Treatment of her3 mutant cancer by administration of Anti-her3 antibody-drug conjugate

Abstract

To provide a therapeutic agent for HER3-mutated cancer containing an anti-HER3 antibody-drug conjugate as an active ingredient.SOLUTION: The present invention provides a therapeutic agent comprising an anti-HER3 antibody-drug conjugate where a drug-linker represented by the following formula is conjugated to an anti-HER3 antibody via a thioether bond, (where A represents a connecting position to the anti-HER3 antibody.)SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for HER3 mutant cancer containing an anti-HER3 antibody-drug conjugate, and / or a method for treating cancer, which is characterized by administering the anti-HER3 antibody-drug conjugate to a patient confirmed to have a HER3 mutant cancer. [Background technology]

[0002] Human epidermal growth factor receptor 3 (HER3; also known as ErbB3) is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases. HER3 is expressed in various types of cancers, such as breast cancer, lung cancer, and colon cancer, and is known to form heterodimers with tyrosine kinase receptors such as HER2 and EGFR, undergo phosphorylation, and induce cancer cell proliferation and apoptosis inhibitory signals (Non-Patent Documents 1 to 3).

[0003] Mutations exist in HER3, and they are known to be one of the driver mutations of cancer (Non-Patent Documents 4 to 6). It has been reported that such HER3 mutant cancers are present in, for example, 4% of breast cancers (Non-Patent Document 7), 10% of gastric cancers (Non-Patent Document 8), 1% of ovarian cancers (Non-Patent Document 9), 1% of colorectal cancers (Non-Patent Document 5), and 1% of head and neck cancers (Non-Patent Document 10).

[0004] It has been reported that anti-HER2 drugs such as trastuzumab, pertuzumab, and lapatinib have shown efficacy against HER3-mutated cancers in vitro and in vivo under conditions in which HER2 is overexpressed (Non-Patent Document 11).

[0005] On the other hand, it has been reported that clinical trials using the anti-HER2 drug neratinib showed no efficacy against HER3-mutated cancers (Non-Patent Document 12).

[0006] One of the reasons why anti-HER2 drugs are ineffective against HER3 mutant cancers is that HER3 mutant cancers function independently of HER2 overexpression. That is, it has been reported that HER3 mutant cancers can induce cancer cell proliferation even when HER2 is not overexpressed (Non-Patent Documents 13 and 14), and it is believed that anti-HER2 drugs cannot exert an antitumor effect on HER3 mutant cancers when HER2 is not overexpressed.

[0007] In addition, a study verifying the effectiveness of anti-HER3 antibodies against HER3-mutated cancers is also known (Non-Patent Document 11). However, there are no known reports showing that anti-HER3 antibodies show clear effectiveness against HER3-mutated cancers, regardless of the presence or absence of HER2 overexpression. In addition, it is generally assumed that the binding ability of anti-HER3 antibodies to HER3 decreases with HER3 mutation, and it is considered difficult to obtain anti-HER3 antibodies that consistently show excellent antitumor activity against various HER3 mutations. Therefore, an effective treatment for HER3-mutated cancers has not yet been established.

[0008] Antibody-drug conjugates (ADCs), which combine a cytotoxic drug with an antibody that can bind to an antigen expressed on the surface of cancer cells and be internalized into the cells, are expected to be able to selectively deliver drugs to cancer cells, thereby accumulating the drugs within the cancer cells and killing the cancer cells (Non-Patent Documents 15 to 19).

[0009] As one of the antibody-drug conjugates, an anti-HER3 antibody-drug conjugate containing an anti-HER3 antibody and a derivative of exatecan, a topoisomerase I inhibitor, as components is known (Patent Document 1). There are no reports on the effectiveness of anti-HER3 antibody-drug conjugates against HER3 mutant cancers. [Prior art documents] [Patent documents]

[0010]

Patent Document 1

Non-licensed literature

[0011]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0012] An objective of the present invention is to provide a therapeutic agent for HER3 mutant cancer containing an anti-HER3 antibody-drug conjugate, and / or a method for treating cancer, which is characterized by administering the anti-HER3 antibody-drug conjugate to a patient confirmed to have a HER3 mutant cancer. [Means for solving the problem]

[0013] Means for Solving the Problems The present inventors conducted extensive studies to solve the above problems and found that an anti-HER3 antibody-drug conjugate exhibits excellent antitumor activity against HER3 mutated cancer, thereby completing the present invention.

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

[85] . [1] A therapeutic agent for HER3-mutated cancer that contains an anti-HER3 antibody-drug conjugate as an active ingredient. [2] The therapeutic agent according to [1], wherein the HER3 mutation in the HER3 mutated cancer is at least one selected from the group consisting of V104L, V104M, A232V, P262H, G284R, D297Y, G325R, T355I, Q809R, S846I, and E928G. [3] The therapeutic agent described in [1], wherein the HER3 mutation in the HER3 mutated cancer is Q809R. [4] The therapeutic agent according to any one of [1] to [3], wherein the HER3 mutated cancer overexpresses HER2. [5] The therapeutic agent according to any one of [1] to [3], wherein the HER3 mutated cancer does not overexpress HER2. [6] The therapeutic agent according to any one of [1] to [5], wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between wild-type HER3 expressing cells and mutant HER3 expressing cells. [7] The therapeutic agent according to any one of [1] to [6], wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between HER3-expressing cells that overexpress HER2 and HER3-expressing cells that do not overexpress HER2. [8] The anti-HER3 antibody-drug conjugate has the formula

[0015] [ka]

[0016] (In the formula, A represents the binding site for the anti-HER3 antibody.) The therapeutic agent according to any one of [1] to [7], which is an anti-HER3 antibody-drug conjugate in which a drug linker represented by the following formula (I) is linked to the anti-HER3 antibody via a thioether bond. [9] The anti-HER3 antibody-drug conjugate has the formula

[0017] [ka]

[0018] (In the formula, the drug linker is bound to the anti-HER3 antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) The therapeutic agent according to any one of [1] to [7], which is an anti-HER3 antibody-drug conjugate represented by the formula:

[10] The therapeutic agent according to any one of [1] to [9], wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[11] The therapeutic agent according to any one of [1] to [9], wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[12] The therapeutic agent according to any one of [1] to [9], wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[13] The therapeutic agent according to

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

[14] The therapeutic agent according to any one of [1] to

[13] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8.

[15] The therapeutic agent according to any one of [1] to

[13] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8.

[16] The therapeutic agent according to any one of [1] to

[15] , wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer.

[17] The therapeutic agent according to any one of [1] to

[15] , wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma.

[18] A method for treating cancer, comprising administering an anti-HER3 antibody-drug conjugate to a patient confirmed to have a HER3 mutated cancer.

[19] The treatment method according to

[18] , wherein the HER3 mutation in the HER3 mutated cancer is at least one selected from the group consisting of V104L, V104M, A232V, P262H, G284R, D297Y, G325R, T355I, Q809R, S846I, and E928G.

[20] The treatment method described in

[18] , wherein the HER3 mutation in the HER3 mutated cancer is Q809R.

[0019] [twenty one] The treatment method according to any one of

[18] to

[20] , wherein the HER3 mutated cancer overexpresses HER2. [twenty two] The treatment method according to any one of

[18] to

[20] , wherein the HER3 mutated cancer does not overexpress HER2. [twenty three] The treatment method according to any one of

[18] to

[22] , wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between wild-type HER3 expressing cells and mutant HER3 expressing cells. [twenty four] The treatment method according to any one of

[18] to

[23] , wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between HER3-expressing cells that overexpress HER2 and HER3-expressing cells that do not overexpress HER2. [twenty five] The anti-HER3 antibody-drug conjugate has the formula

[0020] [ka]

[0021] (In the formula, A represents the binding site for the anti-HER3 antibody.) The method for treating HER3 is an anti-HER3 antibody-drug conjugate in which the drug linker represented by the following formula (I) is linked to the anti-HER3 antibody via a thioether bond.

[26] The anti-HER3 antibody-drug conjugate has the formula

[0022] [ka]

[0023] (In the formula, the drug linker is bound to the anti-HER3 antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) The therapeutic method according to any one of

[18] to

[24] , wherein the anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate represented by the formula:

[27] The treatment method according to any one of

[18] to

[26] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[28] The treatment method according to any one of

[18] to

[26] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[29] The treatment method according to any one of

[18] to

[26] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[30] The treatment method described in

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

[0024]

[31] The treatment method according to any one of

[18] to

[30] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8.

[32] The treatment method according to any one of

[18] to

[30] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8.

[33] The method for treating cancer according to any one of

[18] to

[32] , wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer.

[34] The method for treating cancer according to any one of

[18] to

[32] , wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma.

[35] Anti-HER3 antibody-drug conjugates for the treatment of HER3-mutated cancers.

[36] The anti-HER3 antibody-drug conjugate according to

[35] , wherein the HER3 mutation in the HER3 mutant cancer is at least one selected from the group consisting of V104L, V104M, A232V, P262H, G284R, D297Y, G325R, T355I, Q809R, S846I, and E928G.

[37] The anti-HER3 antibody-drug conjugate described in

[35] , wherein the HER3 mutation in the HER3 mutated cancer is Q809R.

[38] The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[37] , wherein the HER3 mutated cancer overexpresses HER2.

[39] An anti-HER3 antibody-drug conjugate described in any one of

[35] to

[37] , wherein the HER3 mutant cancer does not overexpress HER2.

[40] An anti-HER3 antibody-drug conjugate described in any one of

[35] to

[39] , wherein the lysosomal transport ability of the anti-HER3 antibody-drug conjugate is not substantially different between wild-type HER3 expressing cells and mutant HER3 expressing cells.

[41] An anti-HER3 antibody-drug conjugate described in any one of

[35] to

[40] , wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between HER3-expressing cells that overexpress HER2 and HER3-expressing cells that do not overexpress HER2.

[42] The anti-HER3 antibody-drug conjugate has the formula

[0025] [ka]

[0026] (In the formula, A represents the binding site for the anti-HER3 antibody.) The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[41] , wherein the anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate in which a drug linker represented by the following formula (I) is linked to the anti-HER3 antibody via a thioether bond.

[43] The anti-HER3 antibody-drug conjugate has the formula

[0027] [ka]

[0028] (In the formula, the drug linker is bound to the anti-HER3 antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[41] , wherein the anti-HER3 antibody-drug conjugate is represented by the formula:

[44] The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[43] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[45] The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[43] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[46] The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[43] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[47] An anti-HER3 antibody-drug conjugate described in

[46] , in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[48] An anti-HER3 antibody-drug conjugate according to any one of

[35] to

[47] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8.

[49] An anti-HER3 antibody-drug conjugate according to any one of

[35] to

[47] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8.

[50] The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[49] , wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer.

[0029]

[51] The anti-HER3 antibody-drug conjugate according to any one of

[35] to

[49] , wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma.

[52] Use of an anti-HER3 antibody-drug conjugate for the manufacture of a medicament for the treatment of a HER3 mutated cancer.

[53] The use described in

[52] , wherein the HER3 mutation in the HER3 mutant cancer is at least one selected from the group consisting of V104L, V104M, A232V, P262H, G284R, D297Y, G325R, T355I, Q809R, S846I, and E928G.

[54] The use described in

[52] , wherein the HER3 mutation in the HER3 mutant cancer is Q809R.

[55] The use described in any one of

[52] to

[54] , wherein the HER3 mutant cancer overexpresses HER2.

[56] The use described in any one of

[52] to

[54] , wherein the HER3 mutant cancer does not overexpress HER2.

[57] The use described in any one of

[52] to

[56] , wherein the lysosomal transport ability of the anti-HER3 antibody-drug conjugate is not substantially different between wild-type HER3 expressing cells and mutant HER3 expressing cells.

[58] The use described in any one of

[52] to

[57] , wherein the lysosomal transport ability of the anti-HER3 antibody-drug conjugate is not substantially different between HER3-expressing cells that overexpress HER2 and HER3-expressing cells that do not overexpress HER2.

[59] The anti-HER3 antibody-drug conjugate has the formula

[0030] [ka]

[0031] (In the formula, A represents the binding site for the anti-HER3 antibody.) The use according to any one of

[52] to

[58] , wherein the anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate in which the drug linker represented by the formula:

[60] The anti-HER3 antibody-drug conjugate has the formula

[0032] [ka]

[0033] (In the formula, the drug linker is bound to the anti-HER3 antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) The use according to any one of

[52] to

[58] , wherein the anti-HER3 antibody-drug conjugate is represented by the formula:

[61] The use described in any one of

[52] to

[60] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[62] The use described in any one of

[52] to

[60] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[63] The use described in any one of

[52] to

[60] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[64] The use according to

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

[65] The use described in any one of

[52] to

[64] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8.

[66] The use described in any one of

[52] to

[64] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8.

[67] The use according to any one of

[52] to

[66] , wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer.

[68] The use according to any one of

[52] to

[66] , wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma.

[69] A method for treating a HER3 mutant cancer, comprising administering an anti-HER3 antibody-drug conjugate to a patient in need of treatment for a HER3 mutant cancer.

[70] The treatment method described in

[69] , wherein the HER3 mutation in the HER3 mutant cancer is at least one selected from the group consisting of V104L, V104M, A232V, P262H, G284R, D297Y, G325R, T355I, Q809R, S846I, and E928G.

[0034]

[71] The treatment method described in

[69] , wherein the HER3 mutation in the HER3 mutated cancer is Q809R.

[72] The treatment method according to any one of

[69] to

[71] , wherein the HER3 mutated cancer overexpresses HER2.

[73] The treatment method according to any one of

[69] to

[71] , wherein the HER3 mutated cancer does not overexpress HER2.

[74] The treatment method according to any one of

[69] to

[73] , wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between wild-type HER3 expressing cells and mutant HER3 expressing cells.

[75] The treatment method according to any one of

[69] to

[74] , wherein the lysosomal migration ability of the anti-HER3 antibody-drug conjugate is not substantially different between HER3-expressing cells that overexpress HER2 and HER3-expressing cells that do not overexpress HER2.

[76] The anti-HER3 antibody-drug conjugate has the formula

[0035] [ka]

[0036] (In the formula, A represents the binding site for the anti-HER3 antibody.) The treatment method according to any one of

[69] to

[75] , wherein the anti-HER3 antibody is an anti-HER3 antibody-drug conjugate in which the drug linker represented by the following formula (I) is linked to the anti-HER3 antibody via a thioether bond.

[77] The anti-HER3 antibody-drug conjugate has the formula

[0037] [ka]

[0038] (In the formula, the drug linker is bound to the anti-HER3 antibody via a thioether bond, and n represents the average number of drug linkers bound per antibody.) The therapeutic method according to any one of

[69] to

[75] , wherein the anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate represented by the formula:

[78] The treatment method described in any one of

[69] to

[77] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[79] The treatment method according to any one of

[69] to

[77] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8.

[80] The treatment method according to any one of

[69] to

[77] , wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[81] The treatment method described in

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

[82] The treatment method according to any one of

[69] to

[81] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8.

[83] The treatment method according to any one of

[69] to

[81] , wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8.

[84] The method for treating cancer according to any one of

[69] to

[83] , wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer.

[85] The method for treating cancer according to any one of

[69] to

[83] , wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma.

[0039] The present invention can also be expressed as follows (1) to (48). (1) A treatment for HER3 gene-mutated cancer that contains an anti-HER3 antibody-drug conjugate as an active ingredient. (2) The therapeutic agent described in (1), wherein the HER3 gene mutated cancer overexpresses HER2. (3) The therapeutic agent according to (1), wherein the HER3 gene mutated cancer does not overexpress HER2. (4) The anti-HER3 antibody-drug conjugate has the formula

[0040] [ka]

[0041] (In the formula, A represents the binding site for the anti-HER3 antibody.) The therapeutic agent according to any one of (1) to (3), which is an anti-HER3 antibody-drug conjugate in which a drug linker represented by the following formula (I) is linked to the anti-HER3 antibody via a thioether bond. (5) The therapeutic agent according to any one of (1) to (4), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6. (6) The therapeutic agent according to any one of (1) to (5), wherein the anti-HER3 antibody is an antibody comprising a heavy chain having a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain having a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. (7) The therapeutic agent according to any one of (1) to (6), wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10. (8) The therapeutic agent according to (7), wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted. (9) The therapeutic agent according to any one of (1) to (8), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8. (10) The therapeutic agent according to any one of (1) to (8), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8. (11) The therapeutic agent according to any one of (1) to (10), wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer. (12) The therapeutic agent according to any one of (1) to (10), wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colon cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma. (13) A method for treating cancer, comprising administering an anti-HER3 antibody-drug conjugate to a patient confirmed to have a cancer with a HER3 gene mutation. (14) The method for treatment according to (13), wherein the HER3 gene mutated cancer overexpresses HER2. (15) The method of treatment according to (13), wherein the HER3 gene mutated cancer does not overexpress HER2. (16) The anti-HER3 antibody-drug conjugate has the formula

[0042] [ka]

[0043] (In the formula, A represents the binding site for the anti-HER3 antibody.) and the anti-HER3 antibody is linked via a thioether bond to a drug linker represented by the following formula (1): (17) The method of treatment according to any one of (13) to (16), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6. (18) The method of treatment according to any one of (13) to (17), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. (19) The method for treatment according to any one of (13) to (18), wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10. (20) The therapeutic method according to (19), wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

[0044] (twenty one) The method for treatment according to any one of (13) to (20), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8. (twenty two) The method for treatment according to any one of (13) to (20), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8. (twenty three) The method according to any one of (13) to (22), wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer. (twenty four) The method for treating cancer according to any one of (13) to (22), wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colon cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma. (twenty five) Anti-HER3 antibody-drug conjugate for the treatment of HER3 gene-mutated cancers. (26) The anti-HER3 antibody-drug conjugate according to (25), wherein the HER3 gene mutated cancer overexpresses HER2. (27) The anti-HER3 antibody-drug conjugate according to (25), wherein the HER3 gene mutated cancer does not overexpress HER2. (28) The anti-HER3 antibody-drug conjugate has the formula

[0045] [ka]

[0046] (In the formula, A represents the binding site for the anti-HER3 antibody.) The anti-HER3 antibody-drug conjugate according to any one of (25) to (27), wherein the anti-HER3 antibody is linked to a drug linker represented by the following formula (1): (29) The anti-HER3 antibody-drug conjugate according to any one of (25) to (28), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6. (30) The anti-HER3 antibody-drug conjugate according to any one of (25) to (29), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. (31) The anti-HER3 antibody-drug conjugate according to any one of (25) to (30), wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10. (32) An anti-HER3 antibody-drug conjugate according to (31), in which the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted. (33) The anti-HER3 antibody-drug conjugate according to any one of (25) to (32), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8. (34) The anti-HER3 antibody-drug conjugate according to any one of (25) to (33), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8. (35) The anti-HER3 antibody-drug conjugate according to any one of (25) to (34), wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer. (36) The anti-HER3 antibody-drug conjugate according to any one of (25) to (34), wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma. (37) Use of an anti-HER3 antibody-drug conjugate for the manufacture of a medicament for the treatment of HER3 gene mutated cancer. (38) The use described in (37), wherein the HER3 gene mutated cancer overexpresses HER2. (39) The use described in (37), wherein the HER3 gene mutated cancer does not overexpress HER2. (40) The anti-HER3 antibody-drug conjugate has the formula

[0047] [ka]

[0048] (In the formula, A represents the binding site for the anti-HER3 antibody.) The use according to any one of (37) to (39), wherein the drug linker is an anti-HER3 antibody-drug conjugate in which the anti-HER3 antibody is linked to the drug linker via a thioether bond. (41) The use according to any one of (37) to (40), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6. (42) The use according to any one of (37) to (41), wherein the anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8. (43) The use according to any one of (37) to (42), wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10. (44) The use according to (43), wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (45) The use according to any one of (37) to (44), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7 to 8. (46) The use according to any one of (37) to (44), wherein the average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is in the range of 7.5 to 8. (47) The use according to any one of (37) to (46), wherein the cancer is at least one selected from the group consisting of breast cancer, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, renal cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer. (48) The use according to any one of (37) to (46), wherein the cancer is at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma. Effect of the Invention

[0049] The present invention can provide a method for treating cancer, which is characterized by administering a therapeutic agent for HER3 mutant cancer containing an anti-HER3 antibody-drug conjugate and / or an anti-HER3 antibody-drug conjugate to a patient confirmed to have a HER3 mutant cancer. [Brief description of the drawings]

[0050] [Figure 1] 1 shows the amino acid sequence of the anti-HER3 antibody heavy chain (SEQ ID NO: 9). [Diagram 2] 1 shows the amino acid sequence of the anti-HER3 antibody light chain (SEQ ID NO: 10). [Diagram 3] The results of 1% agarose gel electrophoresis of RT-PCR products from various HER3 stably expressing cells (HER3 expression) are shown. [Figure 4] The results of 1% agarose gel electrophoresis of RT-PCR products from various HER2-overexpressing and HER3-stably expressing cells (HER3 expression) are shown. [Diagram 5] The results of 1% agarose gel electrophoresis of RT-PCR products from various HER2-overexpressing and HER3-stable expressing cells (HER2 expression) are shown. [Figure 6]1 shows the binding of HER3-ADC(1) to HER3 stably expressing cells. [Figure 7] 1 shows the binding of HER3-ADC(1) to HER2-overexpressing, HER3-stable expressing cells. [Figure 8] 1 shows the cell proliferation inhibitory activity of HER3-ADC(1), HER3-Ab(1), and IgG-ADC(1) against empty vector-transfected cells (no HER2 overexpression). [Figure 9] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against wild-type HER3-transfected cells (without HER2 overexpression). [Figure 10] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (V104L)-transfected cells (without HER2 overexpression).

[0051] [Figure 11] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (V104M)-transfected cells (without HER2 overexpression). [Figure 12] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (A232V)-transfected cells (without HER2 overexpression). [Figure 13] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (P262H)-transfected cells (without HER2 overexpression). [Figure 14] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (G284R)-transfected cells (without HER2 overexpression). [Figure 15] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (D297Y)-transfected cells (without HER2 overexpression). [Figure 16] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (G325R)-transfected cells (without HER2 overexpression). [Figure 17] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (T355I)-transfected cells (without HER2 overexpression). [Figure 18] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (S846I)-transfected cells (without HER2 overexpression). [Figure 19] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (E928G)-transfected cells (without HER2 overexpression). [Figure 20] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against empty vector-transfected cells (with HER2 overexpression).

[0052] [Figure 21] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against wild-type HER3-transfected cells (with HER2 overexpression). [Figure 22] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (V104L)-transfected cells (with HER2 overexpression). [Diagram 23] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (V104M)-transfected cells (with HER2 overexpression). [Figure 24]1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (A232V)-transfected cells (with HER2 overexpression). [Diagram 25] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (P262H)-transfected cells (with HER2 overexpression). [Figure 26] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (G284R)-transfected cells (with HER2 overexpression). [Figure 27] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (D297Y)-transfected cells (with HER2 overexpression). [Figure 28] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (G325R)-transfected cells (with HER2 overexpression). [Figure 29] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (T355I)-transfected cells (with HER2 overexpression). [Diagram 30] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (S846I)-transfected cells (with HER2 overexpression).

[0053] [Diagram 31] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (E928G)-transfected cells (with HER2 overexpression). [Diagram 32] 1 shows the cell proliferation inhibitory activity of HER3-ADC(1), HER3-Ab(1), and IgG-ADC(1) against empty vector-transfected cells (no HER2 overexpression). [Diagram 33] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against wild-type HER3-transfected cells (without HER2 overexpression). [Diagram 34] The results of 1% agarose gel electrophoresis of RT-PCR products (HER3 expression) from cells transfected with mutant HER3 (Q809R) (with HER2 overexpression) are shown. [Diagram 35] The results of 1% agarose gel electrophoresis of RT-PCR products (HER2 expression) from cells transfected with mutant HER3 (Q809R) (with HER2 overexpression) are shown. [Diagram 36] 1 shows the binding of HER3-ADC (1) to mutant HER3 (Q809R)-transfected cells (with HER2 overexpression). [Figure 37] 1 shows the cell proliferation inhibitory activity of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against mutant HER3 (Q809R)-transfected cells (with HER2 overexpression). [Figure 38] 1 shows the lysosomal transport of HER3-ADC(1) in empty vector-transfected cells (no HER2 overexpression). [Figure 39] 1 shows the lysosomal transport of HER3-ADC (1) in wild-type HER3-transfected cells (without HER2 overexpression). [Diagram 40] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (V104L)-transfected cells (without HER2 overexpression).

[0054] [Diagram 41] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (V104M)-transfected cells (without HER2 overexpression). [Diagram 42] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (A232V)-transfected cells (without HER2 overexpression). [Diagram 43] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (P262H)-transfected cells (without HER2 overexpression). [Diagram 44] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (G284R)-transfected cells (without HER2 overexpression). [Diagram 45] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (D297Y)-transfected cells (without HER2 overexpression). [Figure 46] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (G325R)-transfected cells (without HER2 overexpression). [Figure 47] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (T355I)-transfected cells (without HER2 overexpression). [Figure 48] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (S846I)-transfected cells (without HER2 overexpression). [Figure 49] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (E928G)-transfected cells (without HER2 overexpression). [Figure 50] 1 shows the lysosomal transport of HER3-ADC(1) in empty vector-transfected cells (with HER2 overexpression).

[0055] [Figure 51] 1 shows the lysosomal transport of HER3-ADC (1) in wild-type HER3-transfected cells (with HER2 overexpression). [Figure 52] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (V104L)-transfected cells (with HER2 overexpression). [Figure 53] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (V104M)-transfected cells (with HER2 overexpression). [Figure 54] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (A232V)-transfected cells (with HER2 overexpression). [Figure 55]1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (P262H)-transfected cells (with HER2 overexpression). [Figure 56] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (G284R)-transfected cells (with HER2 overexpression). [Figure 57] 1 shows the lysosomal migration of HER3-ADC (1) to mutant HER3 (D297Y)-transfected cells (with HER2 overexpression). [Figure 58] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (G325R)-transfected cells (with HER2 overexpression). [Figure 59] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (T355I)-transfected cells (with HER2 overexpression). [Figure 60] This shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (Q809R)-transfected cells (with HER2 overexpression).

[0056] [Figure 61] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (S846I)-transfected cells (with HER2 overexpression). [Figure 62] 1 shows the lysosomal transport of HER3-ADC (1) in mutant HER3 (E928G)-transfected cells (with HER2 overexpression). [Figure 63] 1 shows the lysosomal transport of HER3-ADC(1) in empty vector-transfected cells (no HER2 overexpression). [Figure 64] 1 shows the lysosomal transport of HER3-ADC (1) in wild-type HER3-transfected cells (without HER2 overexpression). [Figure 65] The amino acid sequence of the HER3 protein (sequence number 69) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] A preferred embodiment for carrying out the present invention will be described below. Note that the embodiment described below shows one example of a typical embodiment of the present invention, and the scope of the present invention is not to be interpreted narrowly by this embodiment.

[0058] [Definition] In the present invention, "HER3" is synonymous with human epidermal growth factor receptor 3 (HER3; also known as ErbB3), which is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases along with HER1 (EGFR, ErbB1), HER2 (ErbB2) and HER4 (ErbB4). HER3 is expressed in various types of cancers such as breast cancer, lung cancer, and colon cancer, and is known to form heterodimers with tyrosine kinase receptors such as HER2 and EGFR, undergo phosphorylation, and induce cancer cell proliferation and apoptosis inhibitory signals (Alimandi et al., Oncogene (1995) 10, 1813-1821; deFazio et al., Int. J. Cancer (2000) 87, 487-498; Naidu et al., Br. J. Cancer (1998) 78, 1385-1390).

[0059] In the present invention, the term "HER3 protein" is used synonymously with HER3. The expression of HER3 protein can be detected by a method well known to those skilled in the art, such as immunohistochemistry (IHC). In addition, the expression of HER3 protein can be detected by introducing Flag peptide into HER3 protein and using an anti-Flag peptide antibody.

[0060] The amino acid sequence of the HER3 protein is shown in SEQ ID NO:69 (Figure 65).

[0061] In the present invention, the term "HER3 gene" refers to a gene encoding the HER3 protein. The HER3 protein is a gene product of the HER3 gene.

[0062] In the present invention, "HER3 mutation" means having a mutation in the amino acid sequence of the HER3 protein.

[0063] In the present invention, the term "HER3 mutated cancer" refers to a cancer having a mutation in the amino acid sequence of the HER3 protein. In addition, even if the entire tumor tissue does not have a HER3 mutation, a cancer containing cancer cells having a HER3 mutation is included in the HER3 mutated cancer.

[0064] In the present invention, the term "HER3 gene mutation" means having a mutation in the HER3 gene.

[0065] In the present invention, "HER3 gene mutated cancer" refers to a cancer having a mutation in the HER3 gene. In addition, even if the entire tumor tissue does not have a HER3 gene mutation, if the cancer contains cancer cells having a HER3 gene mutation, it is included in the HER3 gene mutated cancer. The HER3 gene mutation causes a mutation in the amino acid sequence of the HER3 protein, which is the gene product, resulting in a HER3 mutation.

[0066] A specific example of a HER3 mutation is a mutation in which the 104th amino acid in the HER3 protein, V (valine), is replaced with L (leucine) (also called "V104L") (Cancer Cell. 2013 May 13;23(5):603-17, Nat Genet. 2014 Aug;46(8):872-6, Cancer Res. 2014 Nov 1;74(21):6071-81, Cancer. 2016 Sep 1;122(17):2654-62, Nat Med. 2017 Jun;23(6):703-713, and Nature. 2018 Feb 19). 8;554(7691):189-194, etc.), and a mutation in which the 104th amino acid in the HER3 protein, V (valine), is replaced by M (methionine) (also called "V104M") (Hum Mutat. 2008 Mar;29(3):441-50, Cancer Cell. 2013 May 13;23(5):603-17, Genome Biol. 2014 Apr 1;15(4):R55, Cancer Res. 2014 Jun 15;74(12):3238-47, Nat Genet. 2014 Aug;46(8):872-6, and Ann Oncol. 2016 Jan;27(1):127-33, etc.), a mutation in which the 232nd amino acid in the HER3 protein, A (alanine), is replaced by V (valine) (also called "A232V") (Nat Genet. 2013 May;45(5):478-86, Cancer Cell. 2013 May 13;23(5):603-17, Cancer Cell. 2016 Feb 8;29(2):229-40, Cell Rep. 2016 Apr 26;15(4):857-865, etc.), a mutation in which the 262nd amino acid in the HER3 protein, P (proline), is replaced by H (histidine) (also called "P262H") (Cancer Cell. 2013 May 13;23(5):603-17, Gastroenterology. 2014 Feb;146(2):530-38.e5, and Nat Med.2017 Jun;23(6):703-713, etc.), a mutation in which the 284th amino acid in the HER3 protein, G (glycine), is replaced by R (arginine) (also called "G284R") (Nature. 2008 Oct 23;455(7216):1069-75, Cancer Cell. 2013 May 13;23(5):603-17, Nat Genet. 2014 Jun;46(6):573-82, and Ann Oncol. 2015 Aug;26(8):1704-9, etc.), a mutation in which the 297th amino acid in the HER3 protein, D (aspartic acid), is replaced by Y (tyrosine) (also called "D297Y") (PLoS One. 2014 Mar 5;9(3):e90459, Nat Genet. 2014 Jun;46(6):573-82, Nat Genet. 2014 Oct;46(10):1097-102, Ann Oncol. 2016 Jan;27(1):127-33, and Cancer. 2016 Sep 1;122(17):2654-62, etc.), a mutation in which the 325th amino acid in the HER3 protein, G (glycine), is replaced by R (arginine) (also called "G325R") (see Nat Med. 2017 Jun;23(6):703-713, etc.), a mutation in which the 355th amino acid in the HER3 protein, T (threonine), is replaced by I (isoleucine) (also called "T355I") (see Nat Med. 2017 Jun;23(6):703-713, Nature. 2018 Feb 8;554(7691):189-194, etc.), a mutation in which the 809th amino acid in the HER3 protein, Q (glutamine), is replaced by R (arginine) (also called "Q809R") (see Cancer Cell. 2013 May 13;23(5):603-17, Cell Rep. 2016 Apr 26;15(4):857-865, and Nat Med. 2017 Jun;23(6):703-713, etc.), a mutation in which the 846th amino acid in the HER3 protein, S (serine), is replaced by I (isoleucine) (also called "S846I") (Int J Cancer.2006 Dec 15;119(12):2986-7, Nat Genet. 2014 Dec;46(12):1264-6, and Cell Rep. 2016 Apr 26;15(4):857-865, etc.), and a mutation in which the 928th amino acid in the HER3 protein, E (glutamic acid), is replaced by G (glycine) (also called "E928G") (Nat Genet. 2011 Oct 30;43(12):1219-23, Clin Cancer Res. 2016 Apr 1;22(7):1583-91, Cancer. 2016 Sep 1;122(17):2654-62, Clin Cancer Res. 2016 Dec 15;22(24):6061-6068, Cancer Res. 2016 Oct 15;76(20):5954-5961, and PLoS Med. 2016 Dec 27;13(12):e1002201.

[0067] In addition, it has been suggested that cancers with the above-mentioned HER3 mutations, particularly those with Q809R, show strong resistance to existing anti-HER2 drugs and anti-HER3 antibodies (Jaiswal et al., Cancer cell (2013) 23, 603-17).

[0068] The HER3 mutation in the present invention is not particularly limited as long as it has a mutation in the amino acid sequence of the HER3 protein, but preferably includes at least one selected from the group consisting of V104L, V104M, A232V, P262H, G284R, D297Y, G325R, T355I, Q809R, S846I, and E928G, and more preferably includes Q809R.

[0069] The presence or absence of a HER3 mutation can be confirmed, for example, by collecting tumor tissue from a cancer patient and subjecting the formalin-fixed, paraffin-embedded (FFPE) specimen to real-time quantitative PCR (qRT-PCR) or microarray analysis.

[0070] In addition, the presence or absence of HER3 mutations can be confirmed by collecting cell-free circulating tumor DNA (ctDNA) from cancer patients and performing methods such as next-generation sequencing (NGS) (Sergina et al., Nature (2007) 445, 437-41; Jeong et al., Int. J. Cancer (2006) 119, 2986-7; Ding et al., Nature (2008) 455, 1069-75; Kan et al., Nature (2010) 466, 869-73; Wang et al., Nat. Genet. (2011) 43, 1219-23; Greenman et al., Nature (2007) 446, 153-8; Stransky et al., Science (2011) 333, 1157-60; Jaiswal et al., Cancer cell (2013) 23, 603-17, Hyman et al., Cancer Res. (2017) Abstract CT001, Mishra et al., Oncotarget (2017) 69, 114371-114392, Mishra et al., Oncotarget (2018) 45, 27773-27788, etc.).

[0071] In the present invention, the term "HER3 mutation" is used synonymously with HER3 gene mutation.

[0072] In the present invention, "wild-type HER3" refers to a HER3 protein that does not have a HER3 mutation. In the present invention, the wild-type may also be referred to as "WT."

[0073] In the present invention, "mutated HER3" refers to a HER3 protein having a HER3 mutation.

[0074] HER3 stable expressing cells can be produced by chemical gene transfer methods by transfection using cationic lipids, cationic polymers, calcium phosphate, etc., physical gene transfer methods by electroporation, microinjection, sonoporation, laser irradiation, etc., biological gene transfer methods using viral vectors, etc. For example, among biological gene transfer methods, when lentivirus is used, lentivirus protein and envelope protein expression plasmids and HER3 expression plasmids are introduced together into packaging cells such as Lenti-X 293T cells, and then a lentivirus solution is prepared from the culture supernatant and used to culture tumor cells. Here, wild-type or mutant HER3 expressing cells can be produced depending on the type of HER3 expression plasmid used. Mutant HER3 expression plasmids can be produced using HER3 mutation introduction primers.

[0075] In the present invention, "anti-HER3 antibody" refers to an antibody that specifically binds to HER3, and preferably has the activity of being internalized into HER3-expressing cells upon binding to HER3, in other words, an antibody that has the activity of migrating into HER3-expressing cells after binding to HER3.

[0076] In the present invention, "HER2" is synonymous with human epidermal growth factor receptor 2 (also called neu or ErbB-2), and is a transmembrane receptor that belongs to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases along with HER1, HER3, and HER4. HER2 is known to play an important role in cell proliferation, differentiation, and survival in normal and tumor cells by being activated through autophosphorylation of intracellular tyrosine residues upon heterodimerization with HER1, HER3, or HER4.

[0077] In the present invention, the term "HER2 protein" is used interchangeably with HER2.

[0078] In the present invention, "overexpression" of HER2 means that the expression of HER2 is determined to be positive, for example, that the expression of HER2 is determined to be 3+ by immunohistochemistry (IHC), or that the expression of HER2 is determined to be 2+ by immunohistochemistry and that the expression of HER2 is determined to be positive by in situ hybridization (ISH). The immunohistochemistry method and the in situ hybridization method can be performed using methods well known to those skilled in the art, for example, by referring to the HER2 Testing Guide Breast Cancer Edition, Fourth Edition (created by the Breast Cancer HER2 Testing Pathology Committee), etc.

[0079] HER2-overexpressing cells can be produced by chemical gene transfer methods using transfection with cationic lipids, cationic polymers, calcium phosphate, etc., physical gene transfer methods using electroporation, microinjection, sonoporation, laser irradiation, etc., biological gene transfer methods using viral vectors, etc. For example, among biological gene transfer methods, when lentivirus is used, lentivirus protein and envelope protein expression plasmids and HER2 expression plasmids are introduced into packaging cells such as Lenti-X 293T cells, and then a lentivirus solution is prepared from the culture supernatant and used to culture tumor cells. Furthermore, HER2-overexpressing HER3-stably expressing cells can be produced by culturing this HER2-overexpressing cell using the lentivirus solution obtained by the previous HER3 expression plasmid introduction.

[0080] [Anti-HER3 antibody-drug conjugate] In the present invention, the term "antibody-drug conjugate" refers to a complex in which a cytotoxic drug is bound to an antibody via a linker. Examples of the antibody-drug conjugate include those described in U.S. Pat. No. 6,214,345, WO 2002 / 083067, WO 2003 / 026577, WO 2004 / 054622, WO 2005 / 112919, WO 2006 / 135371, WO 2007112193, WO 2008 / 033891, WO 2009 / 100194, WO 2009 / 134976, WO 2009 / 134977, WO 2010 / 093395, WO 2011 / 093396, WO 2012 / 093397, WO 2013 / 093398, WO 2014 / 093399, WO 2015 / 093399, WO 2016 / 093391, WO 2017 / 093392, WO 2018 / 093393, WO 2019 / 093394, WO 2019 / 093395, WO 2019 / 093396, WO 2019 / 093397, WO 2019 / 093398, WO 2019 / 093399, WO 2019 / 093396, WO 2019 / 093399, WO 2019 / 093399, WO 2019 / 093399, WO 20 Examples of the antibody-drug conjugates include those described in International Publication Nos. 11 / 130613, 2011 / 130616, 2013 / 055993, 2014 / 057687, 2014 / 061277, 2014 / 107024, 2014 / 134457, and 2014 / 145090, preferably those described in International Publication Nos. 2014 / 057687 and 2014 / 061277, and more preferably those described in International Publication No. 2014 / 057687. These antibody-drug conjugates can be produced by the methods described in the above-mentioned documents.

[0081] The cytotoxic drug is not particularly limited as long as it has an antitumor effect and has a substituent or partial structure that can be bound to a linker, and examples of the cytotoxic drug include camptothecin, calicheamicin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin, auristatin E, and the like. E), Maytansine, Paclitaxel, Pyrrolobenzodiazepine, and derivatives thereof, preferably camptothecin derivatives, and more preferably exatecan derivatives.

[0082] Exatecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione)) is a topoisomerase I inhibitor having the formula

[0083] [ka]

[0084] It is a compound represented by the formula:

[0085] In the present invention, the term "drug linker" refers to the drug and linker moiety in an antibody-drug conjugate, in other words, a partial structure other than the antibody in an antibody-drug conjugate.

[0086] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate in which the antibody in the antibody-drug conjugate is an anti-HER3 antibody. Examples of the anti-HER3 antibody-drug conjugate include those described in WO 2012 / 019024, WO 2012 / 064733, and WO 2015 / 155998, and preferably those described in WO 2015 / 155998. These anti-HER3 antibody-drug conjugates can be produced by the methods described in the above documents.

[0087] The anti-HER3 antibody-drug conjugate preferably used in the present invention has the formula

[0088] [ka]

[0089] (wherein A represents the binding site to the antibody) The anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate in which the antibody is bound to a drug linker represented by the formula (I) via a thioether bond. This drug linker is bound to a thiol group (in other words, a sulfur atom of a cysteine ​​residue) generated at disulfide bond sites between antibody chains (two sites between heavy chains and two sites between heavy chains and light chains).

[0090] The above-mentioned anti-HER3 antibody-drug conjugate preferably used in the present invention can also be represented by the following formula:

[0091] [ka]

[0092] Here, the drug linker is bound to the anti-HER3 antibody via a thioether bond, and n is synonymous with the so-called average drug binding number (DAR; Drug-to-Antibody Ratio), and indicates the average number of drug linkers bound per antibody.

[0093] The average number of drug linker bonds per antibody in an anti-HER3 antibody-drug conjugate preferably used in the present invention is preferably 2 to 8, more preferably 3 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8.

[0094] The anti-HER3 antibody portion of the anti-HER3 antibody-drug conjugate used in the present invention is preferably an antibody comprising a heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6, More preferably, it is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, and a light chain comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8, Even more preferably, the antibody is an antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10, or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the above antibody is deleted.

[0095] The anti-HER3 antibody-drug conjugate preferably used in the present invention is capable of transporting the antibody into cancer cells and then expressing the formula:

[0096] [ka]

[0097] By releasing the compound represented by the formula (I), an antitumor effect is exerted.

[0098] The above compound is considered to be the main component of the antitumor activity of the above anti-HER3 antibody-drug conjugate preferably used in the present invention, and has been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0099] The above compound is believed to be generated by cleavage of the linker portion of the above anti-HER3 antibody-drug conjugate, and is represented by the formula:

[0100] [ka]

[0101] This is believed to occur due to decomposition of the aminal structure of the compound represented by the following formula:

[0102] [Production of anti-HER3 antibodies] The HER3 protein used in the present invention can be directly purified from human HER3-expressing cells, or when used as an antigen, the cell membrane fraction of the cells can be used as the HER3 protein. HER3 can also be obtained by synthesizing HER3 in vitro or by producing it in a host cell through genetic engineering. In genetic engineering, specifically, HER3 can be synthesized by incorporating HER3 cDNA into an expression vector and then incubating the vector in a solution containing enzymes, substrates and energy substances required for transcription and translation. Alternatively, the protein can be obtained by transforming other prokaryotic or eukaryotic host cells with the vector and expressing HER3. In addition, the HER3-expressing cells produced by genetic engineering or cell lines expressing HER3 can also be used as HER3 protein antigens. The RNA sequence, cDNA sequence, and amino acid sequence of HER3 are published in public databases and can be referenced by accession numbers such as AAA35979 (precursor including a signal sequence consisting of 19 amino acid residues at the amino terminus) and M34309 (NCBI).

[0103] Furthermore, HER3 also includes proteins that have an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence of the above-mentioned HER3 and have biological activity equivalent to that of the said protein.

[0104] The anti-HER3 antibody used in the present invention can be obtained by known means. For example, it can be obtained by immunizing an animal with the antigen HER3 or any polypeptide selected from the amino acid sequence of HER3 using a method commonly used in this field, and collecting and purifying the antibody produced in the body. The origin of the antigen is not limited to humans, and animals can also be immunized with antigens derived from animals other than humans, such as mice and rats. In this case, anti-HER3 antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibody that binds to a heterologous antigen with a human antigen.

[0105] Alternatively, a hybridoma can be established by fusing an antibody-producing cell that produces an antibody against an antigen with a myeloma cell according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)), thereby obtaining a monoclonal antibody.

[0106] The antigen can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce it. Specifically, a vector capable of expressing an antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above-mentioned genetically engineered antigen-expressing cells or cell lines expressing the antigen.

[0107] The anti-HER3 antibody used in the present invention is preferably a genetically engineered antibody that has been artificially modified for the purpose of reducing heterologous antigenicity against humans, such as a chimeric antibody or a humanized antibody, or is preferably an antibody having only the genetic sequence of an antibody derived from humans, i.e., a human antibody. These antibodies can be produced using known methods.

[0108] Chimeric antibodies include antibodies whose variable and constant regions are of different species, such as chimeric antibodies in which the variable region of an antibody derived from a mouse or rat is joined to a constant region derived from a human (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0109] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody has been incorporated into an antibody of human origin (Nature (1986) 321, p. 522-525), antibodies in which not only the CDR sequence of a heterologous antibody but also some framework amino acid residues of the heterologous antibody have been grafted onto a human antibody by CDR grafting (WO 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0110] Examples of human antibodies include antibodies produced using human antibody-producing mice having a human chromosome fragment containing the heavy and light chain genes of a human antibody (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, p.133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be used (see, for example, Wormstone, IM et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109 (3), p. 427-431).

[0111] The anti-HER3 antibody used in the present invention also includes modified antibodies. The modified antibody means an antibody according to the present invention that has been chemically or biologically modified. Chemical modifications include those having a chemical moiety bonded to the amino acid backbone, or a chemical moiety bonded to an N- or O-linked carbohydrate chain. Biological modifications include those that have been post-translationally modified (e.g., addition of an N- or O-linked glycan, processing of the N- or C-terminus, deamidation, isomerization of aspartic acid, oxidation of methionine, etc.), and those that have been expressed using a prokaryotic host cell to add a methionine residue to the N-terminus. In addition, those labeled to enable detection or isolation of the anti-HER3 antibody or antigen used in the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies, are also included in the meaning of such modifications. Such modified anti-HER3 antibodies used in the present invention are useful for improving the stability and blood retention of the antibody, reducing antigenicity, and detecting or isolating the antibody or antigen.

[0112] In addition, antibody-dependent cellular cytotoxicity can be enhanced by regulating the sugar chain modification (glycosylation, defucosylation, etc.) bound to the anti-HER3 antibody used in the present invention. Known techniques for regulating the sugar chain modification of antibodies include, but are not limited to, WO 99 / 54342, WO 00 / 61739, WO 02 / 31140, WO 2007 / 133855, and WO 2013 / 120066. The anti-HER3 antibody used in the present invention also includes antibodies in which the sugar chain modification has been regulated.

[0113] It is known that antibodies produced in cultured mammalian cells have a deletion of the lysine residue at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)), and also have a deletion of two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain, and amidation of the proline residue at the carboxyl terminus (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the anti-HER3 antibody used in the present invention also includes antibodies and functional fragments of the antibodies that have been modified in this way, including deletions in which one or two amino acids have been deleted at the carboxyl terminus of the heavy chain, and deletions that have been amidated (for example, heavy chains in which the proline residue at the carboxyl terminus has been amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletion of the carboxyl terminus of the heavy chain of the anti-HER3 antibody used in the present invention is not limited to the above types. The two heavy chains constituting the anti-HER3 antibody used in the present invention may be any one of heavy chains selected from the group consisting of full-length and the above deletions, or may be a combination of any two of them. The quantitative ratio of each deletion may be influenced by the type and culture conditions of the cultured mammalian cells producing the anti-HER3 antibody used in the present invention, but the anti-HER3 antibody used in the present invention preferably includes one in which one amino acid residue is deleted at the carboxyl terminus of both of the two heavy chains.

[0114] The isotype of the anti-HER3 antibody used in the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG2. Modified versions of these antibodies can also be used as the anti-HER3 antibody of the present invention.

[0115] Anti-HER3 antibodies that can be used in the present invention include Patritumab (U3-1287), U1-59 (WO 2007 / 077028), AV-203 (WO 2011 / 136911), LJM-716 (WO 2012 / 022814), Duligotumab (MEHD-7945A) (WO 2010 / 108127), Istiratumab (MM-141) (WO 2011 / 047180), Lumretuz ... b(RG-7116) (WO 2014 / 108484), Setibantumab (MM-121) (WO 2008 / 100624), REGN-1400 (WO 2013 / 048883), ZW-9 (WO 2013 / 063702), and variants, active fragments, modified forms, etc. thereof can be mentioned, and preferably, Patritumab and U1-59 can be mentioned. These anti-HER3 antibodies can be produced by the methods described in the above documents.

[0116] [Production of anti-HER3 antibody-drug conjugate] The drug linker intermediate used in the production of the anti-HER3 antibody-drug conjugate of the present invention is represented by the following formula:

[0117] [ka]

[0118] The above drug linker intermediate can be represented by the chemical name N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide, and can be produced with reference to the descriptions in WO 2014 / 057687, WO 2015 / 155998, and WO 2019 / 044947.

[0119] The anti-HER3 antibody-drug conjugate used in the present invention can be produced by reacting the above-mentioned drug linker intermediate with an anti-HER3 antibody having a thiol group (also called a sulfhydryl group).

[0120] An anti-HER3 antibody having a sulfhydryl group can be obtained by a method well known to those skilled in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) can be used in an amount of 0.3 to 3 molar equivalents per intra-chain disulfide of the antibody, and reacted with the anti-HER3 antibody in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), to obtain an anti-HER3 antibody having a sulfhydryl group in which the intra-chain disulfide of the antibody has been partially or completely reduced.

[0121] Additionally, 2 to 20 molar equivalents of drug linker intermediate can be used per anti-HER3 antibody bearing a sulfhydryl group to produce anti-HER3 antibody-drug conjugates with 2 to 8 drugs attached per antibody.

[0122] The average number of drugs bound per antibody molecule of the produced anti-HER3 antibody-drug conjugate can be calculated, for example, by a method in which the UV absorbance of the anti-HER3 antibody-drug conjugate and its conjugation precursor are measured at two wavelengths, 280 nm and 370 nm (UV method), or by a method in which the antibody-drug conjugate is treated with a reducing agent, and each of the resulting fragments is quantified by HPLC measurement and then calculated (HPLC method).

[0123] Conjugation of the anti-HER3 antibody to the drug linker intermediate, and calculation of the average number of drugs bound per antibody molecule of the anti-HER3 antibody-drug conjugate can be performed with reference to the descriptions in WO 2015 / 155998, etc.

[0124] [Therapeutic agent and / or treatment method] The therapeutic agent and / or method of the present invention is characterized by administering an anti-HER3 antibody-drug conjugate, and can be used for the treatment of HER3 mutated cancer.

[0125] The HER3 mutant cancer for which the therapeutic agent and / or method of the present invention can be used is preferably at least one selected from the group consisting of breast cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), colon cancer (sometimes referred to as colorectal cancer, including colon cancer and rectal cancer), gastric cancer (sometimes referred to as gastric adenocarcinoma), ovarian cancer, head and neck cancer, glioblastoma multiforme, melanoma, kidney cancer, urothelial cancer, prostate cancer, pancreatic cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, endometrial cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, and penile cancer, and more preferably at least one selected from the group consisting of breast cancer, non-small cell lung cancer, colon cancer, gastric cancer, ovarian cancer, head and neck cancer, glioblastoma multiforme, and melanoma.

[0126] The therapeutic agent and method of the present invention can be suitably used for mammals, and more suitably used for humans.

[0127] The antitumor effect of the therapeutic agent and method of the present invention can be confirmed, for example, by expressing mutant HER3 in tumor cells and measuring the cell proliferation inhibitory activity of an anti-HER3 antibody-drug conjugate in the absence and / or presence of HER2 overexpression. It can also be confirmed by preparing a model in which a tumor expressing mutant HER3 is transplanted into a test animal and administering the therapeutic agent or method of the present invention.

[0128] Furthermore, the antitumor effect of the therapeutic agent and method of the present invention can also be confirmed in clinical trials. That is, the therapeutic agent or method of the present invention can be administered to cancer patients with confirmed HER3 mutation, and can be confirmed by Response Evaluation Criteria in Solid Tumors (RECIST) evaluation method, WHO evaluation method, Macdonald evaluation method, weight measurement, and other methods, and can be judged by indicators such as complete response (CR), partial response (PR), progressive disease (PD), objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), and overall survival (OS).

[0129] By the above-mentioned method, it is possible to confirm the superiority of the therapeutic agent and therapeutic method of the present invention in terms of the antitumor effect against HER3 mutant cancer compared to existing anticancer agents.

[0130] The therapeutic agent and method of the present invention can slow the growth of cancer cells, suppress their proliferation, and even destroy them. These actions can relieve cancer patients from symptoms caused by cancer and improve their QOL, and the therapeutic effect can be achieved while preserving the life of the cancer patient. Even if the destruction of cancer cells is not achieved, the inhibition and control of the proliferation of cancer cells can enable cancer patients to achieve a higher QOL and longer survival.

[0131] The therapeutic agent of the present invention can be applied to patients as a systemic therapy, and also can be locally applied to cancer tissues to be expected to have a therapeutic effect.

[0132] The therapeutic agent of the present invention may be administered as a pharmaceutical composition containing one or more pharma- ceutical compatible components. The pharma-ceutical compatible components may be appropriately selected from formulation additives and others commonly used in this field according to the dosage and administration concentration of the anti-HER3 antibody-drug conjugate used in the present invention. For example, the therapeutic agent of the present invention may be administered as a pharmaceutical composition (hereinafter referred to as the "pharmaceutical composition of the present invention") containing a buffer such as a histidine buffer, an excipient such as sucrose or trehalose, and a surfactant such as polysorbate 80 or 20. The pharmaceutical composition of the present invention may be preferably used as an injection, more preferably as an aqueous injection or a lyophilized injection, and even more preferably as a lyophilized injection.

[0133] When the pharmaceutical composition of the present invention is an aqueous injection, it can be administered intravenously by drip infusion after being diluted with a suitable diluent, such as a glucose solution or physiological saline solution.

[0134] When the pharmaceutical composition of the present invention is a lyophilized injection, it is preferably dissolved in water for injection, and then diluted with a required amount of a suitable diluent, such as a glucose solution or physiological saline solution, and then administered by intravenous drip.

[0135] Examples of introduction routes that can be used to administer the pharmaceutical composition of the present invention include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, and preferably include the intravenous route.

[0136] The anti-HER3 antibody-drug conjugate used in the present invention can be administered to humans preferably at intervals of once every 1, 2, 3, or 4 weeks, and even more preferably at intervals of once every 3 weeks.

[0137] Furthermore, the anti-HER3 antibody-drug conjugate used in the present invention can be administered to humans preferably at a single dose of 1.6 mg / kg to 12.8 mg / kg, more preferably at a single dose of 1.6 mg / kg, 3.2 mg / kg, 4.8 mg / kg, 5.6 mg / kg, 6.4 mg / kg, 8.0 mg / kg, 9.6 mg / kg, or 12.8 mg / kg, and even more preferably at a single dose of 4.8 mg / kg, 5.6 mg / kg, or 6.4 mg / kg.

[0138] The therapeutic agent of the present invention can be administered in combination with a cancer therapeutic agent other than the anti-HER3 antibody-drug conjugate used in the present invention, thereby enhancing the antitumor effect. The other cancer therapeutic agent used for such a purpose may be administered to an individual simultaneously with the therapeutic agent of the present invention, separately, or consecutively, or may be administered at different administration intervals. Such cancer therapeutic agents are not limited as long as they have antitumor activity, and examples thereof include irinotecan (CPT-11), cisplatin, carboplatin, oxaliplatin, fluorouracil (5-FU), gemcitabine, capecitabine, paclitaxel, docetaxel, doxorubicin, epirubicin, cyclophosphamide, mitomycin C, and the like. C), Tegafur / Gimeracil / Oteracil combination drug, Cetuximab, Panitumumab, Bevacizumab, Ramucirumab, Regorafenib, Trifluridine / Tipiracil combination drug, Gefitinib, Erlotinib, Afatinib Afatinib, Osimertinib, Methotrexate, Pemetrexed, Tamoxifen, Toremifene, Fulvestrant, Leuprorelin, Goserelin, Letrozole, Anastrozole, Progesterone formulation, Trastuzumab emtansineAt least one selected from the group consisting of emtansine, trastuzumab, pertuzumab, lapatinib, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, ipilimumab, and tremelimumab may be mentioned.

[0139] The therapeutic agent of the present invention can also be used in combination with radiation therapy. For example, a cancer patient receives radiation therapy before and / or after, or simultaneously with, treatment with the therapeutic agent of the present invention.

[0140] The therapeutic agent of the present invention can also be used as adjuvant chemotherapy in combination with surgery. Surgery is performed, for example, by physically removing all or part of a brain tumor. The therapeutic agent of the present invention can be administered before surgery to reduce the size of the brain tumor (referred to as preoperative adjuvant chemotherapy or neoadjuvant therapy), or after surgery to prevent the recurrence of the brain tumor (referred to as postoperative adjuvant chemotherapy or adjuvant therapy). EXAMPLES

[0141] The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. Furthermore, the examples should not be construed as being limiting in any sense.

[0142] [Example 1] Preparation of anti-HER3 antibody-drug conjugate According to the production method described in WO 2015 / 155998, an anti-HER3 antibody (referred to as "HER3-Ab(1)" in the present invention) comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 9 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10 is used to produce a HER3 antibody having the formula:

[0143] [ka]

[0144] and carrying out a conjugation reaction with a drug linker intermediate represented by the formula:

[0145] [ka]

[0146] (wherein A represents the binding site to the antibody) An anti-HER3 antibody-drug conjugate (referred to as "HER3-ADC(1)" in the present invention) was produced in which an anti-HER3 antibody was linked to a drug linker represented by the following formula via a thioether bond. The average number of drugs bound per antibody in HER3-ADC(1) is in the range of 7 to 8.

[0147] [Example 2] Preparation of HER3 stable expressing cells Example 2-1: Construction of a lentivirus vector for expressing HER3 mutations 1. Preparation of pLVSIN EF1α HER3(WT)Pur HER3 cDNA was used as a template, and amplified with the HER3 IF primer (Fw) shown in SEQ ID NO: 11 and the HER3 IF primer (Rev) shown in SEQ ID NO: 12, and inserted into the XbaI site of pLVSIN EF1α Pur (Takara, #6186) using the InFusion System (CLONTECH) (pLVSIN EF1α HER3(WT)Pur). The inserted sequence was confirmed by DNA sequencing.

[0148] 2. Preparation of pLVSIN EF1α flag-HER3(WT)Pur Using pLVSIN EF1α HER3(WT)Pur as a template, the entire plasmid was amplified with the Flag IF primer (Fw) shown in SEQ ID NO: 13 and the Flag IF primer (Rev) shown in SEQ ID NO: 14, and pLVSIN EF1α flag-HER3(WT)Pur was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(WT) is shown in SEQ ID NO: 15, and the amino acid sequence of Flag-HER3(WT) is shown in SEQ ID NO: 16.

[0149] 3. Preparation of plasmids expressing various HER3 mutants i)pLVSIN EF1α flag-HER3(V104L)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 17 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 18, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(V104L)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(V104L) is shown in SEQ ID NO: 19, and the amino acid sequence of Flag-HER3(V104L) is shown in SEQ ID NO: 20.

[0150] ii)pLVSIN EF1α flag-HER3(V104M)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 21 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 22, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(V104M)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(V104M) is shown in SEQ ID NO: 23, and the amino acid sequence of Flag-HER3(V104M) is shown in SEQ ID NO: 24.

[0151] iii)pLVSIN EF1α flag-HER3(A232V)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 25 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 26, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(A232V)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(A232V) is shown in SEQ ID NO: 27, and the amino acid sequence of Flag-HER3(A232V) is shown in SEQ ID NO: 28.

[0152] iv)pLVSIN EF1α flag-HER3(P262H)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 29 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 30, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(P262H)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(P262H) is shown in SEQ ID NO: 31, and the amino acid sequence of Flag-HER3(P262H) is shown in SEQ ID NO: 32.

[0153] v)pLVSIN EF1α flag-HER3(G284R)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 33 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 34, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(G284R)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(G284R) is shown in SEQ ID NO: 35, and the amino acid sequence of Flag-HER3(G284R) is shown in SEQ ID NO: 36.

[0154] vi)pLVSIN EF1α flag-HER3(D297Y)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 37 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 38, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(D297Y)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(D297Y) is shown in SEQ ID NO: 39, and the amino acid sequence of Flag-HER3(D297Y) is shown in SEQ ID NO: 40.

[0155] vii)pLVSIN EF1α flag-HER3(G325R)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 41 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 42, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(G325R)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(G325R) is shown in SEQ ID NO: 43, and the amino acid sequence of Flag-HER3(G325R) is shown in SEQ ID NO: 44.

[0156] viii)pLVSIN EF1α flag-HER3(T355I)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 45 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 46, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(T355I)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(T355I) is shown in SEQ ID NO: 47, and the amino acid sequence of Flag-HER3(T355I) is shown in SEQ ID NO: 48.

[0157] ix)pLVSIN EF1α flag-HER3(S846I)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 49 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 50, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(S846I)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(S846I) is shown in SEQ ID NO: 51, and the amino acid sequence of Flag-HER3(S846I) is shown in SEQ ID NO: 52.

[0158] x)pLVSIN EF1α flag-HER3(E928G)Pur Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 53 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 54, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(E928G)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(E928G) is shown in SEQ ID NO: 55, and the amino acid sequence of Flag-HER3(E928G) is shown in SEQ ID NO: 56.

[0159] Example 2-2: Construction of HER2-expressing lentivirus vector 1. Preparation of pLVSIN EF1α HER2 Neo HER2 cDNA was used as a template, and amplified with the HER2 IF primer (Fw) shown in SEQ ID NO: 57 and the HER2 IF primer (Rev) shown in SEQ ID NO: 58, and inserted into the NotI site of pLVSIN EF1α Neo (Takara, #6184) (pLVSIN EF1α HER2 Neo) using the InFusion System (CLONTECH). The inserted sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding HER2 is shown in SEQ ID NO: 59, and the amino acid sequence of HER2 is shown in SEQ ID NO: 60.

[0160] Example 2-3: Preparation of HER3 stably expressing cells Lenti-X 293T cells (CLONTECH) at 1x10 6The cells were seeded at 1000 cells / well in a 6-well plate and cultured overnight. ViraPower (Thermofisher) and various HER3 expression plasmids (pLVSIN EF1α flag-HER3(WT)Pur, pLVSIN EF1α flag-HER3(V104L)Pur, pLVSIN EF1α flag-HER3(V104M)Pur, pLVSIN EF1α flag-HER3(A232V)Pur, pLVSIN EF1α flag-HER3(P262H)Pur, pLVSIN EF1α flag-HER3(G284R)Pur, pLVSIN EF1α flag-HER3(D297Y)Pur, pLVSIN EF1α flag-HER3(G325R)Pur, pLVSIN EF1α flag-HER3(T355I)Pur, pLVSIN EF1α flag-HER3(S846I)Pur and pLVSIN EF1α flag-HER3(E928G)Pur) were introduced into Lenti-X 293T cells using Lipofectamine 2000 (Thermofisher). In addition, in order to prepare empty vector-introduced cells, a plasmid (pLVSIN EF1α Pur) was introduced into Lenti-X 293T cells by the same method as above. Two days after the introduction, the culture supernatant was collected and passed through a MILLEX-HP 0.45 UM filter (Merck) to prepare a lentivirus solution. Half of the prepared lentivirus solution was transferred to a RetroNectin Coating plate (12 well plate) and left to stand overnight at 37°C. After washing with PBS, MDA-MB-231 cells were cultured at 1x10 5The cells were seeded at 1000x1000 cells / well and cultured for 3 days. After the culture was completed, the cells were detached and the entire amount was seeded on a 6-well plate, and stable expression cells (polyclone) were obtained using drug resistance in the presence of 0.5 μg / mL puromycin (manufactured by Thermofisher) as an index. Total RNA was extracted from the obtained cells, and reverse transcription reaction was performed using 600 ng of total RNA. Next, PCR reaction was performed using 1 / 20 volume of the reverse transcription reaction product as a template, HER3 primer (Fw) shown in SEQ ID NO: 61, and HER3 primer (Rev) shown in SEQ ID NO: 62. PCR was performed under the conditions of heating at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 1 minute. The RT-PCR product was subjected to 1% agarose gel electrophoresis to confirm HER3 gene expression.

[0161] Example 2-4: Preparation of HER2 overexpression and HER3 stable expression line 1. Generation of HER2-overexpressing cells Lenti-X 293T cells (CLONTECH) at 1x10 6 The cells were seeded in a 6-well plate at 1000 cells / well and cultured overnight. The HER2 expression plasmid (pLVSIN EF1α HER2 Neo) prepared in Example 2-2 was introduced into Lenti-X 293T cells using ViraPower (Thermofisher) and Lipofectamine 2000 (Thermofisher). Two days after the introduction, the culture supernatant was collected and passed through a MILLEX-HP 0.45 UM filter (Merck) to prepare a lentivirus solution. The prepared lentivirus solution was transferred to a plate (6-well plate) coated with RetroNextin (TAKARA) and left to stand overnight at 37°C. After washing with PBS, 4x10 MDA-MB-231 cells were cultured in a 6-well plate at 1000 cells / well. 5The cells were seeded at 1000 x 1000 cells / well and cultured for 3 days. After the culture was completed, the cells were detached and the entire amount was seeded in a T25 flask, and HER2 stable expression cells (polyclone) were obtained using drug resistance in the presence of 800 μg / mL geneticin (manufactured by Thermofisher) as an index. Total RNA was extracted from the obtained cells, and reverse transcription reaction was performed using 600 ng of total RNA. Next, PCR reaction was performed using 1 / 20 volume of the reverse transcription reaction product as a template, HER2 primer (Fw) shown in SEQ ID NO: 63, and HER2 primer (Rev) shown in SEQ ID NO: 64. PCR was performed under the conditions of heating at 94 ° C for 2 minutes, followed by 23 cycles of 98 ° C for 10 seconds and 68 ° C for 1 minute. The RT-PCR product was subjected to 1% agarose gel electrophoresis to confirm HER2 gene expression.

[0162] 2. Creation of HER2-overexpressing HER3-stable cells Lenti-X 293T cells (CLONTECH) at 1x10 6The cells were seeded at 1000 cells / well in a 6-well plate and cultured overnight. ViraPower (Thermofisher) and various HER3 expression plasmids (pLVSIN EF1α flag-HER3(WT)Pur, pLVSIN EF1α flag-HER3(V104L)Pur, pLVSIN EF1α flag-HER3(V104M)Pur, pLVSIN EF1α flag-HER3(A232V)Pur, pLVSIN EF1α flag-HER3(P262H)Pur, pLVSIN EF1α flag-HER3(G284R)Pur, pLVSIN EF1α flag-HER3(D297Y)Pur, pLVSIN EF1α flag-HER3(G325R)Pur, pLVSIN EF1α flag-HER3(T355I)Pur, pLVSIN EF1α flag-HER3(S846I)Pur and pLVSIN EF1α flag-HER3(E928G)Pur) were introduced into Lenti-X 293T cells using Lipofectamine 2000 (Thermofisher). In addition, in order to prepare empty vector-introduced cells, a plasmid (pLVSIN EF1α Pur) was introduced into Lenti-X 293T cells by the same method as above. Two days after the introduction, the culture supernatant was collected and passed through a MILLEX-HP 0.45 UM filter (Merck) to prepare a lentivirus solution. Half of the prepared lentivirus solution was transferred to a plate (12 well plate) coated with RetroNextin (TAKARA) and left to stand overnight at 37°C. After washing with PBS, 1x10 MDA-MB-231 cells overexpressing HER2 were cultured. 5The cells were seeded at 1000x1000 cells / well and cultured for 3 days. After the culture was completed, the cells were detached and the entire amount was seeded on a 6-well plate, and stable expression cells (polyclone) were obtained using drug resistance in the presence of 0.5μg / mL puromycin (manufactured by Thermofisher) as an index. Total RNA was extracted from the obtained cells, and reverse transcription reaction was performed using 600ng of total RNA. Next, PCR reaction was performed using 1 / 20 volume of the reverse transcription reaction product as a template, HER3 primer (Fw) shown in SEQ ID NO: 61, and HER3 primer (Rev) shown in SEQ ID NO: 62. PCR was performed under the conditions of heating at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 1 minute. The RT-PCR product was subjected to 1% agarose gel electrophoresis to confirm HER3 gene expression. HER2 gene expression was also confirmed by the method described above.

[0163] The results are shown in Figures 3 to 5. Figure 3 shows the results of 1% agarose gel electrophoresis of RT-PCR products from various HER3 stably expressing cells, and Figures 4 and 5 show the results of 1% agarose gel electrophoresis of RT-PCR products from various HER2-overexpressing HER3 stably expressing cells.

[0164] In HER3 stable expressing cells, HER3 gene expression was observed in both HER3 wild type and mutant types.In HER2 overexpressing HER3 stable expressing cells, HER2 overexpression was observed, and HER3 gene expression was observed in both HER3 wild type and mutant types.

[0165] Example 2-5: Confirmation of cell surface HER3 expression positivity rate HER3 stable expression MDA-MB-231 cells were cultured in RPMI1640 medium (ThermoFisher) containing 10% FBS (HyClone). The cells were detached from the culture plate using TrypLE (registered trademark) Express Enzyme (ThermoFisher), and the number of live cells was measured by trypan blue treatment. The same number of live cells was added to a 96-well U-bottom plate, the cells were precipitated by centrifugation, and the medium was replaced with Stain Buffer (BD). The cells were again precipitated by centrifugation, and 100 μL of PE anti-DYKDDDDK Tag Antibody (BioLegend, #637310) diluted to 1 μg / mL was added to suspend the cells. Cells treated with Stain Buffer without PE anti-DYKDDDDK Tag Antibody were used as the control group. Each cell was reacted on ice in the dark for 60 minutes, and then washed with Stain Buffer. After resuspending in 100μL of Stain Buffer, an equal volume of 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) was added and reacted in the dark on ice for 20 minutes. After washing with Stain Buffer, the fluorescent signal was measured using an Attune NxT Flow Cytometer (ThermoFisher, Ltd.), and the measurement results were analyzed using FlowJo software (BD, Version 10.5.0). Table 1 shows the HER3 expression positivity rate of various mutant HER3-introduced cell lines treated with PE anti-DYKDDDDK Tag Antibody.

[0166] [Table 1]

[0167] The HER3 expression rate on the cell surface ranged from 65.0% to 83.9%, which was determined to be within a range that would not interfere with comparing the properties of anti-HER3 antibody-drug conjugates between these cells.

[0168] [Example 3] Confirmation of binding of HER3-ADC (1) to wild-type and various mutant HER3-introduced cell lines Wild-type and various mutant HER3-introduced MDA-MB-231 cells were cultured in RPMI1640 medium (ThermoFisher) containing 10% FBS (HyClone). The cells were detached from the culture plate using TrypLE (registered trademark) Express Enzyme (ThermoFisher), and the number of live cells was measured by trypan blue treatment. The same number of live cells was added to a 96-well U-bottom plate, the cells were precipitated by centrifugation, and the medium was replaced with Stain Buffer (BD). The cells were again precipitated by centrifugation and suspended in 100 μL of ice-cold HER3-ADC (1) dilution solution (prepared to 100 nM to 0.1 nM by 1 / 10 serial dilution using Stain Buffer). Cells treated with Stain Buffer without HER3-ADC (1) were used as the control group. Each cell was reacted on ice for 60 minutes, and then washed with Stain Buffer. Further, 100 μL of stain buffer or secondary antibody (Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, ThermoFisher, #A-21445) diluted to 10 μg / mL was added to suspend the cells. After reacting for 60 minutes on ice in the dark, the cells were washed with stain buffer. After suspending in 100 μL of stain buffer, an equal amount of 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) was added and reacted for 20 minutes on ice in the dark. After washing with stain buffer, the fluorescent signal was measured using Attune NxT Flow Cytometer (ThermoFisher), and the measurement results were analyzed using FlowJo software (BD, Version 10.5.0). In addition, in order to quantify the fluorescent signal derived from HER3-ADC (1) in the cells, the value obtained by subtracting the signal from the control group treated with stain buffer was used.

[0169] The results are shown in Figures 6 and 7. The vertical axis shows the binding of HER3-ADC(1) at each concentration to wild-type and various mutant HER3-introduced cell lines (mean fluorescence intensity of HER3-positive cells).

[0170] The binding of HER3-ADC(1) to cells stably expressing various mutant HER3 increased in a concentration-dependent manner, and showed similar binding to wild-type HER3 (Figure 6). The binding of HER3-ADC(1) to cells stably expressing HER2-overexpressing HER3 also increased in a concentration-dependent manner, and showed similar binding to wild-type HER3 (Figure 7). In addition, as a comparison, the binding was again measured using cells without HER2 overexpression, and it was confirmed that there was no difference in the binding of HER3-ADC(1) depending on the HER2 expression level (Figure 7).

[0171] [Example 4] In vitro cell proliferation inhibition by HER3-ADC (1) in wild-type and various mutant HER3-introduced cell lines The growth inhibitory activity of HER3-ADC(1) against wild-type and various mutant HER3-introduced MDA-MB-231 cells was measured in the presence of RPMI1640 medium (ThermoFisher) containing 10% FBS (HyClone). Cell growth was evaluated by measuring adenosine triphosphate (ATP) activity in the untreated, HER3-ADC(1), and HER3-Ab(1)-treated groups. As a negative control, an IgG antibody-drug conjugate (referred to as "IgG-ADC(1)" in the present invention) produced by a conjugation reaction between an IgG antibody and a drug linker intermediate (1) was used.

[0172] Example 4-1: Cell treatment Wild-type and various HER3 mutant-transduced MDA-MB-231 cell lines were seeded in a 96-microwell plate (Corning, #3904, black-walled transparent bottom) as a cell suspension of 500 cells / 100 μL / well. The next day, 10-fold concentrated solutions of HER3-ADC(1), HER3-Ab(1), and IgG-ADC(1) as a negative control were prepared in RPMI1640 medium containing 10% FBS, 10 μL of each solution was added, and the cells were cultured at 37°C under 5% CO2 for 6 days. Only 10 μL of medium was added to the control wells. Measurements were performed in 3 or 5 wells per condition.

[0173] Example 4-2: Evaluation of cell proliferation inhibitory effect The cytostatic activity of various drugs during 6 days of culture was evaluated based on ATP activity in metabolically active live cells. 100 μL of CellTiter-Glo (registered trademark) reagent (Promega, #G7573) or 100 μL of ATPlite 1step Luminescence Assay System (PerkinElmer, #6016739) was added to each well of a 96-microwell plate and measured with an EnVision Workstation (Ultra-sensitive luminescence measurement protocol, h=0.9). To measure the decrease in ATP activity, the average luminescence value of 3 or 5 wells per condition was calculated. The luminescence residual rate (%) was calculated by comparing with the cells of the untreated group, and this value was interpreted as the cell viability (%).

[0174] The results are shown in Figures 8 to 33. Figures 8 to 19 are figures showing the cell proliferation inhibitory activity of various drugs in wild-type and various mutant HER3-introduced cells without HER2 overexpression. Figures 20 to 31 are figures showing the cell proliferation inhibitory activity of various drugs in wild-type and various mutant HER3-introduced cells with HER2 overexpression. It was found that HER3-ADC (1) exhibits cell proliferation inhibitory activity against various mutant HER3-introduced cells as well as wild-type HER3-introduced cells (Figures 8 to 19). It was also found that HER3-ADC (1) exhibits cell proliferation inhibitory activity against various mutant HER3-introduced cells as well as wild-type HER3-introduced cells in HER2 overexpression HER3 stable expressing cells (Figures 20 to 31). In addition, as a comparison, the cell proliferation inhibitory activity of various drugs in cells without HER2 overexpression was measured again (Figures 32 and 33), and it was confirmed that there was no difference in the cell proliferation inhibitory activity of HER3-ADC (1) depending on the HER2 expression level.

[0175] [Example 5] Preparation of mutant HER3 (Q809R)-transfected cells and pharmacological evaluation Example 5-1: Preparation of HER3 mutant expression plasmid Using pLVSIN EF1α flag-HER3(WT) as a template, the entire plasmid was amplified with the HER3 mutation introduction primer (Fw) shown in SEQ ID NO: 65 and the HER3 mutation introduction primer (Rev) shown in SEQ ID NO: 66, and the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(Q809R)Pur) was obtained by self-ligation using the InFusion System (CLONTECH). The sequence was confirmed by DNA sequencing. The nucleotide sequence of the cDNA encoding Flag-HER3(Q809R) is shown in SEQ ID NO: 67, and the amino acid sequence of Flag-HER3(Q809R) is shown in SEQ ID NO: 68.

[0176] Example 5-2: Preparation of mutant HER3 (Q809R)-transfected cells (with HER2 overexpression) Using the HER3 mutant expression plasmid (pLVSIN EF1α flag-HER3(Q809R)Pur) prepared in Example 5-1, mutant HER3(Q809R)-introduced cells were obtained as HER2-overexpressing HER3 stable expressing cells (polyclone) in the same manner as in Example 2-4. Total RNA was extracted from the obtained cells in the same manner as in Example 2-4, and the RT-PCR product was subjected to 1% agarose gel electrophoresis to confirm HER3 gene expression (Figure 34) and HER2 gene expression (Figure 35).

[0177] Example 5-3: Confirmation of cell surface HER3 expression positivity rate For the mutant HER3 (Q809R)-transfected cells (with HER2 overexpression) prepared in Example 5-2, the cell surface HER3 expression positivity rate was confirmed by the same method as in Example 2-5. The cell surface HER3 expression rate was 58.8%, which was determined to be a reasonable value for comparing the properties of anti-HER3 antibody-drug conjugates.

[0178] Example 5-4: Confirmation of binding to mutant HER3 (Q809R)-transfected cells (with HER2 overexpression) For the mutant HER3 (Q809R)-introduced cells (with HER2 overexpression) prepared in Example 5-2, the binding of HER3-ADC (1) to wild-type and mutant HER3 (Q809R)-introduced cell lines at each concentration was evaluated by the same method as in Example 3. The binding of HER3-ADC (1) to the mutant HER3 (Q809R)-introduced cell line increased in a concentration-dependent manner, and showed binding similar to that of wild-type HER3 (Figure 36).

[0179] Example 5-5: In vitro cell proliferation inhibition in mutant HER3 (Q809R)-transfected cells (with HER2 overexpression) For the mutant HER3 (Q809R)-introduced cells (with HER2 overexpression) prepared in Example 5-2, in vitro cell proliferation inhibition evaluation of HER3-ADC (1), HER3-Ab (1), and IgG-ADC (1) against wild-type and mutant HER3 (Q809R)-introduced cell lines was performed in the same manner as in Example 4. It was found that HER3-ADC (1) exhibited cell proliferation inhibition activity against mutant HER3 (Q809R)-introduced cells as well as wild-type HER3-introduced cells (FIG. 37).

[0180] [Example 6] Confirmation of lysosomal migration of HER3-ADC(1) in cells transfected with wild-type and various mutant HER3 Lysosomal translocation of HER3-ADC(1) in wild-type and various mutant HER3-transfected cells was measured in the presence of RPMI1640 medium (ThermoFisher) containing 10% FBS (HyClone). HER3-ADC(1) was labeled with the pH-sensitive fluorescent dye pHrodo, and the fluorescent signal emitted by lysosomal translocation was measured.

[0181] Example 6-1: Preparation of pHrodo-labeled HER3-ADC (1) For pHrodo labeling, pHrodo iFL Microscale Protein Labeling Kit was used. HER3-ADC(1) was prepared at 1 mg / mL with 10 mM acetate / 5% sorbitol (pH 5.5, Nacalai Tesque). 100 μL (equivalent to 100 μg) was added to the reaction tube. pHrodo iFL Red STP ester was dissolved in DMSO, and an amount equivalent to 10 times the protein molar amount of HER3-ADC(1) was added to the reaction tube and mixed, and then reacted at room temperature for 60 minutes. The reaction solution was centrifuged using a Zeba-spin desalting column (ThermoFisher) and Amicon Ultra-0.5 (Merk-Millipore) to purify pHrodo-labeled HER3-ADC(1). The absorbance at 280 nm and 566 nm was measured using a NanoDrop® 8000 Spectrophotometer, and the labeling efficiency was calculated.

[0182] Example 6-2: Lysosomal migration in cultured cells Wild-type and various mutant HER3-transfected cells were cultured at 5x10 4The cells were seeded in a 96-microwell plate (Cellcarrier-96 Ultra, ThermoFisher) as a cell suspension of 100 cells / 100 μL / well. The next day, the medium was aspirated, and 50 μL of Hoechst 33342 (Life Technologies) diluted to 100 ng / mL in RPMI1640 medium containing 10% FBS was added. After 30 minutes, 50 μL of a 2x concentrated solution of pHrodo-labeled HER3-ADC(1) prepared in RPMI1640 medium containing 10% FBS containing 100 ng / mL Hoechst 33342 was added, and medium was added instead of pHrodo-labeled HER3-ADC(1) for the negative control. Fluorescent signals were captured using an Opera Phenix™ high-throughput, high-content imaging system (Perkin Elmer) to obtain live cell imaging images every 30 minutes for 10 hours. Measurements were performed at 3 spots / well per condition. The acquired images were quantitatively analyzed using image analysis software Harmony (registered trademark) (Perkin Elmer). The quantitative value was expressed as the value (Traffiking index) obtained by multiplying the number of fluorescent dots per cell by the fluorescent intensity per dot.

[0183] The results are shown in Figures 38 to 64. Figures 38 to 49 show the results for cells stably expressing HER3 (without HER2 overexpression), and Figures 50 to 62 show the results for cells stably expressing HER3 (with HER2 overexpression).

[0184] pHrodo-labeled HER3-ADC(1) showed concentration-dependent lysosomal migration in various mutant HER3-transfected cells as well as in wild-type HER3-transfected cells (Figures 38 to 49). This demonstrated that the lysosomal migration of HER3-ADC(1) is not substantially different between wild-type HER3-expressing cells and mutant HER3-expressing cells.

[0185] It was also found that pHrodo-labeled HER3-ADC(1) exhibited concentration-dependent lysosomal migration even in HER2-overexpressing HER3-stable cells (Figures 50 to 62). As a comparison, the lysosomal migration of HER3-ADC(1) in cells without HER2 overexpression was confirmed again (Figures 63 and 64). This revealed that the lysosomal migration of HER3-ADC(1) was not substantially different between HER3-expressing cells that overexpress HER2 and HER3-expressing cells that did not overexpress HER2.

[0186] These results demonstrated that HER3-ADC(1) exhibits cell proliferation inhibitory activity against various HER3 mutant-transfected cells, regardless of the presence or absence of HER2 overexpression.

[0187] Clinical trials of HER3-ADC(1) are currently underway, and it has been suggested that the drug may be as effective in treating cases of mutant HER3 as it is in treating cases of wild-type HER3. [Sequence List Free Text]

[0188] SEQ ID NO: 1: Amino acid sequence of CDRH1 of anti-HER3 antibody SEQ ID NO: 2: Amino acid sequence of CDRH2 of anti-HER3 antibody SEQ ID NO: 3: Amino acid sequence of CDRH3 of anti-HER3 antibody SEQ ID NO: 4: Amino acid sequence of CDRL1 of anti-HER3 antibody SEQ ID NO: 5: Amino acid sequence of CDRL2 of anti-HER3 antibody SEQ ID NO: 6: Amino acid sequence of CDRL3 of anti-HER3 antibody SEQ ID NO: 7: Amino acid sequence of the heavy chain variable region of the anti-HER3 antibody SEQ ID NO: 8: Amino acid sequence of the light chain variable region of the anti-HER3 antibody SEQ ID NO: 9: Amino acid sequence of the heavy chain of the anti-HER3 antibody SEQ ID NO: 10: Amino acid sequence of the light chain of anti-HER3 antibody SEQ ID NO: 11: Nucleotide sequence of HER3 IF primer (Fw) SEQ ID NO: 12: Nucleotide sequence of HER3 IF primer (Rev) SEQ ID NO: 13: Nucleotide sequence of Flag IF primer (Fw) SEQ ID NO: 14: Nucleotide sequence of Flag IF primer (Rev) SEQ ID NO: 15: Nucleotide sequence of cDNA encoding Flag-HER3 (WT) SEQ ID NO: 16: Amino acid sequence of Flag-HER3 (WT) SEQ ID NO: 17: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 18: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 19: Nucleotide sequence of cDNA encoding Flag-HER3(V104L) SEQ ID NO: 20: Amino acid sequence of Flag-HER3(V104L) SEQ ID NO: 21: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 22: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 23: Nucleotide sequence of cDNA encoding Flag-HER3(V104M) SEQ ID NO: 24: Amino acid sequence of Flag-HER3 (V104M) SEQ ID NO: 25: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 26: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 27: Nucleotide sequence of cDNA encoding Flag-HER3(A232V) SEQ ID NO: 28: Amino acid sequence of Flag-HER3(A232V) SEQ ID NO: 29: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 30: Nucleotide sequence of HER3 mutagenesis primer (Rev)

[0189] SEQ ID NO: 31: Nucleotide sequence of cDNA encoding Flag-HER3 (P262H) SEQ ID NO: 32: Amino acid sequence of Flag-HER3 (P262H) SEQ ID NO: 33: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 34: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 35: Nucleotide sequence of cDNA encoding Flag-HER3(G284R) SEQ ID NO: 36: Amino acid sequence of Flag-HER3(G284R) SEQ ID NO: 37: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 38: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 39: Nucleotide sequence of cDNA encoding Flag-HER3(D297Y) SEQ ID NO: 40: Amino acid sequence of Flag-HER3(D297Y) SEQ ID NO: 41: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 42: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 43: Nucleotide sequence of cDNA encoding Flag-HER3(G325R) SEQ ID NO: 44: Amino acid sequence of Flag-HER3(G325R) SEQ ID NO: 45: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 46: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 47: Nucleotide sequence of cDNA encoding Flag-HER3(T355I) SEQ ID NO: 48: Amino acid sequence of Flag-HER3(T355I) SEQ ID NO: 49: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 50: Nucleotide sequence of HER3 mutagenesis primer (Rev)

[0190] SEQ ID NO: 51: Nucleotide sequence of cDNA encoding Flag-HER3 (S846I) SEQ ID NO: 52: Amino acid sequence of Flag-HER3 (S846I) SEQ ID NO: 53: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 54: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 55: Nucleotide sequence of cDNA encoding Flag-HER3 (E928G) SEQ ID NO: 56: Amino acid sequence of Flag-HER3 (E928G) SEQ ID NO: 57: Nucleotide sequence of HER2 IF primer (Fw) SEQ ID NO: 58: Nucleotide sequence of HER2 IF primer (Rev) SEQ ID NO: 59: Nucleotide sequence of the cDNA encoding HER2 SEQ ID NO: 60: Amino acid sequence of HER2 SEQ ID NO: 61: Nucleotide sequence of HER3 primer (Fw) SEQ ID NO: 62: Nucleotide sequence of HER3 primer (Rev) SEQ ID NO: 63: Nucleotide sequence of HER2 primer (Fw) SEQ ID NO: 64: Nucleotide sequence of HER2 primer (Rev) SEQ ID NO: 65: Nucleotide sequence of HER3 mutagenesis primer (Fw) SEQ ID NO: 66: Nucleotide sequence of HER3 mutagenesis primer (Rev) SEQ ID NO: 67: Nucleotide sequence of cDNA encoding Flag-HER3(Q809R) SEQ ID NO: 68: Amino acid sequence of Flag-HER3(Q809R) SEQ ID NO: 69: Amino acid sequence of HER3 protein

Claims

[Claim 1] The inventions described herein.

Images