Method for treating EGFR-TKI-resistant non-small cell lung cancer by administration of Anti-her3 antibody-drug conjugate

Abstract

[Problem] To provide a therapeutic agent and a therapeutic method for EGFR-TKI-resistant non-small cell lung cancer, wherein EGFR-TKI-resistant non-small cell lung cancer is resistant to osimertinib. [Solution] Provided are: a therapeutic agent that contains an anti-HER3 antibody-drug conjugate as an active ingredient, and a therapeutic method characterized by administering an anti-HER3 antibody-drug conjugate in combination with a second drug (osimertinib).

Description

Method for treating EGFR-TKI-resistant non-small cell lung cancer by administering an anti-HER3 antibody-drug conjugate

[0001] The present invention relates to a therapeutic agent and method for EGFR-TKI-resistant non-small cell lung cancer, which comprises administering an anti-HER3 antibody-drug conjugate.

[0002] Treatment using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) is effective against non-small cell lung cancer (NSCLC) with EGFR gene mutations. However, as treatment continues, the target cancer often develops resistance to the inhibitors, resulting in disease progression. Approximately half of cancers resistant to the first-generation EGFR-TKIs gefitinib and erlotinib and the second-generation EGFR-TKI afatinib harbor the T790M mutation in the EGFR gene. Osimertinib, a third-generation EGFR-TKI, is known to be effective against NSCLCs harboring the EGFR T790M mutation (Non-Patent Document 1). However, no optimal drug has yet been approved for osimertinib-resistant NCLC. Furthermore, no optimal drug has yet been approved for non-small cell lung cancer that is resistant to EGFR-TKIs and has been confirmed to be negative for the EGFR T790M mutation.

[0003] 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. Increased expression of HER3 in cancer cells is known to be associated with the development of resistance to EGFR-TKIs (Non-Patent Document 2). Studies are also being conducted to evaluate the effects of anti-HER3 antibodies on non-small cell lung cancer (Non-Patent Document 3).

[0004] Antibody-drug conjugates (ADCs), which combine a cytotoxic drug with an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into the cells, are expected to be able to selectively deliver drugs to cancer cells, thereby accumulating the drug within the cancer cells and killing the cancer cells (Non-Patent Documents 4 to 8).

[0005] One known antibody-drug conjugate is one that comprises an anti-HER3 antibody and exatecan, a topoisomerase I inhibitor, as components (Patent Document 1).

[0006] International Publication No. 2015 / 155998

[0007] I. Sullivan et al. Ther Adv Respir Dis 2016, Vol. 10(6) 549-565.NV Sergina et al. Nature 2007 January 25; 445(7126): 437-441.K Yonesaka et al., Oncogene (2016) 35, 878-886.Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13.Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537.Damle NK Expert Opin. Biol. Ther. (2004) 4, 1445-1452.Senter PD, et al., Nature Biotechnology (2012) 30, 631-637.Howard A. et al., J Clin Oncol 29: 398-405.

[0008] An objective of the present invention is to provide a therapeutic agent and a therapeutic method for EGFR-TKI-resistant non-small cell lung cancer.

[0009] The present inventors have found that anti-HER3 antibody-drug conjugates exhibit excellent antitumor effects against EGFR-TKI-resistant non-small cell lung cancer.

[0010] That is, the present invention provides: [1] a therapeutic agent for EGFR-TKI-resistant non-small cell lung cancer, which comprises an anti-HER3 antibody-drug conjugate as an active ingredient;

[0011] [2] The therapeutic agent according to [1], wherein the non-small cell lung cancer is EGFR T790M mutation-negative non-small cell lung cancer.

[0012] [3] The therapeutic agent according to [1] or [2], wherein the EGFR-TKI is gefitinib, erlotinib, afatinib, or osimertinib.

[0013] [4] The therapeutic agent according to [1] or [2], wherein the EGFR-TKI is osimertinib.

[0014] [5] The therapeutic agent according to [2], wherein the EGFR-TKI is gefitinib, erlotinib, or afatinib.

[0015] [6] The therapeutic agent according to [2], wherein the EGFR-TKI is gefitinib or erlotinib.

[0016] [7] The therapeutic agent according to any one of [1] to [6], wherein the non-small cell lung cancer expresses HER3.

[0017] [8] The anti-HER3 antibody-drug conjugate is represented by the formula

[0018] (wherein A represents the binding site to the anti-HER3 antibody), and the anti-HER3 antibody is linked via a thioether bond to a drug linker represented by the formula:

[0019] [9] The therapeutic agent according to any one of [1] to [8], 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.

[0020]

[10] The therapeutic agent according to any one of [1] to [8], 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.

[0021]

[11] The therapeutic agent according to any one of [1] to [8], 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.

[0022]

[12] The therapeutic agent according to

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

[0023]

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

[12] , 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.

[0024]

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

[12] , 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.

[0025]

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

[14] , which is administered in combination with a second drug.

[0026]

[16] The therapeutic agent according to

[15] , wherein the second drug is gefitinib, erlotinib, afatinib, or osimertinib.

[0027]

[17] The therapeutic agent according to

[16] , wherein the second drug is erlotinib.

[0028]

[18] The therapeutic agent according to

[16] , wherein the second drug is osimertinib.

[0029]

[19] A method for treating EGFR-TKI-resistant non-small cell lung cancer, comprising administering an anti-HER3 antibody-drug conjugate.

[0030]

[20] The method of treatment according to

[19] , wherein the non-small cell lung cancer is EGFR T790M mutation-negative non-small cell lung cancer.

[0031]

[21] The method of treatment according to

[19] or

[20] , wherein the EGFR-TKI is gefitinib, erlotinib, afatinib, or osimertinib.

[0032]

[22] The method of treatment according to

[19] or

[20] , wherein the EGFR-TKI is osimertinib.

[0033]

[23] The method of treatment according to

[20] , wherein the EGFR-TKI is gefitinib, erlotinib, or afatinib.

[0034]

[24] The method of treatment according to

[20] , wherein the EGFR-TKI is gefitinib or erlotinib.

[0035]

[25] The method of any one of

[19] to

[24] , wherein the non-small cell lung cancer expresses HER3.

[0036]

[26] The anti-HER3 antibody-drug conjugate is represented by the formula

[0037] (wherein A represents the binding site to the anti-HER3 antibody), and the anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate in which the drug linker represented by the formula:

[0038]

[27] The method of any one of

[19] 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.

[0039]

[28] The method of any one of

[19] 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.

[0040]

[29] The method of any one of

[19] 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.

[0041]

[30] The method of treatment according to

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

[0042]

[31] The method of any one of

[19] 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.

[0043]

[32] The method of any one of

[19] 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.

[0044]

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

[19] to

[32] , wherein the anti-HER3 antibody-drug conjugate is administered in combination with a second drug.

[0045]

[34] The method of treatment according to

[33] , wherein the second drug is gefitinib, erlotinib, afatinib, or osimertinib.

[0046]

[35] The method of treatment according to

[34] , wherein the second drug is erlotinib.

[0047]

[36] The method of treatment according to

[34] , wherein the second drug is osimertinib.

[0048]

[37] Anti-HER3 antibody-drug conjugate for the treatment of EGFR-TKI-resistant non-small cell lung cancer.

[0049]

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

[37] , wherein the non-small cell lung cancer is EGFR T790M mutation-negative non-small cell lung cancer.

[0050]

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

[37] or

[38] , wherein the EGFR-TKI is gefitinib, erlotinib, afatinib, or osimertinib.

[0051]

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

[37] or

[38] , wherein the EGFR-TKI is osimertinib.

[0052]

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

[38] , wherein the EGFR-TKI is gefitinib, erlotinib, or afatinib.

[0053]

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

[38] , wherein the EGFR-TKI is gefitinib or erlotinib.

[0054]

[43] The anti-HER3 antibody-drug conjugate of any one of

[37] to

[42] , wherein the non-small cell lung cancer expresses HER3.

[0055]

[44] The anti-HER3 antibody-drug conjugate is represented by the formula

[0056] (wherein A represents the binding site to the anti-HER3 antibody), and the anti-HER3 antibody is linked to the drug linker via a thioether bond.

[0057]

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

[37] to

[44] , wherein the anti-HER3 antibody comprises 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.

[0058]

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

[37] to

[44] , 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.

[0059]

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

[37] to

[44] , 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.

[0060]

[48] ​​The anti-HER3 antibody-drug conjugate according to

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

[0061]

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

[37] to

[48] , 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.

[0062]

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

[37] to

[48] , 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.

[0063]

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

[37] to

[50] , which is administered in combination with a second drug.

[0064]

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

[51] , wherein the second drug is gefitinib, erlotinib, afatinib, or osimertinib.

[0065]

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

[52] , wherein the second drug is erlotinib.

[0066]

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

[52] , wherein the second drug is osimertinib.

[0067]

[55] Use of an anti-HER3 antibody-drug conjugate for the manufacture of a medicament for the treatment of EGFR-TKI-resistant non-small cell lung cancer.

[0068]

[56] The use according to

[55] , wherein the non-small cell lung cancer is EGFR T790M mutation-negative non-small cell lung cancer.

[0069]

[57] The use according to

[55] or

[56] , wherein the EGFR-TKI is gefitinib, erlotinib, afatinib, or osimertinib.

[0070]

[58] The use according to

[55] or

[56] , wherein the EGFR-TKI is osimertinib.

[0071]

[59] The use according to

[56] , wherein the EGFR-TKI is gefitinib, erlotinib, or afatinib.

[0072]

[60] The use according to

[56] , wherein the EGFR-TKI is gefitinib or erlotinib.

[0073]

[61] The use according to any one of

[55] to

[60] , wherein the non-small cell lung cancer expresses HER3.

[0074]

[62] The anti-HER3 antibody-drug conjugate is represented by the formula

[0075] (wherein A represents the binding site to the anti-HER3 antibody) and the anti-HER3 antibody are linked via a thioether bond.

[0076]

[63] The use according to any one of

[55] to

[62] , 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.

[0077]

[64] The use according to any one of

[55] to

[62] , 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.

[0078]

[65] The use according to any one of

[55] to

[62] , 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.

[0079]

[66] The use according to

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

[0080]

[67] The use according to any one of

[55] to

[66] , 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.

[0081]

[68] The use according to any one of

[55] to

[66] , 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.

[0082]

[69] The use according to any one of

[55] to

[68] , wherein the anti-HER3 antibody-drug conjugate is administered in combination with a second drug.

[0083]

[70] The use according to

[69] , wherein the second drug is gefitinib, erlotinib, afatinib, or osimertinib.

[0084]

[71] The use according to

[70] , wherein the second drug is erlotinib.

[0085]

[72] The use according to

[70] , wherein the second drug is osimertinib.

[0086] The present invention can provide a therapeutic agent and method for EGFR-TKI-resistant non-small cell lung cancer, which is characterized by administering an anti-HER3 antibody-drug conjugate.

[0087] 1 shows the heavy chain amino acid sequence of anti-HER3 antibody (1). 2 shows the light chain amino acid sequence of anti-HER3 antibody (1). 3 shows the cytostatic activity of HER3-ADC (1) against HCC827 cell line and HCC827GR5 cell line. Error bars in the figure indicate standard error (n=6). 4 shows the HER3 mRNA levels in HCC827 cell line and HCC827GR5 cell line. 5 shows the cytostatic activity of HER3-ADC (1) alone, erlotinib alone, or a combination of HER3-ADC (1) and erlotinib against the HCC827GR5 cell line. Error bars in the figure indicate standard error (n=6). 6 shows the antitumor effect of HER3-ADC (1) alone, erlotinib alone, or a combination of HER3-ADC (1) and erlotinib on the HCC827GR5 cell line implanted in nude mice. Error bars in the figure indicate standard deviation (n=10). Figure 1 shows the cytostatic activity of osimertinib against PC9 cell lines and PC9AZDR7 cell lines. Error bars in the figure indicate standard error (n=6). Figure 2 shows the protein levels of HER3 in PC9 cell lines and PC9AZDR7 cell lines. Error bars in the figure indicate standard deviation (n=3). Figure 3 shows the antitumor effect of HER3-ADC(1) alone against PC9 cell lines transplanted into nude mice. Error bars in the figure indicate standard error (control group n=8, HER3-ADC(1) group n=9), and arrows indicate drug administration. Figure 4 shows the antitumor effect of HER3-ADC(1) alone against PC9AZDR7 cell lines transplanted into nude mice. Error bars in the figure indicate standard error (control group n=8, HER3-ADC(1) group n=9), and arrows indicate drug administration. This figure shows the antitumor effect of the combination of HER3-ADC(1) and osimertinib on the PC9AZDR7 cell line transplanted into nude mice. The error bars in the figure indicate standard error (control group n = 11, HER3-ADC(1) monotherapy group n = 12, osimertinib monotherapy group n = 12, HER3-ADC(1) and osimertinib combination group n = 10), and the arrows indicate drug administration.

[0088] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention should not be construed as being narrow.

[0089] In the present invention, "EGFR-TKI" refers to an EGFR tyrosine kinase inhibitor, and examples thereof include gefitinib, erlotinib, afatinib, and osimertinib.

[0090] In the present invention, gefitinib and erlotinib may be referred to as first-generation EGFR-TKIs, afatinib as second-generation EGFR-TKIs, and osimertinib as third-generation EGFR-TKIs.

[0091] In the present invention, "EGFR-TKI-resistant non-small cell lung cancer" refers to non-small cell lung cancer that has been confirmed to be resistant to EGFR-TKI, as well as non-small cell lung cancer that can be reasonably recognized or predicted to be resistant to EGFR-TKI.

[0092] In the present invention, the term "EGFR T790M mutation" refers to a mutation in which the 790th amino acid, threonine, located in the gatekeeper region of the ATP-binding site of the EGFR tyrosine kinase domain is converted to methionine (Pao W, et al., PLoS Med. 2(3):e73, 2005; Kobayashi S, et al., N Engl J Med. 352(8):786-792, 2005). The presence or absence of the EGFR T790M mutation can be confirmed by collecting tissue or plasma samples from patients with non-small cell lung cancer and using methods such as real-time PCR.

[0093] In the present invention, "EGFR T790M mutation-positive non-small cell lung cancer" refers to non-small cell lung cancer that has been confirmed to be EGFR T790M mutation-positive, as well as non-small cell lung cancer that can be reasonably recognized or predicted to be EGFR T790M mutation-positive.

[0094] EGFR T790M mutation-positive non-small cell lung cancer is thought to exhibit resistance to first-generation and second-generation EGFR-TKIs due to mechanisms such as steric hindrance caused by changes in the conformation of the ATP-binding site of EGFR, and this is observed in approximately half of the cases of resistance to first-generation and second-generation EGFR-TKIs.Osimertinib, a third-generation EGFR-TKI, is known to be an effective drug for EGFR T790M mutation-positive non-small cell lung cancer.

[0095] The optimal drug for osimertinib-resistant non-small cell lung cancer has not yet been approved, and there is an unmet medical need.

[0096] An example of a cell line corresponding to osimertinib-resistant non-small cell lung cancer is the PC9AZDR7 cell line. The PC9AZDR7 cell line is derived from the PC9 human non-small cell lung cancer cell line as a parent line and has acquired resistance to osimertinib. The PC9AZDR7 cell line can be established by the method described in Example 3-1 of the present specification.

[0097] The antitumor effect of a drug against osimertinib-resistant non-small cell lung cancer can be confirmed by testing the in vitro cell growth inhibitory activity against the above cell lines, and the in vivo tumor growth inhibition rate in a model in which the above cell lines are transplanted into nude mice.

[0098] In the present invention, "EGFR T790M mutation-negative non-small cell lung cancer" refers to non-small cell lung cancer that has been confirmed to be EGFR T790M mutation-negative, as well as non-small cell lung cancer that can be reasonably recognized or predicted to be EGFR T790M mutation-negative.

[0099] In cases of EGFR-TKI resistance, cases other than EGFR T790M mutation-positive NSCLC correspond to EGFR T790M mutation-negative NSCLC. Such EGFR T790M mutation-negative NSCLC is thought to have acquired resistance through mutations other than EGFR T790M (e.g., MET gene amplification), but the existence of unknown resistance mechanisms has also been suggested.

[0100] There is currently no approved optimal drug for EGFR T790M mutation-negative non-small cell lung cancer (NSCLC) resistant to EGFR-TKIs, and there is an unmet medical need.

[0101] An example of a cell line corresponding to EGFR T790M mutation-negative non-small cell lung cancer that exhibits resistance to EGFR-TKIs is the HCC827GR5 cell line (Engelman JA et al., Science 2007, 316(5827), 1039-1043). The HCC827GR5 cell line is derived from the human non-small cell lung cancer cell line HCC827 as a parent line and has acquired resistance to the EGFR-TKI gefitinib. In addition, the 11-18 cell line (Proc Natl Acad Sci US A. 2012 Jul 31;109(31):E2127-33) and the Ma70GR cell line (K Yonesaka et al., Oncogene (2016) 35, 878-886) can also be used as cell lines corresponding to EGFR T790M mutation-negative non-small cell lung cancer that is resistant to EGFR-TKIs.

[0102] The antitumor effect of a drug against EGFR T790M mutation-negative non-small cell lung cancer that is resistant to EGFR-TKIs can be confirmed by testing the in vitro cell growth inhibitory activity against the above cell lines, or the in vivo tumor growth inhibition rate in a nude mouse model in which the above cell lines are transplanted.

[0103] In the present invention, "HER3" is synonymous with human epidermal growth factor receptor 3 (sometimes referred to as ErbB3), which is a transmembrane receptor that, together with HER1, HER2, and HER4, belongs to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases. HER3 is expressed in several types of cancer, including breast cancer, gastrointestinal cancer, and pancreatic cancer, and is known to form heterodimers with tyrosine kinase receptors such as EGFR and HER2, thereby undergoing phosphorylation and inducing signals to inhibit cancer cell proliferation and apoptosis.

[0104] 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 synthesized in vitro or produced in host cells by genetic engineering. Specifically, genetic engineering involves incorporating HER3 cDNA into an expression vector and then incubating the vector in a solution containing the enzymes, substrates, and energy sources necessary for transcription and translation to synthesize HER3. Alternatively, other prokaryotic or eukaryotic host cells can be transformed with the vector to express HER3, thereby obtaining the protein. Furthermore, the genetically engineered HER3-expressing cells or cell lines expressing HER3 can also be used as HER3 protein antigens.

[0105] 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 (a precursor containing a signal sequence consisting of 19 amino acid residues at the amino terminus) and M34309 (NCBI).

[0106] 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 that have biological activity equivalent to that of the protein.

[0107] In the present invention, the term "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.

[0108] The anti-HER3 antibodies used in the present invention can be obtained by known means. For example, they can be obtained by immunizing an animal with the antigen HER3 or any polypeptide selected from the amino acid sequence of HER3, using methods commonly used in this field, and collecting and purifying the antibodies produced in the body. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, anti-HER3 antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0109] Alternatively, a monoclonal antibody can be obtained by fusing antibody-producing cells that produce an antibody against an antigen with myeloma cells to establish a hybridoma according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0110] Antigens can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce it. Specifically, a vector capable of expressing the antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above-mentioned genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[0111] 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 to humans, such as a chimeric antibody or a humanized antibody, or is preferably an antibody that has only the genetic sequence of a human-derived antibody, i.e., a human antibody. These antibodies can be produced using known methods.

[0112] Chimeric antibodies include antibodies whose variable and constant regions are heterologous, such as chimeric antibodies in which the variable region of a mouse- or rat-derived antibody is joined to a human-derived constant region (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0113] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody has been incorporated into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies in which not only the CDR sequence of a heterologous antibody but also some framework amino acid residues of the heterologous antibody have been grafted onto a human antibody using a CDR grafting method (WO 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0114] Examples of human antibodies include antibodies produced using human antibody-producing mice carrying human chromosomal fragments containing human antibody heavy and light chain genes (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be used (see, for example, Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427-431).

[0115] The anti-HER3 antibodies used in the present invention also include modified antibodies. The term "modified antibody" refers to an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include those with a chemical moiety attached to the amino acid backbone or an N- or O-linked carbohydrate chain. Biological modifications include those that have undergone post-translational modification (e.g., addition of an N- or O-linked glycan, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, etc.), and those that have been expressed in prokaryotic host cells to add a methionine residue to the N-terminus. Modified antibodies also include those labeled to enable detection or isolation of the anti-HER3 antibodies or antigens used in the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified anti-HER3 antibodies used in the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens.

[0116] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the anti-HER3 antibody used in the present invention. Techniques for modulating antibody sugar chain modification are known, including, but not limited to, those disclosed in International Publication Nos. 99 / 54342, 00 / 61739, and 02 / 31140. Anti-HER3 antibodies used in the present invention also include antibodies with modified sugar chain modification.

[0117] It is known that antibodies produced in cultured mammalian cells have deletions of lysine residues at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (e.g., complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the anti-HER3 antibodies used in the present invention also include antibodies and functional fragments of such antibodies that have undergone such modifications, including deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and amidated deletions (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function are maintained, the carboxyl-terminal deletion 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 type of heavy chain selected from the group consisting of full-length and the above-mentioned deletions, or a combination of any two types. The quantitative ratio of each deletion may be affected by the type and culture conditions of the cultured mammalian cells producing the 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 each of the two heavy chains.

[0118] The isotype of the anti-HER3 antibody used in the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), preferably IgG1 or IgG2. Modified versions of these antibodies can also be used as anti-HER3 antibodies according to the present invention.

[0119] 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), and lumretuzumab. Examples of anti-HER3 antibodies include 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, and modified forms thereof, with patritumab and U1-59 being preferred. These anti-HER3 antibodies can be produced by the methods described in the above-mentioned documents.

[0120] 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 antibody-drug conjugates 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 / 093399, WO 2017 / 093391, WO 2018 / 093392, WO 2019 / 093393, WO 2019 / 093394, WO 2019 / 093395, WO 2019 / 093396, WO 2019 / 093397, WO 2019 / 093398, WO 2019 / 093399, WO 2019 / 093399, WO 2019 / 093391, WO 2019 / 093392, WO 20 Examples of antibody-drug conjugates include those described in WO 2011 / 11 / 130613, WO 2011 / 130616, WO 2013 / 055993, WO 2014 / 057687, WO 2014 / 061277, WO 2014 / 107024, WO 2014 / 134457, and WO 2014 / 145090, preferably those described in WO 2014 / 057687 and WO 2014 / 061277, and more preferably those described in WO 2014 / 057687. These antibody-drug conjugates can be produced by the methods described in the above-mentioned documents.

[0121] The cytotoxic drug is not particularly limited as long as it has an antitumor effect and a substituent or partial structure that can be bound to a linker, but examples thereof include camptothecin, calicheamicin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin, auristatin E, maytansine, paclitaxel, pyrrolobenzodiazepine, and derivatives thereof, with camptothecin derivatives being preferred, and exatecan derivatives being more preferred. The topoisomerase I inhibitor exatecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione)) has the formula

[0122] It is a compound represented by the formula:

[0123] In the present invention, the term "drug linker" refers to the drug and linker moiety in an antibody-drug conjugate, in other words, the partial structure other than the antibody in an antibody-drug conjugate.

[0124] 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 anti-HER3 antibody-drug conjugates 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.

[0125] The anti-HER3 antibody-drug conjugate more preferably used in the present invention is represented by the formula

[0126] (wherein A represents the binding site to the anti-HER3 antibody) and the anti-HER3 antibody are bound via a thioether bond. This drug linker is bound to thiol groups (in other words, sulfur atoms of cysteine ​​residues) generated at disulfide bond sites between antibody chains (two sites between heavy chains and two sites between heavy chains and light chains).

[0127] The above-mentioned anti-HER3 antibody-drug conjugate, which is more preferably used in the present invention, can also be represented by the following formula:

[0128]

[0129] Here, the drug linker is bound to the antibody via a thioether bond, and n is synonymous with the so-called average drug binding number (DAR; Drug-to-Antibody Ratio), which indicates the average number of drug linkers bound per antibody.

[0130] The anti-HER3 antibody-drug conjugate more preferably used in the present invention is one in which the linker moiety is cleaved after the conjugate is transferred into tumor cells, and the conjugate is represented by the formula

[0131] The compound represented by the formula:

[0132] The above compound is believed to be the main component of the antitumor activity of the above anti-HER3 antibody-drug conjugate, which is more preferably used in the present invention, and has been confirmed to have topoisomerase I inhibitory activity (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0133] 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 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; and even more preferably 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 a lysine residue is deleted at the carboxyl terminus of the heavy chain of the above antibody.

[0134] The drug linker intermediate used in the preparation of the above anti-HER3 antibody-drug conjugate is represented by the following formula:

[0135]

[0136] 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 and WO 2015 / 155998, etc.

[0137] The anti-HER3 antibody-drug conjugate preferably used in the present invention can be produced by reacting the aforementioned drug linker intermediate with an anti-HER3 antibody having a thiol group (also referred to as a sulfhydryl group).

[0138] Anti-HER3 antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, G. T., Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, an anti-HER3 antibody can be reacted with a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in an amount of 0.3 to 3 molar equivalents per antibody interchain disulfide in a buffer containing a chelating agent such as ethylenediaminetetraacetic acid (EDTA), thereby obtaining an anti-HER3 antibody having sulfhydryl groups in which the antibody interchain disulfides have been partially or completely reduced.

[0139] Furthermore, 2 to 20 molar equivalents of a drug linker intermediate can be used per anti-HER3 antibody having a sulfhydryl group to produce anti-HER3 antibody-drug conjugates with 2 to 8 drugs bound per antibody.

[0140] The average number of drugs bound per antibody molecule in the produced anti-HER3 antibody-drug conjugate can be calculated, for example, by measuring the UV absorbance of the anti-HER3 antibody-drug conjugate and its conjugation precursor at two wavelengths, 280 nm and 370 nm (UV method), or by treating the antibody-drug conjugate with a reducing agent, and quantifying each of the resulting fragments by HPLC measurement (HPLC method).

[0141] Conjugation of an anti-HER3 antibody to a drug linker intermediate and calculation of the average number of drugs bound per antibody molecule in an anti-HER3 antibody-drug conjugate can be performed with reference to the descriptions in WO 2015 / 155998, etc.

[0142] The average number of drug linkers bonded per antibody in the anti-HER3 antibody-drug conjugate 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.

[0143] The therapeutic agent and method of the present invention are characterized by administering an anti-HER3 antibody-drug conjugate and can be used to treat EGFR-TKI-resistant non-small cell lung cancer. The "non-small cell lung cancer" may be EGFR T790M mutation-negative or EGFR T790M mutation-positive non-small cell lung cancer.

[0144] The "EGFR-TKI" in the "EGFR-TKI-resistant non-small cell lung cancer" is preferably gefitinib, erlotinib, afatinib, or osimertinib, and more preferably osimertinib. When the "non-small cell lung cancer" in the "EGFR-TKI-resistant non-small cell lung cancer" is EGFR T790M mutation-negative non-small cell lung cancer, the "EGFR-TKI" is preferably gefitinib, erlotinib, or afatinib, and more preferably gefitinib or erlotinib.

[0145] The "EGFR-TKI-resistant non-small cell lung cancer" preferably expresses HER3, more preferably highly expresses HER3. HER3 expression can be confirmed, for example, by detecting the HER3 gene product (protein) level using immunohistochemistry (IHC), a flow cytometer, Western blot analysis, or the like, or by detecting the gene transcription level using in situ hybridization (ISH) or quantitative PCR (q-PCR). Whether HER3 is highly expressed can be determined using methods well known to those skilled in the art.

[0146] The therapeutic agents and methods of the present invention may contain one or more other drugs (e.g., second drugs) in addition to the anti-HER3 antibody-drug conjugate used in the present invention. That is, the therapeutic agents of the present invention or the anti-HER3 antibody-drug conjugate used in the present invention can be administered in combination with other drugs, thereby enhancing the anti-cancer effect. The other drugs used for such purposes may be administered to an individual simultaneously with the anti-HER3 antibody-drug conjugate used in the present invention, separately, or sequentially, or may be administered at different administration intervals. The other drugs or second drugs are preferably cancer therapeutic agents. Such a cancer therapeutic agent is not limited as long as it is a drug having antitumor activity, but may be, for example, at least one selected from the group consisting of EGFR-TKI, cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, gemcitabine, capecitabine, irinotecan (CPT-11), etoposide, cyclophosphamide, doxorubicin, vinblastin, and vincristine, and is preferably an EGFR-TKI.

[0147] The above-mentioned EGFR-TKI is preferably gefitinib, erlotinib, afatinib, or osimertinib, more preferably erlotinib or osimertinib, and even more preferably osimertinib.

[0148] The therapeutic agent and method of the present invention can be selected and used as a drug for drug therapy, which is the main treatment for cancer, and as a result, can slow the growth of cancer cells, suppress their proliferation, and even destroy them. These effects can relieve cancer patients from cancer-related symptoms, improve their quality of life, and achieve therapeutic effects while preserving the lives of cancer patients. Even if cancer cells are not destroyed, the inhibition and control of cancer cell proliferation can enable cancer patients to achieve a higher quality of life and longer survival.

[0149] In addition to being used as a drug alone in such drug therapy, the therapeutic agent and method of the present invention can also be used as a drug to be combined with other therapies in adjuvant therapy, and can be combined with surgery, radiation therapy, hormone therapy, etc. Furthermore, it can also be used as a drug for drug therapy in neoadjuvant therapy.

[0150] In addition to the therapeutic uses described above, the therapeutic agent and method of the present invention can also be expected to have a preventive effect, such as suppressing the growth of and even destroying micrometastatic cancer cells. For example, they can be expected to have an effect of suppressing and destroying cancer cells present in body fluids during the metastasis process, and to suppress and destroy microscopic cancer cells immediately after implantation in any tissue. Therefore, they can be expected to have an effect of suppressing and preventing cancer metastasis, particularly after surgical removal of cancer.

[0151] The therapeutic agent and method of the present invention can be applied to patients as a systemic therapy, and also can be applied locally to cancer tissues to be expected to have a therapeutic effect.

[0152] The therapeutic agent and method of the present invention can be preferably used in mammals, and more preferably in humans.

[0153] The therapeutic agents of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. The substances used in the pharmaceutical compositions of the present invention can be appropriately selected from formulation additives and other agents commonly used in this field, depending on the dosage and administration concentration. For example, the pharmaceutical composition typically contains one or more pharmaceutical carriers (e.g., a sterile liquid). Liquids include, for example, water and oils (petroleum, animal, plant, or synthetic). Oils may be, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients can be appropriately selected from those known in the art. The pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation corresponds to the mode of administration.

[0154] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. Routes of introduction can include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, administration of the antibody-drug conjugate is by infusion. Parenteral administration is a preferred route of administration.

[0155] In a representative embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the pharmaceutical composition is in a form to be administered by injection, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical-grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.

[0156] The single dose of the anti-HER3 antibody-drug conjugate used in the present invention is preferably in the range of 1.6 mg / kg to 12.4 mg / kg, more preferably 3.2 mg / kg, 4.8 mg / kg, 6.4 mg / kg, 8 mg / kg, 9.6 mg / kg, or 12.4 mg / kg, and even more preferably 4.8 mg / kg, 6.4 mg / kg, 8 mg / kg, 9.6 mg / kg, or 12.4 mg / kg.

[0157] Furthermore, when the anti-HER3 antibody-drug conjugate used in the present invention is used in combination with a second drug (preferably an EGFR-TKI, more preferably erlotinib or osimertinib, and even more preferably osimertinib), the single dose of the anti-HER3 antibody-drug conjugate used in the present invention is preferably in the range of 0.8 mg / kg to 12.4 mg / kg, more preferably 1.6 mg / kg, 3.2 mg / kg, 4.8 mg / kg, 6.4 mg / kg, 8 mg / kg, 9.6 mg / kg, or 12.4 mg / kg, and even more preferably 3.2 mg / kg, 4.8 mg / kg, 6.4 mg / kg, 8 mg / kg, 9.6 mg / kg, or 12.4 mg / kg.

[0158] The administration interval of the anti-HER3 antibody-drug conjugate used in the present invention is preferably once every week (q1w), once every two weeks (q2w), once every three weeks (q3w), or once every four weeks (q4w), and more preferably once every three weeks (q3w).

[0159] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples. Furthermore, these examples should not be construed as limiting in any sense.

[0160] Example 1: Preparation of antibody-drug conjugate

[0049] According to the production method described in WO 2015 / 155998, an antibody-drug conjugate of the formula:

[0161] An anti-HER3 antibody-drug conjugate (referred to as "HER3-ADC(1)" in the present invention) was produced in which the anti-HER3 antibody was linked to a drug linker represented by the formula: (wherein A represents the binding site to the antibody) via a thioether bond. The average number of drugs bound per antibody in HER3-ADC(1) was 7.6.

[0162] Example 2: Sensitivity test of HER3-ADC(1) against HCC827GR5 cell line

[0163] Example 2-1: Cytostatic activity against HCC827 cell line and HCC827GR5 cell line The HCC827 cell line was cultured in RPMI1640 medium (Sigma) containing R10 medium (10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.)). The HCC827GR5 cell line was cultured in the above medium supplemented with gefitinib at a final concentration of 1 μM. It has been reported that the HCC827GR5 cell line does not exhibit strong sensitivity to single-agent erlotinib or single-agent treatment with anti-HER3 antibody (1) (the antibody moiety of HER3-ADC (1)) (K Yonesaka et al., Oncogene (2016) 35, 878-886). HCC827 and HCC827GR5 cell lines were cultured, detached by trypsinization, and harvested. The cell count was determined and the cells were suspended in RPMI 1640 medium containing 10% fetal bovine serum at a concentration of 100,000 cells / mL. 50 μL of each cell suspension was added to each well of a Sumilon 96-well plate (Sumitomo Bakelite) at 5,000 cells / well. After 3 days of culture, HER3-ADC(1) diluted in R10 medium or R10 medium containing no drug as a negative control was added and cultured (final volume per well: 100 μL, final concentrations of culture medium: 0, 0.0033, 0.01, 0.033, 0.1, 0.33, 1, 3.3, and 10 μg / mL). On the seventh day after treatment, 50 μL of CellTiter Glo (Promega) was added to each well, mixed for 2 minutes using a plate mixer, and then allowed to stand for 30 minutes in the dark. 120 μL of each well was then transferred to a black microplate and the luminescence intensity was measured using a luminometer.

[0164] The cytostatic activity of each drug (% Control) was calculated using the following formula.

[0165] % Control = (mean luminescence value in sample-added wells ÷ mean luminescence value in negative control wells) x 100

[0166] The experiment was carried out in 6 wells for each group.

[0167] The results are shown in Figure 3. Cytostatic activity against the HCC827GR5 cell line was 41.3%, 40.0%, and 50.0%, respectively, in the HER3-ADC(1)-treated groups at 10, 3.3, and 1 μg / mL. On the other hand, no cytostatic activity was observed against the HCC827 cell line at concentrations of 3.3 μg / mL or less.

[0168] These results demonstrate that HER3-ADC(1) exhibits cell growth inhibitory activity against the HCC827GR5 cell line.

[0169] Example 2-2: HER3 mRNA Expression in HCC827 and HCC827GR5 Cell Lines 1. Preparation of Total RNA Total RNA was prepared using the Rneasy Mini Kit (Qiagen). The HCC827 cell line was cultured in RPMI1640 medium (Sigma) containing R10 medium (10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.)). The HCC827GR5 cell line was cultured in the above medium supplemented with gefitinib at a final concentration of 1 μM. After culturing, the HCC827 and HCC827GR5 cell lines were detached by trypsinization, collected, and the cell number in the cell suspension was determined. The cells were then suspended in RPMI1640 medium containing 10% fetal bovine serum. Five million cells of each type were collected and centrifuged. After centrifugation, 600 μL of Buffer RLT (containing 100:1 β-mercaptoethanol) was added, vortexed for 30 seconds, and stored at -80°C. The resulting solution was thawed, loaded into a QIAShredder, and centrifuged at 15,000 rpm for 2 minutes. 600 μL of 70% ethanol was added to the extract, vortexed, and then loaded into a spin column and centrifuged at 12,000 rpm for 15 seconds. 80 μL of DNase(+) (70 μL Buffer RDD, 10 μL DNase I stock solution) was added to the spin column and allowed to stand at room temperature for 15 minutes. 700 μL of Buffer RW was added and centrifuged at ≥10,000 rpm for 15 seconds. The collection tube was replaced, 500 μL of Buffer RPE was added, and the column was centrifuged at ≥10,000 rpm for 15 seconds. The extract was discarded. After adding 500 μL of Buffer RPE and centrifuging for 2 minutes, the tube was transferred to a collection tube, and 100 μL of RNase-free water was added to the spin column, which was then left to stand for 5 minutes. After centrifuging at 12,000 rpm for 15 seconds, the total RNA content in the collection tube was measured.

[0170] 2. cDNA Preparation cDNA was prepared using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). 2 μg of the RNA prepared above was added to a solution prepared with 2x RT Buffer, 20x RT Enzyme Mix, and Nuclease-free H2O to prepare a total volume of 20 μL. After centrifugation to remove air bubbles, the mixture was loaded into a thermal cycler and incubated at 37°C for 60 minutes, then at 95°C for 5 minutes, followed by cooling to 4°C to perform the reverse transcription reaction and prepare cDNA.

[0171] 3. Quantitative PCR Reaction: Quantitative polymerase chain reaction (qPCR) reactions were performed using a MicroAmp Optical 96-well Reaction Plate. 50 ng of the cDNA prepared above was added to the plate, followed by 12.5 μL of Soraris qPCR Master Mix (2x) (Thermo Fisher Scientific), 12.5 μL of Soraris Primer / Probe set (20x) (Thermo Fisher Scientific) for HER3 mRNA amplification, and distilled water. To generate a standard curve for calculating mRNA levels, the same procedure was performed on 200, 100, and 20 ng of cDNA prepared in the same manner from human colon cancer cells HCT116. The plates containing the various samples were loaded into an ABI 7900HT (Applied Biosystems) and incubated at 95°C for 15 minutes, followed by 60 cycles of incubation at 95°C for 15 seconds and 60°C for 60 seconds. After cooling at 4°C for 10 minutes, the fluorescence intensity of each well was measured and the amount of PCR product was quantified to determine the amount of mRNA in each sample.

[0172] The results are shown in Figure 4. The amount of HER3 mRNA in the HCC827GR5 cell line was significantly higher than the amount of HER3 mRNA derived from the HCC827 cell line (student t-test, p<0.05).

[0173] Example 2-3: Cytostatic activity against HCC827GR5 cell line. The HCC827GR5 cell line was cultured in RPMI1640 medium (Sigma) containing R10 medium (10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.)) supplemented with gefitinib at a final concentration of 1 μM. After culturing, the HCC827GR5 cell line was detached by trypsinization, harvested, and the cell number in the cell suspension was determined. The cell suspension was then suspended in RPMI1640 medium containing 10% fetal bovine serum and adjusted to a concentration of 100,000 cells / mL. 50 μL of the cell suspension was added to each well of a Sumilon 96-well plate (Sumitomo Bakelite Co., Ltd.) (5,000 cells / well) and cultured. After 3 days of culture, HER3-ADC (1) diluted solution dissolved in R10 medium (final concentration of culture solution: 0, 0.0033, 0.01, 0.033, 0.1, 0.33, 1, 3.3, 10 μg / mL), erlotinib diluted solution (final concentration of culture solution: 0, 0.0033, 0.01, 0.033, 0.1, 0.33, 1, 3.3, 10 μM), erlotinib (final concentration of culture solution 1 μM) dissolved in R10 medium containing HER3-ADC (1) diluted solution (final concentration of culture solution: 0, 0.0033, 0.01, 0.033, 0.1, 0.33, 1, 3.3, 10 μg / mL), and drug-free R10 medium as a negative control were added, and the final culture volume of each well was 100 μL, and culture was performed. On the seventh day after treatment, 50 μL of CellTiter Glo (Promega) was added to each well, mixed for 2 minutes using a plate mixer, and then allowed to stand for 30 minutes in the dark. 120 μL of each well was then transferred to a black microplate and the luminescence intensity was measured using a luminometer.

[0174] The cytostatic activity (% Control) of each drug against the HCC827GR5 cell line was calculated using the following formula.

[0175] % Control = (mean luminescence value in sample-added wells ÷ mean luminescence value in negative control wells) x 100

[0176] The experiment was carried out in 6 wells for each group.

[0177] The results are shown in Figure 5. Against the HCC827GR5 cell line, HER3-ADC(1) at 10, 3.3, and 1 μg / mL inhibited cell growth by 41.3, 40.0, and 50.0%, respectively, while erlotinib at 10, 3.3, and 1 μg / mL inhibited cell growth by 31.7, 52.6, and 69.2%, respectively. Meanwhile, the combined use of erlotinib at 1 μM and HER3-ADC(1) at 10, 3.3, and 1 μg / mL inhibited cell growth by 3.1, 3.2, and 3.0%, respectively.

[0178] From the above results, HER3-ADC(1) showed higher cell growth inhibitory activity in combination with erlotinib against the HCC827GR5 cell line compared to treatment with HER3-ADC(1) or erlotinib alone.

[0179] Example 2-4: Antitumor effect on HCC827GR5 cell line transplanted into nude mice. The HCC827GR5 cell line was cultured in RPMI1640 medium (Sigma) containing R10 medium (10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.)) and erlotinib at a final concentration of 1 μM. The cells were then detached by trypsin treatment, recovered, and the number of cells in the cell suspension was measured. The cells were then suspended in R10 medium and adjusted to a concentration of 75,000,000 cells / mL. After subcutaneous transplantation of 100 μL (7,500,000 cells) of the prepared cell suspension into the ventral flank of 6-week-old female nude mice (BALB / cAJcl-nu / nu), the average estimated tumor volume of the transplanted tumor was 110 mm. 2Seven days after tumor implantation, mice were divided into groups and drug administration began (Day 0). HER3-ADC(1) was dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg / mL. Erlotinib was dissolved in hydroxypropyl methylcellulose (HPMC) solution at a concentration of 2.75 mg / mL. From Day 0 through Day 49, the monotherapy groups received 200 μL / mouse (10 mg / kg) of the prepared HER3-ADC(1) intraperitoneally once a week (7 doses in total) and 0.18 mL / mouse (25 mg / kg) of the prepared erlotinib orally six times a week (19 doses in total). In the combination therapy group, HER3-ADC(1) and erlotinib were administered at the same doses and schedules as the monotherapy groups from Day 0. A control group was also administered no treatment. Each group consisted of 10 mice. After the start of administration, tumor diameters (longer and shorter diameters) were measured twice a week (days 0, 3, 7, 10, 14, 17, 21, 24, 28, 31, 35, 38, 41, 45, and 49), and the estimated tumor volume for each group was calculated according to the formula below. The mean estimated tumor volume for each group was then calculated.

[0180] Estimated tumor volume (Volume, mm 3 ) = major axis (mm) × minor axis (mm) 2 ÷2

[0181] In addition, the tumor growth inhibition rate of each group compared with the control group was calculated according to the formula shown below.

[0182] Tumor growth inhibition rate (%) = 100 - (mean estimated tumor volume in the treatment group ÷ mean estimated tumor volume in the control group × 100)

[0183] In the control group and the erlotinib monotherapy group, the estimated tumor volume was between 1200 and 1500 mm on day 21. ３ Therefore, the tumor growth inhibition rate (%) was calculated up to the 21st day.

[0184] The results are shown in Tables 1, 2, and Figure 6. Erlotinib was not effective against the HCC827GR5 cell line. In contrast, significant antitumor effects were observed in the HER3-ADC(1) treatment group and the HER3-ADC(1) and erlotinib combination treatment group (Dunnett's Multiple Comparison test p<0.001 at Day 21).

[0185] These results demonstrate that HER3-ADC(1), when used alone or in combination with erlotinib, demonstrated significantly greater antitumor effects against the HCC827GR5 cell line implanted in nude mice than did untreated or erlotinib alone (Dunnett's Multiple Comparison test p<0.001 at Day 21).

[0186]

[0187]

[0188] The results of Example 2 confirmed the antitumor effect of HER3-ADC(1) on the HCC827GR5 cell line. The HCC827GR5 cell line is a cell line derived from the human non-small cell lung cancer cell line HCC827 as a parent line, that has acquired resistance to EGFR-TKIs, and corresponds to non-small cell lung cancer negative for the EGFR T790M mutation.

[0189] These results demonstrate that administration of an anti-HER3 antibody-drug conjugate can provide a therapeutic agent and method for EGFR-TKI-resistant, EGFR T790M mutation-negative non-small cell lung cancer.

[0190] Example 3: Sensitivity test of HER3-ADC(1) on PC9 cell line and PC9AZDR7 cell line

[0191] Example 3-1: Creation of an osimertinib-resistant PC9 cell line The non-small cell lung cancer line PC9 was cultured in RPMI-1640 medium (Sigma) containing 10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.). After adding 1 nM osimertinib to the culture medium, the cells were cultured, and the osimertinib concentration was gradually increased while the cells were subcultured. Finally, the cells were cultured in a medium containing 100 nM osimertinib, thereby establishing an osimertinib-resistant PC9 cell line (PC9AZDR7).

[0192] Example 3-2: Cytostatic Activity Against PC9 and PC9AZDR7 Cell Lines. PC9 and PC9AZDR7 cell lines were cultured in RPMI1640 medium (Sigma) containing 10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.). PC9AZDR7 was cultured with the addition of 100 nM osimertinib. After culturing, PC9 and PC9AZDR7 cell lines were detached by trypsinization, harvested, and the cell number in the cell suspension was measured. The cells were then suspended in RPMI1640 medium containing 2% fetal bovine serum. 50 μL of each cell suspension was added to each well of a Sumilon 96-well plate (Sumitomo Bakelite Co., Ltd.) (10,000 cells / well) and cultured. One day after the start of culture, osimertinib diluted solutions prepared at various concentrations or drug-free RPMI-1640 medium as a negative control were added, and the cells were cultured. The final concentrations of osimertinib were 0, 0.001, 0.0033, 0.01, 0.033, 0.1, 0.33, 1, and 3.3 μM. On day 3 of treatment, 50 μL of CellTiter Glo (Promega) was added to each well, mixed for 2 minutes using a plate mixer, and then allowed to stand for 30 minutes in the dark. 120 μL of aliquots were taken from each well and transferred to a black microplate, and luminescence was measured using a luminometer.

[0193] The cytostatic activity (% of control) of each drug was calculated using the following formula.

[0194] % of Control = (mean luminescence value in sample-added wells ÷ mean luminescence value in negative control wells) x 100

[0195] The experiment was carried out in 6 wells for each group.

[0196] The results are shown in Figure 7. Strong cytostatic activity was observed against the PC9 cell line when osimertinib was added at concentrations of 0.01 μM or higher. On the other hand, no cytostatic activity was observed against the PC9AZDR7 cell line at concentrations of 1 μM or lower.

[0197] These results demonstrate that PC9AZDR7 exhibits drug resistance to osimertinib.

[0198] Example 3-3: HER3 Protein Expression in PC9 and PC9AZDR7 Cell Lines HER3 protein expression in PC9 and PC9AZDR7 cell lines was measured using QIFIKIT (Dako). PC9 and PC9AZDR7 cells were cultured, then cultured with mouse anti-human HER3 antibody (Clone 1B4C3, Dako) or mouse IgG2a isotype control antibody, followed by incubation with FITC-conjugated anti-mouse IgG antibody (Dako). The expression level of HER3 protein in each cell line was measured by measuring the fluorescence intensity of each sample using an LSRFortessaX-20 (BD Biosciences).

[0199] The results are shown in Figure 8. The HER3 protein expression level in PC9 was 5,088 cells / cell, while the expression level in PC9AZDR7 was 15,469 cells / cell, demonstrating more than three times higher HER3 protein expression than in PC9 (unpaired T test, p<0.001).

[0200] These results demonstrate that the expression level of HER3 protein in PC9AZDR7, which was established from the non-small cell lung cancer cell line PC9 and is resistant to osimertinib, is significantly higher than that of the parent cell line PC9.

[0201] Example 3-4: Antitumor effect of HER3-ADC(1) on PC9 and PC9AZDR7 cell lines transplanted into nude mice. PC9 and PC9AZDR7 cell lines were cultured in RPMI1640 medium (Sigma) containing 10% fetal bovine serum and 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.). The PC9AZDR7 cell line was cultured in medium containing osimertinib at a final concentration of 100 nM. After culture, the cells were detached by trypsin treatment and collected. The number of cells in the cell suspension was measured, and each cell suspension was prepared. 100 μL (5,000,000 cells) of each prepared cell suspension was transplanted subcutaneously into the ventral side of 6-week-old female nude mice (BALB / cAJcl-nu / nu), and the average estimated tumor volume of the transplanted tumors was approximately 200 mm. 3 Once the tumor volume reached 100 μL, the mice were divided into groups and drug administration began (Day 0). HER3-ADC(1) was dissolved in phosphate-buffered saline (PBS) to a concentration of 0.6 mg / mL. The prepared HER3-ADC(1) was administered intraperitoneally in a single dose of 100 μL / mouse (3 mg / kg) on ​​Day 0. A control group received PBS alone. The control group consisted of eight mice, and the HER3-ADC(1) group consisted of nine mice. After the start of administration, tumor diameters (longer and shorter diameters) were measured twice weekly, and the estimated tumor volume for each group was calculated according to the formula below. The mean estimated tumor volume for each group was then calculated.

[0202] Estimated tumor volume (Volume, mm 3 ) = major axis (mm) × minor axis (mm) 2 ÷2

[0203] In addition, the tumor growth inhibition rate of each group compared with the control group was calculated according to the formula shown below.

[0204] Tumor growth inhibition rate (%) = 100 - (mean estimated tumor volume in the treatment group ÷ mean estimated tumor volume in the control group × 100)

[0205] The antitumor effect of HER3-ADC(1) on the PC9 cell line is shown in Table 3 and Figure 9, and the antitumor effect of HER3-ADC(1) on the PC9AZDR7 cell line is shown in Table 4 and Figure 10. HER3-ADC(1) was not effective against the PC9 cell line (tumor growth inhibition rate of 17% on Day 21). In contrast, a significant antitumor effect (tumor growth inhibition rate of 72% on Day 18) was observed in the HER3-ADC(1)-treated PC9AZDR7 group (unpaired t-test p<0.05 on Day 18).

[0206]

[0207]

[0208] These results demonstrate that HER3-ADC(1) exhibits significantly greater antitumor activity against the PC9AZDR7 cell line implanted in nude mice than the control group.

[0209] These findings demonstrate that administration of an anti-HER3 antibody-drug conjugate can provide a therapeutic agent and method for osimertinib-resistant non-small cell lung cancer.

[0210] Example 3-5: Antitumor Effect of Combination Use of HER3-ADC(1) and Osimertinib on PC9AZDR7 Cell Line Transplanted in Nude Mice The PC9AZDR7 cell line was cultured in RPMI1640 medium (Sigma) containing 10% fetal bovine serum, 1% penicillin-streptomycin B (Wako Pure Chemical Industries, Ltd.), and 100 nM osimertinib. After culturing, the PC9AZDR7 cell line was detached by trypsin treatment, the cells were collected, the number of cells in the cell suspension was measured, and each cell suspension was prepared. After subcutaneous transplantation of 100 μL (34,000,000 cells) of each prepared cell suspension into the ventral flank of 6-week-old female nude mice (BALB / cAJcl-nu / nu), the average estimated tumor volume of the transplanted tumors was approximately 60 mm. 3Once the HER3-ADC(1) concentration reached 100μL / mouse, mice were assigned to groups (Day 0), and drug administration began the day after group assignment (Day 1). HER3-ADC(1) was dissolved in phosphate-buffered saline (PBS) at a concentration of 0.1 mg / mL. Osimertinib was dissolved in distilled water for injection containing 0.1% dimethyl sulfoxide and 30% polyethylene glycol 300 at a concentration of 0.2 mg / mL. In the single-agent groups, HER3-ADC(1) was administered intraperitoneally at 200μL / mouse (1mg / kg) on ​​Day 1, and osimertinib was administered orally at 100μL / mouse (1mg / kg) on ​​Days 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 19. The HER3-ADC(1) and osimertinib combination group received each drug at the same dose and schedule as the single-agent groups. A control group received no treatment. The control group consisted of 11 mice, the HER3-ADC(1) single-agent group consisted of 12 mice, the osimertinib single-agent group consisted of 12 mice, and the HER3-ADC(1) and osimertinib combination group consisted of 10 mice. After the start of treatment, tumor diameters (longer and shorter diameters) were measured twice a week, and the estimated tumor volume for each group was calculated according to the formula below. The mean estimated tumor volume for each group was then calculated.

[0211] Estimated tumor volume (Volume, mm 3 ) = major axis (mm) × minor axis (mm) 2 ÷2

[0212] In addition, the tumor growth inhibition rate of each group compared with the control group was calculated according to the formula shown below.

[0213] Tumor growth inhibition rate (%) = 100 - (mean estimated tumor volume in the treatment group ÷ mean estimated tumor volume in the control group × 100)

[0214] The results are shown in Table 5 and Figure 11. The HER3-ADC(1) 1 mg / kg and osimertinib 1 mg / kg monotherapy groups did not demonstrate significant efficacy against the PC9AZDR7 cell line (tumor growth inhibition rate at Day 21: HER3-ADC(1) 25.3%, osimertinib 27.7%). In contrast, the HER3-ADC(1) 1 mg / kg and osimertinib 1 mg / kg combination treatment group demonstrated a significant antitumor effect (tumor growth inhibition rate at Day 21: 66.6%; p = 0.0057 vs. the HER3-ADC(1) monotherapy group and p = 0.0092 vs. the osimertinib monotherapy group at Day 21, Dunnett's test).

[0215]

[0216]

[0217] These results demonstrate that combined treatment with HER3-ADC(1) and osimertinib exhibits significantly greater antitumor effects against the PC9AZDR7 cell line transplanted in nude mice than treatment with HER3-ADC(1) or osimertinib alone.

[0218] SEQ ID NO: 1: Amino acid sequence of CDRH1 of anti-HER3 antibody (1) SEQ ID NO: 2: Amino acid sequence of CDRH2 of anti-HER3 antibody (1) SEQ ID NO: 3: Amino acid sequence of CDRH3 of anti-HER3 antibody (1) SEQ ID NO: 4: Amino acid sequence of CDRL1 of anti-HER3 antibody (1) SEQ ID NO: 5: Amino acid sequence of CDRL2 of anti-HER3 antibody (1) SEQ ID NO: 6: Amino acid sequence of CDRL3 of anti-HER3 antibody (1) SEQ ID NO: 7: Amino acid sequence of the heavy chain variable region of anti-HER3 antibody (1) SEQ ID NO: 8: Amino acid sequence of the light chain variable region of anti-HER3 antibody (1) SEQ ID NO: 9: Amino acid sequence of the heavy chain of anti-HER3 antibody (1) SEQ ID NO: 10: Amino acid sequence of the light chain of anti-HER3 antibody (1)

Claims

1. A therapeutic agent for treating EGFR-TKI-resistant non-small cell lung cancer, containing an anti-HER3 antibody-drug conjugate as an active ingredient.

2. The therapeutic agent according to claim 1, wherein the non-small cell lung cancer is an EGFR T790M mutation-negative non-small cell lung cancer.

3. The therapeutic agent according to claim 1 or 2, wherein the EGFR-TKI is gefitinib, erlotinib, afatinib, or osimertinib.

4. The therapeutic agent according to claim 1 or 2, wherein the EGFR-TKI is osimertinib.

5. The therapeutic agent according to claim 2, wherein the EGFR-TKI is gefitinib, erlotinib, or afatinib.

6. The therapeutic agent according to claim 2, wherein the EGFR-TKI is gefitinib or erlotinib.

7. The therapeutic agent according to any one of claims 1 to 6, wherein the non-small cell lung cancer expresses HER3.

8. The anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate in which a drug linker represented by the formula (wherein A represents the binding position to the anti-HER3 antibody) and the anti-HER3 antibody are bound by a thioether bond. The therapeutic agent according to any one of claims 1 to 7.

9. The anti-HER3 antibody is an antibody comprising a heavy chain containing CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, and a light chain containing CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:

6. The therapeutic agent according to any one of claims 1 to 8.

10. The anti-HER3 antibody is an antibody comprising a heavy chain containing a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 7 and a light chain containing a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:

8. The therapeutic agent according to any one of claims 1 to 8.

11. The therapeutic agent according to any one of claims 1 to 8, wherein the anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 9 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

10.

12. The therapeutic agent according to claim 11, wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

13. The therapeutic agent according to any one of claims 1 to 12, wherein the average number of drug linkers per antibody in the anti-HER3 antibody-drug conjugate ranges from 7 to 8.

14. The therapeutic agent according to any one of claims 1 to 12, wherein the average number of drug linkers per antibody in the anti-HER3 antibody-drug conjugate ranges from 7.5 to 8.

15. The therapeutic agent according to any one of claims 1 to 14, which is characterized by being administered in combination with a second agent.

16. The therapeutic agent according to claim 15, wherein the second agent is gefitinib, erlotinib, afatinib, or osimertinib.

17. The therapeutic agent according to claim 16, wherein the second agent is erlotinib.

18. The therapeutic agent according to claim 16, wherein the second agent is osimertinib.

19. A method for treating EGFR-TKI-resistant non-small cell lung cancer, which comprises administering an anti-HER3 antibody-drug conjugate.

20. The method for treatment according to claim 19, wherein the non-small cell lung cancer is non-small cell lung cancer negative for the EGFR T790M mutation.

21. The method for treatment according to claim 19 or 20, wherein the EGFR-TKI is gefitinib, erlotinib, afatinib, or osimertinib.

22. The method for treatment according to claim 19 or 20, wherein the EGFR-TKI is osimertinib.

23. The method for treatment according to claim 20, wherein the EGFR-TKI is gefitinib, erlotinib, or afatinib.

24. The method for treatment according to claim 20, wherein the EGFR-TKI is gefitinib or erlotinib.

25. The treatment method according to any one of claims 19 to 24, wherein non-small cell lung cancer expresses HER3.

26. The anti-HER3 antibody-drug conjugate is an anti-HER3 antibody-drug conjugate in which a drug linker represented by the formula (wherein A represents a binding position with the anti-HER3 antibody) and the anti-HER3 antibody are bound by a thioether bond. The treatment method according to any one of claims 19 to 25.

27. The anti-HER3 antibody comprises a heavy chain comprising CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 3, and a light chain comprising CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:

6. The treatment method according to any one of claims 19 to 26.

28. The anti-HER3 antibody is an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 7 and a light chain comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:

8. The treatment method according to any one of claims 19 to 26.

29. The anti-HER3 antibody is an antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 9 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

10. The treatment method according to any one of claims 19 to 26.

30. The treatment method according to claim 29, wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

31. The treatment method according to any one of claims 19 to 30, wherein the average number of drug linkers per antibody in the anti-HER3 antibody-drug conjugate ranges from 7 to 8.

32. The treatment method according to any one of claims 19 to 30, wherein the average number of drug linkers per antibody in the anti-HER3 antibody-drug conjugate ranges from 7.5 to 8.

33. The treatment method according to any one of claims 19 to 32, wherein the anti-HER3 antibody-drug conjugate is administered in combination with a second agent.

34. The treatment method according to claim 33, wherein the second agent is gefitinib, erlotinib, afatinib, or osimertinib.

35. The treatment method according to claim 34, wherein the second agent is erlotinib.

36. The treatment method according to claim 34, wherein the second agent is osimertinib.

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