8 antibody monotherapy group did not show a significant reduction in tumor volume compared to the control group. On the other hand, the group receiving combination therapy with anti-human CCR8 antibody and 5-FU showed a significant reduction in tumor volume from 8 days after tumor transplantation compared to the control group, the anti-human CCR8 antibody monotherapy group, and the 5-FU monotherapy group. Based on these results, it became clear that co-administration of anti-human CCR8 antibody and 5-FU, which do not significantly affect tumor volume when administered alone, shows a synergistic or greater antitumor effect compared to each monotherapy group.

[0116] Example 13: Evaluation of antitumor effects of combination therapy with anti-mCCR8 antibody using mouse cell lines derived from mammary cancer, lung cancer, colorectal cancer, renal cancer, sarcoma, liver cancer, bladder cancer, and ovarian cancer. Mouse mammary cancer, lung cancer, colorectal cancer, renal cancer, sarcoma, liver cancer, bladder cancer, and ovarian cancer-derived cell lines are transplanted intradermally into the dorsal skin of Balb / c mice or C57BL / 6 mice, respectively. After tumor transplantation, rat anti-mouse CD198 (CCR8) antibody (clone SA214G2, BioLegend) is administered in combination with chemotherapy. The chemotherapy agents administered intravenously include carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide, or doxorubicin. Isotype control antibodies are administered to the control group. Tumor volume is measured every 3-4 days after tumor transplantation, and the tumor volume (mm²) is measured. 3 ) is measured using the formula: major diameter (mm) × minor diameter (mm) × minor diameter (mm) / 2. As a result, we will investigate whether a synergistic effect is observed when both anti-mouse CCR8 antibody and chemotherapy are administered in combination, compared to when they are administered alone or alone.

[0117] Example 14 Evaluation of the antitumor effect of combination therapy with anti-human CCR8 antibody and chemotherapy in human CCR8 knock-in mice transplanted with cancer-derived cell lines (hereinafter referred to as hCCR8-KI(KI / KI) mice). In Test Example 10, cell lines derived from breast cancer, lung cancer, colorectal cancer, renal cancer, sarcoma, liver cancer, bladder cancer, or ovarian cancer were transplanted intradermally into the dorsal surface of hCCR8-KI (KI / KI) mice, respectively. After tumor transplantation, anti-human CCR8 antibody administration and chemotherapy were performed in combination. As chemotherapy agents, carboplatin, cisplatin, oxaliplatin, paclitaxel, docetaxel, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide, or doxorubicin are administered intravenously in combination. A control is administered as a medium (phosphate-buffered saline). Tumor volume is measured every 3-4 days after tumor transplantation, and the tumor volume (mm³) is measured. 3 ) is measured using the formula: major diameter (mm) × minor diameter (mm) × minor diameter (mm) / 2. As the anti-human CCR8 antibody, use the anti-CCR8 antibody described in any of (14) to (18), the humanized monoclonal antibody described in any of (19) to (27), or an antibody fragment thereof. For example, use the antibody described in (26). As a result, we will verify whether a synergistic effect is observed when both are administered in combination, compared to when anti-human CCR8 antibodies are administered alone or chemotherapy is administered alone. [Industrial applicability]

[0118] The combination of the anti-CCR8 antibody and chemotherapeutic agent of the present invention is extremely useful for the treatment or prevention of cancer.

Claims

1. A pharmaceutical composition for cancer treatment, which contains an anti-CCR8 antibody and is used together with a chemotherapeutic agent, wherein the anti-CCR8 antibody is 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 Heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 (Here, it may have one or two of the following substitutions, A) The 10th asparagine in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with leucine or arginine. C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7; 3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 11; (Here, it is possible to have one of the following permutations: A) The second serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. B) The third serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. C) The fifth serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. D) The second glutamine in the amino acid sequence of SEQ ID NO: 10 is replaced with asparagine. E) The first threonine in the amino acid sequence of Sequence ID No. 8 is replaced with serine. F) The 14th alanine in the amino acid sequence of sequence number 6 is replaced with valine. G) The 18th lysine in the amino acid sequence of Sequence ID No. 6 is replaced with arginine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO: 6 is replaced with glutamic acid. I) The fifth asparagine in the amino acid sequence of SEQ ID NO: 11 is replaced with glutamine. J) The 12th tyrosine in the amino acid sequence of SEQ ID NO: 11 is substituted with phenylalanine; 4) CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 and the amino acid sequence of SEQ ID NO: 15 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 16 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 17, CDR2 and the amino acid sequence of SEQ ID NO: 18 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 19; or 5) CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 22 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 Heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 25 A monoclonal antibody or antibody fragment containing and having neutralizing activity with an IC50 value of 2 nM or less, The aforementioned chemotherapeutic agent is a drug containing platinum in its structure that induces apoptosis of tumor cells by binding to DNA, such as a taxane, gemcitabine, or fluorouracil. A pharmaceutical composition for cancer treatment.

2. A pharmaceutical composition for cancer treatment that contains a chemotherapeutic agent and is used together with an anti-CCR8 antibody, wherein the anti-CCR8 antibody is 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 Heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 (Here, it may have one or two of the following substitutions, A) The 10th asparagine in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with leucine or arginine. C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7; 3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 11; (Here, it is possible to have one of the following permutations: A) The second serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. B) The third serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. C) The fifth serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. D) The second glutamine in the amino acid sequence of SEQ ID NO: 10 is replaced with asparagine. E) The first threonine in the amino acid sequence of Sequence ID No. 8 is replaced with serine. F) The 14th alanine in the amino acid sequence of sequence number 6 is replaced with valine. G) The 18th lysine in the amino acid sequence of Sequence ID No. 6 is replaced with arginine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO: 6 is replaced with glutamic acid. I) The fifth asparagine in the amino acid sequence of SEQ ID NO: 11 is replaced with glutamine. J) The 12th tyrosine in the amino acid sequence of SEQ ID NO: 11 is substituted with phenylalanine; 4) CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 and the amino acid sequence of SEQ ID NO: 15 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 16 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 17, CDR2 and the amino acid sequence of SEQ ID NO: 18 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 19; or 5) CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 22 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 Heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 25 A monoclonal antibody or antibody fragment containing and having neutralizing activity with an IC50 value of 2 nM or less, The aforementioned chemotherapeutic agent is a drug containing platinum in its structure that induces apoptosis of tumor cells by binding to DNA, such as a taxane, gemcitabine, or fluorouracil. A pharmaceutical composition for cancer treatment.

3. The pharmaceutical composition according to claim 1 or 2, for the treatment of cancer in which treatment with anti-PD-1 antibody or anti-PD-L1 antibody is ineffective.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cancer is breast cancer, lung cancer, colorectal cancer, kidney cancer, sarcoma, liver cancer, bladder cancer, or ovarian cancer.

5. The pharmaceutical composition according to claim 4, wherein the cancer is breast cancer, lung cancer, bladder cancer, or colorectal cancer.

6. A pharmaceutical composition according to any one of claims 1 to 5, wherein the chemotherapeutic agent is gemcitabine.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the chemotherapeutic agent is a drug that has platinum in its structure and induces apoptosis of tumor cells by binding to DNA, and the drug is carboplatin, cisplatin, or oxaliplatin.

8. The pharmaceutical composition according to claim 7, wherein the drug having platinum in its structure and inducing apoptosis of tumor cells by binding to DNA is carboplatin.

9. The pharmaceutical composition according to any one of claims 1 to 5, wherein the chemotherapeutic agent is a taxane, and the taxane is paclitaxel or docetaxel.

10. A pharmaceutical composition according to any one of claims 1 to 5, wherein the chemotherapeutic agent is fluorouracil.

11. Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 It includes a heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7, The pharmaceutical composition according to any one of claims 1 to 10, wherein the monoclonal antibody or antibody fragment is such that the fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamine, and the eleventh glycine in the amino acid sequence of SEQ ID NO: 2 is further substituted with arginine.

12. Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 It includes a heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, The pharmaceutical composition according to any one of claims 1 to 10, wherein the monoclonal antibody or antibody fragment is a monoclonal antibody or antibody fragment in which the 18th lysine of the amino acid sequence of SEQ ID NO: 6 is substituted with arginine.

13. Anti-CCR8 antibody, CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 22 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 25; A pharmaceutical composition according to any one of claims 1 to 10, comprising a monoclonal antibody or antibody fragment.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the anti-CCR8 antibody is a humanized monoclonal antibody or an antibody fragment thereof.

15. Humanized monoclonal antibodies or their antibody fragments, 1) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 2) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 3) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 4) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 5) A light chain variable region having the amino acid sequence of Sequence ID No. 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 6) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 41; 7) A light chain variable region having the amino acid sequence of SEQ ID NO: 40, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 8) A light chain variable region having the amino acid sequence of SEQ ID NO: 42, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 9) A light chain variable region having the amino acid sequence of SEQ ID NO: 43, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 10) A light chain variable region having the amino acid sequence of SEQ ID NO: 44, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; 11) A light chain variable region having the amino acid sequence of SEQ ID NO: 45, and A heavy chain variable region having the amino acid sequence of SEQ ID NO: 46; or 12) A light chain variable region having the amino acid sequence of SEQ ID NO: 47, and Heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 Includes, It may have one or two of the following substitutions: A) The 33rd asparagine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with lysine; B) The 34th glycine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with leucine or arginine; C) The 58th asparagine in the amino acid sequence of SEQ ID NO: 40 or 42 is replaced with glutamine. The pharmaceutical composition according to claim 14.

16. The pharmaceutical composition according to claim 14 or 15, wherein the humanized monoclonal antibody or antibody fragment thereof has a light chain variable region having the amino acid sequence of SEQ ID NO: 59 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

41.

17. The pharmaceutical composition according to claim 14, wherein the humanized monoclonal antibody or antibody fragment thereof has a light chain variable region having the amino acid sequence of SEQ ID NO: 56 and a heavy chain variable region having the amino acid sequence of SEQ ID NO:

60.

18. Humanized monoclonal antibodies or their antibody fragments, Furthermore, a light chain constant region having the amino acid sequence of SEQ ID NO: 52, and It has a heavy chain constant region having the amino acid sequence of SEQ ID NO: 53, The pharmaceutical composition according to any one of claims 14 to 17, wherein lysine may or may not be added to the C-terminus of sequence number 53.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the anti-CCR8 antibody is a neutralizing antibody.

20. The pharmaceutical composition according to any one of claims 1 to 18, wherein the anti-CCR8 antibody has ADCC activity.

21. The pharmaceutical composition according to any one of claims 1 to 18, wherein the anti-CCR8 antibody is an IgG antibody.

22. A pharmaceutical for cancer treatment comprising an anti-CCR8 antibody and a chemotherapeutic agent, wherein the anti-CCR8 antibody is 1) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, CDR2 and the amino acid sequence of SEQ ID NO: 6 Heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 (Here, it may have one or two of the following substitutions, A) The 10th asparagine in the amino acid sequence of Sequence ID No. 2 is replaced with lysine. B) The 11th glycine in the amino acid sequence of SEQ ID NO: 2 is replaced with leucine or arginine. C) The fourth asparagine in the amino acid sequence of SEQ ID NO: 3 is replaced with glutamine; 2) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 3 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 4 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3, which consists of the amino acid sequence of SEQ ID NO: 7; 3) CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 and the amino acid sequence of SEQ ID NO: 9 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 8, CDR2 and the amino acid sequence of SEQ ID NO: 6 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 11; (Here, it is possible to have one of the following permutations: A) The second serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. B) The third serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. C) The fifth serine in the amino acid sequence of Sequence ID No. 2 is replaced with threonine. D) The second glutamine in the amino acid sequence of SEQ ID NO: 10 is replaced with asparagine. E) The first threonine in the amino acid sequence of Sequence ID No. 8 is replaced with serine. F) The 14th alanine in the amino acid sequence of sequence number 6 is replaced with valine. G) The 18th lysine in the amino acid sequence of Sequence ID No. 6 is replaced with arginine. H) The 19th aspartic acid in the amino acid sequence of SEQ ID NO: 6 is replaced with glutamic acid. I) The fifth asparagine in the amino acid sequence of SEQ ID NO: 11 is replaced with glutamine. J) The 12th tyrosine in the amino acid sequence of SEQ ID NO: 11 is substituted with phenylalanine; 4) CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, CDR2 and the amino acid sequence of SEQ ID NO: 15 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 16 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 17, CDR2 and the amino acid sequence of SEQ ID NO: 18 A heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 19; or 5) CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 and the amino acid sequence of SEQ ID NO: 21 Light chain variable region including CDR3 consisting of the amino acid sequence of SEQ ID NO: 22 and CDR1, consisting of the amino acid sequence of SEQ ID NO: 23, CDR2 and the amino acid sequence of SEQ ID NO: 24 Heavy chain variable region containing CDR3 consisting of the amino acid sequence of SEQ ID NO: 25 A monoclonal antibody or antibody fragment containing and having neutralizing activity with an IC50 value of 2 nM or less, The aforementioned chemotherapeutic agent is a drug containing platinum in its structure that induces apoptosis of tumor cells by binding to DNA, such as a taxane, gemcitabine, or fluorouracil. A medicine used to treat cancer.

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