Anti-her3 antibody-drug conjugate

Abstract

To provide an antitumor drug having excellent therapeutic effect, which is excellent in terms of antitumor effect and safety.SOLUTION: Provided is an antibody-drug conjugate in which an antitumor compound represented by the following formula is conjugated to an anti-HER3 antibody via a linker having a specific structure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antibody-drug conjugate useful as an anti-tumor drug, in which an anti-HER3 antibody and an anti-tumor drug are linked via a linker structure moiety. [Background technology]

[0002] Antibody-drug conjugates (ADCs), which are constructed by conjugating a cytotoxic drug to an antibody that binds to an antigen that is expressed on the surface of cancer cells and can be internalized into the cells (the antibody bound to the antigen can also be internalized into the cells), are expected to be able to selectively deliver the drug to cancer cells, thereby accumulating the drug within the cancer cells and killing the cancer cells (see Non-Patent Documents 1 to 3). For example, Mylotarg (registered trademark; gemtuzumab ozogamicin), an ADC in which calicheamicin is conjugated to an anti-CD33 antibody, has been approved as a therapeutic agent for acute myeloid leukemia. In addition, Adcetris (registered trademark; brentuximab vedotin), an anti-CD30 antibody conjugated to auristatin E, has recently been approved as a therapeutic agent for Hodgkin's lymphoma and anaplastic large cell lymphoma (see Non-Patent Document 4). Drugs contained in ADCs approved to date target DNA or tubulin.

[0003] Camptothecin derivatives, which are known as antitumor low-molecular-weight compounds that inhibit topoisomerase I and exhibit antitumor activity, are compounds represented by the following formula:

[0004] [ka]

[0005] The antitumor compound represented by the formula (exatecan, 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 water-soluble camptothecin derivative (Patent Documents 1 and 2). Unlike irinotecan, which is currently used clinically, this compound does not require enzyme activation to exhibit its antitumor effect. Furthermore, it inhibits topoisomerase I more effectively than SN-38, the active ingredient of irinotecan, or topotecan, which is also used clinically. It has a strong inhibitory activity and exhibits stronger cytotoxicity against various cancer cells in vitro. It was particularly effective against cancer cells that are resistant to SN-38 and other drugs due to the expression of P-glycoprotein. Furthermore, it has shown a strong antitumor effect in a mouse model in which a human tumor was subcutaneously transplanted, and although clinical trials have been conducted, it has not yet been launched on the market (see Non-Patent Documents 5 to 10). It was unclear whether this would be effective.

[0006] DE-310 is a conjugate in which exatecan is bound to a biodegradable carboxymethyldextran polyalcohol polymer via a GGFG peptide spacer (Patent Document 3). By converting exatecan into a polymeric prodrug, it is possible to maintain high blood retention and, furthermore, DE-310 passively targets tumor sites by utilizing the enhanced permeability of tumor neovasculature and tumor tissue retention. DE-310 utilizes enzymatic cleavage of the peptide spacer, resulting in sustained release of the active compound exatecan and exatecan with glycine attached to the amino group, resulting in improved pharmacokinetics. In non-clinical studies of various tumor models, DE-310 demonstrated greater efficacy than exatecan monotherapy, despite a lower total exatecan content compared to exatecan monotherapy. Clinical trials of DE-310 have demonstrated efficacy in some cases, with reports demonstrating that the active compound accumulates in tumors relative to normal tissues. However, other reports have shown that the accumulation of DE-310 and the active compound in tumors is not significantly different from that in normal tissues, and passive targeting was not observed in humans (see Non-Patent Documents 11-14). As a result, DE-310 was not marketed, and it was unclear whether exatecan would function effectively as a targeting drug.

[0007] As a related compound of DE-310, the structural part represented by -NH-(CH2)4-C(=O)- is replaced with a -GGFG-spacer A complex is also known in which -GGFG-NH-(CH2)4-C(=O)- is inserted between exatecan and exatecan as a spacer structure (Patent Document 4), but the antitumor effect of this complex is completely unknown.

[0008] Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor protein tyrosine kinase that belongs to the EGFR subfamily of receptor protein tyrosine kinases, along with HER1 (EGFR, also known as epidermal growth factor receptor), HER2, and HER4 (see Non-Patent Documents 15-17). Like typical epidermal growth factor receptors, the transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and a carboxyl-terminal phosphorylation domain. In addition to these domains, HER1, HER2, and HER4 possess an intracellular protein tyrosine kinase domain (TKD), whereas HER3 lacks this domain and is not capable of autophosphorylation. The ligand heregulin (HRG) binds to the extracellular domain of HER3 and promotes dimerization with other human epidermal growth factor receptor (HER) family members and transphosphorylation of the intracellular domain, thereby activating the receptor-mediated signaling pathway. Dimerization with HER family members enhances the signaling potential of HER3, providing a means for not only signal diversification but also signal amplification. For example, HER2 / HER3 heterodimers induce one of the most important growth signals in the HER family. HER3 is overexpressed in several types of cancer, including breast, gastrointestinal, and pancreatic cancers. Interestingly, a correlation has been shown between HER2 / HER3 expression and progression from the noninvasive to the invasive stage (see Non-Patent Documents 18-20). Therefore, substances that inhibit HER3-mediated signaling are desired. Anti-HER3 antibodies and their immunoconjugates have been reported in Patent Documents 5-10, respectively. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Publication No. 5-59061 [Patent Document 2] Japanese Patent Application Publication No. 8-337584 [Patent Document 3] International Publication No. 1997 / 46260

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Patent document 8

Patent Document 9

Patent document 10

Non-licensed literature

[0010] [Non-licensed document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-licensed document 2] Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [Non-licensed document 3] Damle NK Expert Opin. Biol. Ther. (2004) 4, 1445-1452.

Non-licensed Document 4

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

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Non-licensed Document 16

[0011] In antibody-based tumor treatment, even if an antibody recognizes an antigen and binds to tumor cells, the antitumor effect may be insufficient, and more effective antitumor antibodies may be required. Furthermore, many antitumor low-molecular-weight compounds have safety issues, such as side effects and toxicity, even if they have excellent antitumor effects, and therefore it is an issue to further improve safety and achieve better therapeutic effects. In other words, an issue of the present invention is to obtain and provide an antitumor drug that has excellent antitumor effect and safety, and has excellent therapeutic effects. [Means for solving the problem]

[0012] The present inventors have found that anti-HER3 antibodies are capable of targeting tumor cells, i.e., antibodies that have the properties of recognizing tumor cells, binding to tumor cells, being internalized by tumor cells, or having cytocidal activity against tumor cells. Therefore, they have developed antibody-drug conjugates in which the antitumor compound exatecan is bound to the antibody via a linker structure. By converting the compound into a HER3 antibody, it is possible to more reliably transport the antitumor compound to tumor cells, thereby enabling the compound's antitumor effect to be exerted specifically on tumor cells, thereby ensuring the antitumor effect and enhancing the cytocidal effect of the anti-HER3 antibody. Furthermore, it is possible to reduce the dose of the antitumor compound compared to when the compound is administered alone, which means that the effect of the antitumor compound on normal cells can be alleviated, thereby achieving greater safety. To this end, the inventors created a linker with a specific structure, and succeeded in obtaining an antibody-drug conjugate in which an anti-HER3 antibody and exatecan are linked via this linker. They also found that this conjugate exhibits excellent antitumor effects, thereby completing the present invention.

[0013] That is, the present invention is [1] The following formula [ka] and an anti-HER3 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- or -L 1 -L 2 -L P - The present invention relates to an antibody-drug conjugate characterized in that the antibody-drug conjugate is linked via a thioether bond formed at the disulfide bond present in the hinge region of the anti-HER3 antibody via a linker having the structure shown in

[0014] Here, the anti-HER3 antibody is L 1 The antitumor compound binds to the nitrogen atom of the amino group at position 1, and the ... 2 A carbonyl group in the -C(=O)- moiety or L P At the C-terminus of Combine. In the formula, n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L 1 is -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-, where n 3 denotes an integer from 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P indicates a peptide residue consisting of 2 to 7 amino acids, L a represents -O- or a single bond, -(Succinimid-3-yl-N)- has the following formula: [ka] The structure is represented by the following formula: wherein the 3-position of this structure binds to the anti-HER3 antibody, and the 1-position nitrogen atom binds to the methylene group in the linker structure containing this.

[0015] Furthermore, the present invention also relates to the following: [2]L P The antibody-drug conjugate according to [1], wherein the peptide residue is a peptide residue consisting of an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. [3]L Pis a peptide residue selected from the group consisting of: -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-; Here, "(D-)D" refers to D-aspartic acid. [4]L P is a peptide residue consisting of 4 or 5 amino acids. [5]L P is -GGFG- or -DGGFG-. Conjugates. [6]L P The antibody-drug conjugate according to any one of [1] to [4], wherein -GGFG-.

[0016] [7]n 3 is an integer from 2 to 5, and L 2 [6] The antibody-drug conjugate according to any one of [1] to [6], wherein [8] The linker is -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 The antibody-drug conjugate according to any one of [1] to [7], wherein -C(=O)-. [9]n 3 is an integer from 2 to 5, and L 2 -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4The antibody-drug conjugate according to [8], wherein R is 2 or 4.

[10] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is a moiety with a chain length of 4 to 7 atoms The antibody-drug conjugate according to [8] or [9].

[11] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is a moiety having a chain length of 5 or 6 atoms The antibody-drug conjugate according to [8] or [9].

[12] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is -NH-CH2CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or

[10] or

[11] , wherein the antibody-drug conjugate is -NH-CH2CH2-O-CH2-C(=O)-. to.

[13] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)- The antibody-drug conjugate according to

[12] ,

[14] The linker is -L 1 -L 2 -L P The antibody-drug conjugate according to any one of [1] to [5],

[15] LP The antibody-drug conjugate according to

[14] , wherein is -DGGFG-.

[16] n 3 is an integer from 2 to 5, and L 2

[15] The antibody-drug conjugate according to

[15] , wherein

[0017]

[17] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- or -L 1 -L 2 -L P The antibody-drug conjugate according to [1], wherein the drug-linker structure moiety linking the antibody to the antibody-drug conjugate is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0018] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the following formula: wherein the 3-position of this structure binds to the anti-HER3 antibody, and the 1-position nitrogen atom binds to the methylene group in the linker structure containing this. -(NH-DX) is represented by the following formula: [ka] This represents a group in which the nitrogen atom of the amino group at position 1 is the binding site. -GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly-, and -DGGFG- represents a pentapeptide residue of -Asp-Gly-Gly-Phe-Gly-.

[0019]

[18] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 The antibody-drug conjugate according to [1], wherein the drug-linker structure moiety in which the drug is bound to -C(=O)- is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0020] Here, -(Succinimid-3-yl-N)-, -(NH-DX), -GGFG-, and -DGGFG- are as defined above.

[0021]

[19] The following formula: [ka] and an anti-HER3 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- An antibody-drug conjugate characterized in that the antibody-drug conjugate is linked via a thioether bond formed at the disulfide bond present in the hinge region of the anti-HER3 antibody via a linker having the structure shown in Here, the anti-HER3 antibody is L 1 The antitumor compound binds to the -(CH2)n 2 It bonds to the carbonyl group of the -C(=O)- moiety. In the formula, n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L 1 is -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-, where n 3 denotes an integer from 2 to 8, L 2 is -NH-(CH2CH2-O)n 4represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P represents the tetrapeptide residue of -GGFG-, L a represents -O- or a single bond, -(Succinimid-3-yl-N)- has the following formula: [ka] The structure is represented by the following formula: wherein the 3-position of this structure binds to the anti-HER3 antibody, and the 1-position nitrogen atom binds to the methylene group in the linker structure containing this.

[0022]

[20] n 1 is 3 and n 2 is 0 and n 3 is 2 and L 2 -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4 is 2 and L a is a single bond or n 1 is 1 and n 2 is 1 and n 3 is 5 and L 2 is a single bond, and L a is -O-, or teeth n 1 is 2 and n 2 is 1 and n 3 is 5 and L 2 is a single bond, and L a An antibody-drug conjugate according to

[0019] , wherein is -O-.

[21] n 3 is 2 or 5, and L 2 The antibody-drug conjugate according to

[19] or

[20] , wherein

[22] n 3 is 2 or 5, and L 2 -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n4 The antibody-drug conjugate according to

[19] or

[20] , wherein

[23] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)- The antibody-drug conjugate according to any one of

[19] to

[22] ,

[0023]

[24] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 The antibody-drug conjugate according to any one of

[19] to

[23] , wherein the drug-linker structure moiety in which the drug is bound to -C(=O)- is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX);

[0024] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the following formula: wherein the 3-position of this structure binds to the anti-HER3 antibody, and the 1-position nitrogen atom binds to the methylene group in the linker structure containing this. -(NH-DX) is represented by the following formula: [ka] This represents a group in which the nitrogen atom of the amino group at position 1 is the binding site. -GGFG- indicates the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[0025]

[25] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 The antibody-drug conjugate according to any one of

[19] to

[23] , wherein the drug-linker structure moiety in which the drug is bound to -C(=O)- is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). Here, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- are as defined above.

[0026]

[26] The antibody-drug conjugate according to any one of [1] to

[25] , wherein the average number of selected drug-linker structures bound per antibody is in the range of 1 to 10.

[27] The antibody-drug conjugate according to any one of [1] to

[25] , wherein the average number of selected drug-linker structures bound per antibody is in the range of 2 to 8.

[28] The antibody-drug conjugate according to any one of [1] to

[25] , wherein the average number of selected drug-linker structures bound per antibody is in the range of 3 to 8.

[0027]

[29] A pharmaceutical comprising the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof, or a hydrate thereof.

[30] An antitumor drug and / or anticancer drug comprising the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof, or a hydrate thereof.

[31] The antitumor drug and / or anticancer drug according to

[30] , for use in lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulva cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[32] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof, or a hydrate thereof as an active ingredient, and a pharmaceutically acceptable formulation ingredient.

[33] The pharmaceutical composition according to

[32] , for use in lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulva cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[34] A method for treating tumors and / or cancers, comprising administering the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof, or a hydrate thereof.

[35] The pharmaceutical composition according to

[29] , which is administered in combination with other drugs, or the antitumor drug and / or anticancer drug according to

[30] or

[31] , or the pharmaceutical composition according to

[32] or

[33] . Or the treatment method described in

[34] .

[36] The pharmaceutical composition according to

[32] or

[33] , which also contains another drug as an active ingredient.

[0028]

[35] A compound represented by the formula: (maleimid-N-yl)-(CH2)n 3-C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) or (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -(NH-DX) with an anti-HER3 antibody or a reactive derivative thereof to form a thioether bond at the disulfide bond present in the hinge region of the antibody, thereby binding a drug-linker moiety to the antibody.

[0029] In the formula, n 3 denotes the integers 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L a represents -O- or a single bond, (maleimid-N-yl)- is a group having the following formula: [ka] The nitrogen atom is the bonding site. -(NH-DX) is represented by the following formula: [ka] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in

[0030]

[36] The method of producing according to

[35] , wherein the method of binding the drug-linker moiety to the anti-HER3 antibody is a method of converting the antibody into a reactive derivative by reduction treatment.

[0031]

[37] The method of producing according to

[35] or

[36] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 1 to 10.

[38] The method of producing according to

[35] or

[36] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 2 to 8.

[39] The method of producing according to

[35] or

[36] , wherein the average number of selected drug-linker structures bound per antibody is in the range of 3 to 8.

[40] An antibody-drug conjugate obtained by any one of the production methods described in

[35] to

[39] .

[0032]

[41] A method for producing a thioester at the disulfide bond in the hinge region of an anti-HER3 antibody, characterized by treating the antibody under reducing conditions and then reacting it with a compound selected from the following group of compounds: Antibody-drug conjugates obtained by forming ether bonds: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0033] where (maleimid-N-yl)- is a group represented by the following formula: [ka] The nitrogen atom is the bonding site. -(NH-DX) is represented by the following formula: [ka] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in -GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly-, and -DGGFG- represents a pentapeptide residue of -Asp-Gly-Gly-Phe-Gly-.

[0034]

[42] An antibody-drug conjugate obtained by treating an anti-HER3 antibody under reducing conditions and then reacting the antibody with a compound selected from the following group of compounds to form a thioether bond at the disulfide bond in the hinge region of the antibody: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX). Here, (maleimid-N-yl)-, -(NH-DX), and -GGFG- are as defined above.

[0035]

[43] The antibody-drug conjugate according to

[41] or

[42] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 1 to 10.

[44] The antibody-drug conjugate according to

[41] or

[42] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 2 to 8.

[45] The antibody-drug conjugate according to

[41] or

[42] , wherein the average number of selected drug-linker structures bound per antibody is in the range of 3 to 8.

[46] A pharmaceutical comprising the antibody-drug conjugate according to any one of

[40] to

[45] , a salt thereof, or a hydrate thereof.

[47] An antitumor drug and / or anticancer drug comprising the antibody-drug conjugate according to any one of

[40] to

[45] , a salt thereof, or a hydrate thereof.

[48] ​​The antitumor and / or anticancer agent according to

[47] , for use in lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulva cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[49] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of

[40] to

[45] , a salt thereof, or a hydrate thereof as an active ingredient, and a pharmaceutically acceptable formulation ingredient.

[50] The pharmaceutical composition according to

[49] , for use in treating lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulva cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[51] A method for treating tumors and / or cancers, comprising administering the antibody-drug conjugate, a salt thereof, or a hydrate thereof according to any one of

[40] to

[45] .

[52] The pharmaceutical composition according to

[46] , which is administered in combination with other drugs, or the antitumor drug and / or anticancer drug according to

[47] or

[48] , or the pharmaceutical composition according to

[49] or

[50] . Or the treatment method described in

[51] .

[53] The pharmaceutical composition according to

[49] or

[50] , which also contains another drug as an active ingredient. [Effects of the Invention]

[0036] Anti-HER3 antibody-drug conjugates in which the antitumor compound exatecan is bound via a linker with a specific structure can achieve excellent antitumor efficacy and safety. [Brief explanation of the drawings]

[0037] [Figure 1] 1 shows the full-length amino acid sequence of the anti-HER3 human antibody U1-59 heavy chain (SEQ ID NO: 583). [Figure 2] 1 shows the full-length amino acid sequence of the anti-HER3 human antibody U1-59 light chain (SEQ ID NO: 584). [Figure 3]The mean fluorescence intensity of HCC1569 treated with serial dilutions of U1-59 or antibody-drug conjugates is shown. KD and Bmax values ​​were calculated using GraphPad Prism Software. [Figure 4] A549 cells were cultured with U1-59 or various antibody-drug conjugates for 2 days. HER3 or phosphorylated HER3 was assessed by Western blotting. Pan-Actin was detected as a migration control. [Figure 5] The graph shows the mean reduction in HER3 expression on the surface of HCC1569 cells by treatment with U1-59 or antibody-drug conjugate (37°C, 1 hour). [Figure 6] This shows the results of a test to test the inhibition of proliferation and survival signals by HER3 antibody-drug conjugates in a human breast cancer cell line (HCC 1569). A: Cell proliferation and survival induced by antibody-drug conjugates in the presence of 10% FBS. Data show the mean ± standard error of triplicates. The vertical axis shows the luminescence value, which represents the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. B: The percentage decrease in luminescence due to antibody-drug conjugate treatment is shown, with the untreated group set at 100%. [Figure 7] This shows the results of a test to test the inhibition of proliferation and survival signals by HER3 antibody-drug conjugates in a human breast cancer cell line (MDA-MB 453). A: Cell proliferation and survival induced by antibody-drug conjugates in the presence of 10% FBS. The vertical axis shows the luminescence value, which represents the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. B: The percentage reduction in luminescence due to antibody-drug conjugate treatment is shown, with the untreated group set at 100%. [Figure 8]This shows the results of a test to test the inhibition of proliferation and survival signals by HER3 antibody-drug conjugates in a human melanoma cell line (A375). A: Cell proliferation and survival induced by antibody-drug conjugates in the presence of 10% FBS. The vertical axis shows the luminescence value, which represents the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. B: The percentage reduction in luminescence due to antibody-drug conjugate treatment is shown, with the untreated group set at 100%. [Figure 9] This shows the results of a test to test the inhibition of proliferation and survival signals by HER3 antibody-drug conjugates in a human colon cancer cell line (HT 29). A: Cell proliferation and survival induced by antibody-drug conjugates in the presence of 10% FBS. The vertical axis shows the luminescence value, which represents the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. B: The percentage reduction in luminescence due to antibody-drug conjugate treatment is shown, with the untreated group set at 100%. [Figure 10] This shows the results of a test to test the inhibition of proliferation and survival signals by HER3 antibody-drug conjugates in a human lung cancer cell line (A549). A: Cell proliferation and survival induced by antibody-drug conjugates in the presence of 10% FBS. The vertical axis shows the luminescence value, which represents the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. B: The percentage reduction in luminescence due to antibody-drug conjugate treatment is shown, with the untreated group set at 100%. [Figure 11] The graph shows a comparison of the cell proliferation and viability inhibition rates of antibody-drug conjugate (3) and antibody-drug conjugate (4). The left graph shows the cell proliferation and viability inhibition rates induced by the antibody-drug conjugate in the presence of 10% FBS. The vertical axis shows the luminescence indicating the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. The right graph compares the rate of luminescence reduction due to antibody-drug conjugate treatment between high drug loading (HDL) and medium drug loading (MDL), with the untreated group set at 100%. [Figure 12]The graph shows a comparison of the cell proliferation and viability inhibition rates of antibody-drug conjugate (10) and antibody-drug conjugate (11). The left graph shows the cell proliferation and viability inhibition rates induced by the antibody-drug conjugate in the presence of 10% FBS. The vertical axis shows the luminescence indicating the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. The right graph compares the rate of luminescence reduction due to antibody-drug conjugate treatment between high drug loading (HDL) and medium drug loading (MDL), with the untreated group set at 100%. [Figure 13] The graph shows a comparison of the cell proliferation and viability inhibition rates of antibody-drug conjugate (13) and antibody-drug conjugate (14). The left graph shows the cell proliferation and viability inhibition rates induced by the antibody-drug conjugate in the presence of 10% FBS. The vertical axis shows the luminescence indicating the ATP activity of each sample, and the horizontal axis shows the concentration of each antibody-drug conjugate. Data are shown as the mean ± standard error of triplicates. The right graph compares the rate of luminescence reduction due to antibody-drug conjugate treatment between high drug loading (HDL) and medium drug loading (MDL), with the untreated group set at 100%. [Figure 14] The results of an antitumor study of human breast cancer (HCC1569) using antibody-drug conjugates (3), (10), or (13) are shown. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 15] The results of an antitumor study of human melanoma (HT-144) using antibody-drug conjugates (3), (10), or (13) are shown. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 16]The results of an antitumor study of human breast cancer (MDA-MB-453) using antibody-drug conjugates (3), (10), or (13) are shown. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days since administration. All values ​​are expressed as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 17] The results of an antitumor test using antibody-drug conjugates (3), (10), or (13) against a human colon cancer cell line (HT-29) are shown. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days since administration. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 18] The results of an antitumor test using antibody-drug conjugates (3), (10), or (13) against a human lung cancer cell line (A549) are shown. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are expressed as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 19] This figure shows the results of an antitumor study of the antibody-drug conjugate (13) against a human triple-negative breast cancer cell line (MDA-MB-468). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 20] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human luminal breast cancer line (MCF-7). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 21]This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human melanoma line (WM-266-4). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 22] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human ovarian cancer line (OVCAR-8). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 23] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human bladder cancer line (SW-780). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 24] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human breast cancer line (MDA-MB-453). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 25] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human breast cancer line (MDA-MB-453). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 26]The results of an antitumor study of the antibody-drug conjugate (15) against a human breast cancer line (JIMT-1) are shown. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell implantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 27] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human lung cancer cell line (PC9). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 28] This figure shows the results of an antitumor study of antibody-drug conjugate (16a) against a human triple-negative breast cancer cell line (MDA-MB-468). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 29] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) against a human head and neck cancer line (Fadu). The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). [Figure 30] This figure shows the results of an antitumor test using antibody-drug conjugate (16a) on tumor fragments (NIBIO-G016) derived from a human gastric cancer patient. The vertical axis represents the average tumor volume, and the horizontal axis represents the number of days from cell transplantation. All values ​​are shown as mean ± standard error. Initial tumor volume and initial mouse weight were analyzed using Microsoft Excel 2009 using descriptive data (mean and standard error). DETAILED DESCRIPTION OF THE INVENTION

[0038] Preferred embodiments of the present invention will now be described with reference to the drawings. 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.

[0039] The present invention provides a HER3-binding protein-drug conjugate. The HER3-binding protein of the present invention is preferably a scaffold protein or an antibody having antibody-like binding activity, i.e., an anti-HER3 antibody.

[0040] The anti-HER3 antibody-drug conjugate of the present invention is an antitumor drug in which an antitumor compound is bound to an anti-HER3 antibody via a linker structure moiety, and will be described in detail below. Within the context of the present invention, the term "scaffold protein" as used herein means a polypeptide or protein with an exposed surface that is highly tolerant of amino acid insertions, substitutions, or deletions. An example of a scaffold protein that can be used in accordance with the present invention is the Staphylococcus aureus scaffold protein. Protein A obtained from Pieris brassicae ), bilin-binding proteins or other lipocalins, ankyrin repeat proteins, and human fibronectin (reviewed in Binz and Pluckthun, Curr Opin Biotechnol. 16, 459-69). Engineering of scaffold proteins involves the addition of structural frameworks onto or to the structure of stably folded proteins. The term "scavenger" can be considered as grafting or incorporating affinity functions into a synthetic framework. By affinity function is meant protein binding affinity according to the present invention. The scaffold is structurally separable from the amino acid sequence that confers binding specificity. In general, proteins that appear suitable for the development of such artificial affinity reagents can be obtained by inference or by combinatorial protein engineering techniques (these techniques are known in the art (Skerra, J. Mol. Recog., 2000; Binz and Pluckthun, 2005)) against binders of in vitro displayed artificial scaffold libraries, such as panning, most commonly against HER3 (either purified protein or protein displayed on the cell surface). Furthermore, it is possible to develop scaffolds with antibody-like binding activity. The field protein can be derived from an acceptor polypeptide containing a scaffold domain, and the binding domain of the donor polypeptide can be grafted onto the acceptor polypeptide to confer the binding specificity of the donor polypeptide onto the scaffold domain containing the acceptor polypeptide. The inserted binding domain can be, for example, the complementarity-determining region (CDR) of an antibody, particularly an anti-HER3 antibody. Insertion can be accomplished by various methods known to those skilled in the art, such as polypeptide synthesis, nucleic acid synthesis of the encoding amino acid, or various forms of recombinant methods well known to those skilled in the art.

[0041] [antibody] Furthermore, the term "antibody" or "anti-HER3 antibody" as used herein refers to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody (Jones et al., Nature 321(1986),522-525; Riechmann ... t al., Nature 332(1988),323-329; and Presta, Curr. Op. Struct. Biol.2(1992),593-596), chimeric antibodies (Morrison et al., Proc. Natl. Acad. Sci. USA 81(1984),6851-6855), human antibodies and fully human antibodies (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, 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, pp. 722-727; WO 2007 / 077028, etc.), a multispecific antibody (e.g., a bispecific antibody) formed from at least two antibodies, or an antibody fragment thereof. The term "antibody fragment" includes any part of the above-mentioned antibody, preferably the antigen-binding region or variable region thereof. Examples of antibody fragments include Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, diabodies, etc. Hollinger et al., Proc.Natl.Acad.Sci.U .SA90(1993), 6444-6448), single chain antibody molecules (Pluckthun in:The Pharmacology of Monoclonal Antib odies 113, Rosenburg and Moore, EDS, Springer Verlag, NY (1994), 269-315) and the desired targeting of HER3 Other fragments are included as long as they exhibit binding ability.

[0042] Furthermore, the term "antibody" or "anti-HER3 antibody" as used herein may include antibody-like molecules containing engineered subdomains of antibodies or naturally occurring antibody variants. These antibody-like molecules may be obtained from natural sources such as camelids (Muyldermans et al., Reviews in Molecular Biotechnology 74, 277-302), or through in vitro display of libraries derived from humans, camelids, or other species. (Holt et al.,Trends Biotechnol.,21,484-90 ) It may be a single domain antibody, such as a VH-only or VL-only domain.

[0043] In the present invention, an "Fv fragment" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. The three CDRs of each variable domain are represented by the V H -V L It is in this configuration that they interact to define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although generally with lower affinity than the entire binding site. The "Fab fragment" also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The "Fab fragment" is similar to the "Fab' fragment" in that it has a few additional residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. "F(ab')2 fragments" are different from "Fab' fragments." "F(ab')2 fragments" are initially produced as pairs of "Fab' fragments" that have hinge cysteines between them. Such antibody fragments can be obtained by papain or pepsin digestion, etc. Methods for preparation are known to those skilled in the art.

[0044] In another preferred embodiment of the present invention, the anti-HER3 antibody of the present invention is an anti-HER3 antibody directed against the extracellular domain (ECD) of HER3.

[0045] The anti-HER3 antibody used in the anti-HER3 antibody-drug conjugate of the present invention may be derived from any species, but preferably includes human, rat, mouse, and rabbit. When derived from a species other than human, it is preferable to chimerize or humanize it using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but monoclonal antibodies are preferred. Anti-HER3 antibodies are antibodies that can target tumor cells; that is, they have the properties of being able to recognize tumor cells, being able to bind to tumor cells, being taken up and internalized within tumor cells, and having cytocidal activity against tumor cells. Antibody-drug conjugates can be formed by binding to compounds with anti-tumor activity via a linker. The binding of antibodies to tumor cells can be confirmed using flow cytometry. The uptake of antibodies into tumor cells can be assessed using two assays: (1) an assay in which a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody is used to visualize the antibody taken up into the cells using a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761); and (2) an assay in which a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody is used to visualize the antibody taken up into the cells. (3) an assay that measures the amount of fluorescence incorporated into the cells (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (4) an immunotoxin that binds to the therapeutic antibody. The Mab-ZAP assay (BioTechniques 28:162-165, January 2016) demonstrated that when the toxin is taken up into cells, it is released and inhibits cell proliferation. 2000). A recombinant conjugate protein consisting of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin. The antitumor activity of an antibody can be confirmed in vitro by measuring its inhibitory activity against cell proliferation. For example, cancer cell lines overexpressing the antibody's target protein are cultured, and the antibody is added to the culture system at various concentrations to measure the inhibitory activity against focus formation, colony formation, and spheroid growth. In vivo, for example, tumor cells overexpressing the target protein can be cultured. Antitumor activity can be confirmed by administering the antibody to nude mice transplanted with the cell line and measuring changes in the cancer (tumor) cells. Because antibody-drug conjugates are conjugated with compounds that exert antitumor effects, it is preferable, but not essential, that the antibody itself possess antitumor effects. For the purpose of specifically and selectively exerting the cytotoxicity of antitumor compounds on tumor cells, it is important and preferable that the antibody has the property of being internalized and transported into tumor cells.

[0046] Anti-HER3 antibodies can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies that can be applied to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens. 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)). 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. The anti-HER3 antibody can be obtained by known means.

[0047] There are no particular limitations on the anti-HER3 antibodies that can be used in the present invention, but for example, those having the following properties are desirable. (1) An anti-HER3 antibody characterized by the following properties: (a) Binds specifically to HER3. (b) The antibody has the activity of being internalized into HER3-expressing cells by binding to HER3. (2) The antibody according to (1) above, which binds to the extracellular domain of HER3. (3) The antibody according to (1) or (2) above, which is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, or a human or fully human (monoclonal) antibody. (6) An antibody according to any one of (1) to (5) above, which is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 2. (7) The antibody according to any one of (1) to (6) above, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted. (8) The antibody described in (7) above, comprising a heavy chain variable region represented by the amino acid sequence set forth in SEQ ID NO: 70 and a light chain variable region represented by the amino acid sequence set forth in SEQ ID NO: 72. (9) An antibody obtained by a method for producing the antibody, which comprises the steps of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody described in any one of (1) to (8) above, and collecting the antibody of interest from the culture obtained in the step.

[0048] The anti-HER3 antibody used in the present invention will be described below. In this specification, the terms "cancer" and "tumor" are used interchangeably. As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA and its cRNA. As used herein, the term "polynucleotide" is used interchangeably with nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers. In this specification, the terms "polypeptide" and "protein" are used interchangeably. As used herein, the term "cells" includes cells within an animal body and cultured cells. As used herein, "HER3" is used synonymously with HER3 protein. As used herein, "CDR" refers to a complementarity determining region (CDR). The term "CDR" refers to a CDR (Correlation Determining Region). It is known that the heavy and light chains of an antibody molecule each have three CDRs. CDRs, also known as hypervariable domains, are located in the variable regions of the heavy and light chains of an antibody and are regions with particularly high variability in their primary structure, and are separated into three regions in the primary structure of the heavy and light polypeptide chains. In this specification, with regard to antibody CDRs, the heavy chain CDRs are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence, and the light chain CDRs are referred to as CDRL1, CDRL2, and CDRL3 from the amino-terminal side of the light chain amino acid sequence. These regions are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind. In the present invention, "hybridizing under stringent conditions" means hybridizing at 68°C in a commercially available hybridization solution, ExpressHyb Hybridization Solution (Clontech), or hybridizing using a DNA-immobilized filter at 68°C in the presence of 0.7-1.0 M NaCl, followed by washing at 68°C using a 0.1-2x SSC solution (1x SSC consists of 150 mM NaCl and 15 mM sodium citrate), under conditions that allow identification, or hybridizing under equivalent conditions.

[0049] 1.HER3 HER3, human epidermal growth factor receptor 3 (also known as HER3 or ErbB3), is a receptor protein tyrosine kinase that, along with HER1, HER2, and HER4, belongs to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases. HER3 is a transmembrane receptor and contains an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD), and a carboxyl-terminal phosphorylation domain. HER3 is overexpressed in several types of cancer, including breast, gastrointestinal, and pancreatic cancers. HER2-HER3 expression correlates with progression from noninvasive to invasive cancers. The HER3 protein used in the present invention can be directly purified from HER3-expressing cells of humans or non-human mammals (rats, mice, etc.), or can be prepared as a cell membrane fraction of the cells. HER3 can also be synthesized in vitro or produced in host cells by genetic engineering. Specifically, genetic engineering can be used to synthesize HER3. After inserting the cDNA into an expression vector, the protein can be obtained by synthesizing it in a solution containing the enzymes, substrates, and energy sources necessary for transcription and translation, or by expressing HER3 by transforming other prokaryotic or eukaryotic host cells. HER3-expressing cells or cell lines expressing HER3 obtained by the above-mentioned genetic manipulation can also be used as the HER3 protein. 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). Furthermore, HER3 also includes proteins that have the same biological activity as the above-mentioned HER3 protein and are formed by substituting, deleting, adding, and / or inserting one or more amino acids in the amino acid sequence of HER3.

[0050] 2. Preparation of anti-HER3 antibodies The antibodies against HER3 of the present invention can be obtained by immunizing an animal with HER3 or any polypeptide selected from the amino acid sequence of HER3, and collecting and purifying the antibodies produced in the body, according to methods commonly used in this field. The species of HER3 that serves as the antigen is not limited to humans; animals can also be immunized with HER3 derived from animals other than humans, such as mice or rats. In this case, antibodies that are applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous HER3 with human HER3. Cut. Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against HER3 with myeloma cells 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)), and a monoclonal antibody can be obtained. The antigen HER3 can be obtained by expressing the HER3 gene in host cells through genetic manipulation. Specifically, a vector capable of expressing the HER3 gene is prepared, introduced into host cells to express the gene, and the expressed HER3 is purified. Furthermore, the above-mentioned genetically engineered HER3-expressing cells or cell lines expressing HER3 can also be used as the HER3 protein. A method for obtaining an antibody against HER3 will now be specifically described.

[0051] (1) Antigen preparation Antigens for producing anti-HER3 antibodies include HER3 or a polypeptide consisting of a partial sequence of at least 6 consecutive amino acids thereof, or derivatives of these to which any amino acid sequence or carrier has been added. HER3 can be used after direct purification from human tumor tissues or tumor cells, or it can be obtained by synthesizing HER3 in vitro or by producing it in host cells by genetic engineering. Specifically, in genetic engineering, the antigen can be obtained by inserting HER3 cDNA into an expression vector and then synthesizing it in a solution containing the enzymes, substrates, and energy materials necessary for transcription and translation, or by transforming other prokaryotic or eukaryotic host cells to express HER3. It is also possible to obtain an antigen as a secreted protein by expressing a fusion protein linking the extracellular domain of HER3, a membrane protein, with the constant domain of an antibody in an appropriate host-vector system. HER3 cDNA can be obtained, for example, by the so-called PCR method, in which a cDNA library expressing HER3 cDNA is used as a template and primers that specifically amplify HER3 cDNA are used to perform the polymerase chain reaction (hereinafter referred to as "PCR"; see Saiki, RK, et al., Science (1988) 239, pp. 487-489). Examples of in vitro synthesis of polypeptides include, but are not limited to, the Rapid Translation System (RTS) manufactured by Roche Diagnostics. Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector containing a replicon, i.e., origin of replication, and regulatory sequences derived from a species compatible with the host. Furthermore, vectors preferably contain sequences that can confer selectable phenotypes to transformed cells. Eukaryotic host cells include cells of vertebrates, insects, yeast, and the like. Examples of vertebrate cells include monkey COS cells (Gluzman, Y. Cell (1981) 23, pp. 175-182, ATCC CRL-1650; ATCC: American Type Culture Collection), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1999) 23, pp. 175-182). 80)77, pp.4126-4220) are commonly used, but are not limited to these. The transformant obtained as described above can be cultured according to a method commonly used in this field, and the desired polypeptide is produced intracellularly or extracellularly by the culture. The medium used for the culture can be appropriately selected from various commonly used mediums depending on the host cells used. In the case of Escherichia coli, for example, LB medium can be used with the addition of antibiotics such as ampicillin or IPMG as necessary. The recombinant protein produced inside or outside the cells of the transformant by the above-mentioned culture can be separated and purified by various known separation procedures that utilize the physical and chemical properties of the protein. Specific examples of such methods include treatment with a conventional protein precipitant, ultrafiltration, various liquid chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, dialysis, and combinations of these. Furthermore, recombinant proteins can be efficiently purified using a nickel affinity column by linking them to a six-residue histidine tag, or by linking them to the Fc region of IgG, allowing efficient purification using a protein A column. By combining the above methods, the desired polypeptide can be easily produced in large quantities with high yield and purity. The above-mentioned transformants themselves can be used as antigens, or cell lines expressing HER3 can be used as antigens.

[0052] (2) Production of anti-HER3 monoclonal antibodies An example of an antibody that specifically binds to HER3 is a monoclonal antibody that specifically binds to HER3, and the method for obtaining such an antibody is as described below. The production of a monoclonal antibody generally requires the following steps: That is, (a) Purification of a biopolymer to be used as an antigen or preparation of antigen-expressing cells; (b) immunizing an animal by injecting the antigen, collecting blood, testing the antibody titer, determining the time for spleen removal, and then preparing antibody-producing cells; (c) preparation of myeloma cells (hereinafter referred to as "myeloma"); (d) cell fusion between antibody-producing cells and myeloma; (e) Selection of hybridomas producing the desired antibody; (f) division into single cell clones (cloning); (g) In some cases, culturing hybridomas or rearing animals implanted with hybridomas for the large-scale production of monoclonal antibodies; (h) Examination of the physiological activity and binding specificity of the monoclonal antibody thus produced, or testing of its properties as a labeling reagent. etc. The method for producing a monoclonal antibody will be described in detail below along the above steps, but the method for producing the antibody is not limited to this, and for example, antibody-producing cells other than splenocytes and myelomas can also be used.

[0053] (a) Antigen purification As the antigen, HER3 or a part thereof prepared by the above-mentioned method can be used. Alternatively, membrane fractions prepared from HER3-expressing recombinant cells, or HER3-expressing recombinant cells themselves, or partial peptides of the protein of the present invention chemically synthesized using methods well known to those skilled in the art can also be used as antigens. Furthermore, HER3-expressing cell lines can also be used as antigens.

[0054] (b) Preparation of antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant or an adjuvant such as potassium alum, and the mixture is used as an immunogen to immunize a laboratory animal. Another method involves immunizing a laboratory animal with antigen-expressing cells as an immunogen. Any animal used in known hybridoma production methods can be used as the laboratory animal without any problems. Specifically, for example, mice, rats, goats, sheep, cattle, horses, etc. can be used. However, from the viewpoint of ease of obtaining myeloma cells to be fused with the isolated antibody-producing cells, it is preferable to use mice or rats as the immunized animals. There are no particular limitations on the strains of mice and rats that can be used. In the case of mice, for example, each strain A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, 129, etc., and in the case of rats, Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer, etc. can be used. These mice and rats can be obtained from laboratory animal breeding distributors such as CLEA Japan, Inc. and Charles River Japan, Inc. As the animal to be immunized, BALB / c strain mice and Wistar and Low strain rats are particularly preferred, taking into consideration the compatibility of fusion with myeloma cells described below. Furthermore, taking into consideration the homology of antigens between humans and mice, it is also preferable to use mice in which the biological mechanism for eliminating autoantibodies has been reduced, ie, mice with autoimmune diseases. The age of these mice or rats at the time of immunization is preferably 5 to 12 weeks, more preferably 6 to 8 weeks. To immunize animals with HER3 or a recombinant thereof, see, for example, Weir, DM, Handbook of Experimental Immunology Vol. I.II.III., Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C. A known method, which is described in detail in Thomas Publishers, Springfield, Illinois (1964), etc., can be used. Among these immunization methods, the preferred method in the present invention is specifically shown below, for example. That is, first, the membrane protein fraction as the antigen or cells expressing the antigen is administered intradermally or intraperitoneally to an animal. However, to enhance the immune efficiency, it is preferable to use both in combination, and the immune efficiency can be particularly enhanced by administering the first half intradermally and the second half or the final half intraperitoneally. The antigen administration schedule varies depending on the type of animal to be immunized, individual differences, etc., but in general, the antigen is preferably administered 3 to 6 times at administration intervals of 2 to 6 weeks, and more preferably 3 to 4 times at administration intervals of 2 to 4 weeks. The amount of antigen administered varies depending on the type of animal, individual differences, etc., but is generally about 0.05 to 5 mg, and preferably about 0.1 to 0.5 mg. Booster immunization is performed 1 to 6 weeks, preferably 1 to 4 weeks, and more preferably 1 to 3 weeks after the antigen administration described above. When the immunogen is cells, the booster immunization is performed using 1 × 10 6 〜1×10 7 Use cells. The amount of antigen administered for booster immunization varies depending on the type and size of the animal, but is generally about 0.05 to 5 mg, preferably 0.1 to 0.5 mg, and more preferably 0.1 to 0.2 mg for mice. When the immunogen is cells, it is about 1 × 10 6 ~ 1×10 7 Use cells. 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days after the booster immunization First, spleen cells or lymphocytes containing antibody-producing cells are aseptically extracted from the immunized animal. The antibody titer is measured at this stage, and if an animal with a sufficiently high antibody titer is used as a source of antibody-producing cells, the efficiency of subsequent procedures can be improved. The antibody titer measurement method used here includes, but is not limited to, the RIA method or the ELISA method. The antibody titer measurement in the present invention can be carried out, for example, by the ELISA method according to the following procedure. First, a purified or partially purified antigen is adsorbed onto a solid surface such as a 96-well ELISA plate. The solid surface to which the antigen is not adsorbed is then covered with a protein unrelated to the antigen, such as bovine serum albumin (hereinafter referred to as "BSA"). After washing the surface, the plate is contacted with a serially diluted sample (e.g., mouse serum) as the first antibody, allowing the antibody in the sample to bind to the antigen. Furthermore, an enzyme-labeled antibody against the mouse antibody is added as a second antibody and allowed to bind to the mouse antibody. After washing, a substrate for the enzyme is added, and the antibody titer is calculated by measuring the change in absorbance due to color development based on substrate decomposition. Isolation of antibody-producing cells from spleen cells or lymphocytes of an immunized animal can be carried out according to known methods (e.g., Kohler et al., Nature (1975) 256, p. 495; Kohler et al., Eur. J. Immunol. (1977) 6, p. 511; Milstein et al., Nature (1977) 266, p. 550; Walsh, Nature, (1977) 266, p. 495). For example, in the case of spleen cells, a common method can be used in which the spleen is minced, the cells are filtered through a stainless steel mesh, and then the cells are suspended in Eagle's minimum essential medium (MEM) to isolate antibody-producing cells.

[0055] (c) Preparation of myeloma cells (hereinafter referred to as "myeloma"). There are no particular limitations on the myeloma cells used for cell fusion, and any known cell line can be appropriately selected for use. However, considering the convenience of selecting hybridomas from fused cells, it is preferable to use a hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient strain, for which selection procedures have been established. These include mouse-derived X63-Ag8 (X63), NS1-ANS / 1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2 / 0-Ag14 (SP2 / 0), MPC11-45.6TG1.7 (45.6TG), FO, S149 / 5XXO, and BU.1; rat-derived 210.RSY3.Ag.1.2.3 (Y3); and human-derived U266AR (SKO-007), GM1500·GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), and 8226AR / NIP4-1 (NP41). These HGPRT-deficient strains can be obtained from, for example, ATCC. These cell lines are subcultured in an appropriate medium, such as 8-azaguanine medium (RPMI-1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter referred to as "FBS"), to which 8-azaguanine has been added), Iscove's Modified Dulbecco's Medium (IMDM), or Dulbecco's Modified Eagle Medium (hereinafter referred to as "DMEM"). Three to four days before cell fusion, they are subcultured in a normal medium (e.g., ASF104 medium (Ajinomoto Co., Inc.) containing 10% FCS) and on the day of fusion, 2 × 10 7 Ensure that the above number of cells is maintained.

[0056] (d) Cell fusion Fusion of antibody-producing cells with myeloma cells can be carried out by known methods (Weir, DM, Handbook of Experimental Immunology Vol. I.II.III., Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Ex The immunoprecipitation can be carried out appropriately according to the method described in "Peripheral Immunochemistry, Charles C. Thomas Publisher, Springfield, Illinois (1964)" under conditions that do not excessively reduce the viability of the cells. Such methods include, for example, a chemical method in which antibody-producing cells and myeloma cells are mixed in a high-concentration polymer solution such as polyethylene glycol, a physical method using electrical stimulation, etc. Specific examples of the chemical method are as follows: That is, when polyethylene glycol is used as the high-concentration polymer solution, antibody-producing cells and myeloma cells are mixed in a polyethylene glycol solution having a molecular weight of 1500 to 6000, preferably 2000 to 4000, at a temperature of 30 to 40°C, preferably 35 to 38°C, for 1 to 10 minutes, preferably 5 to 8 minutes.

[0057] (e) Selection of hybridoma population There are no particular limitations on the method for selecting hybridomas obtained by the above cell fusion, but the HAT (hypoxanthine-aminopterin-thymidine) selection method (Kohler et al., Nature (1975) 256, p. 495; Milstein et al., Nature (1977) 266, p. 550) is usually used. This method is effective for obtaining hybridomas using HGPRT-deficient myeloma cells, which cannot survive on aminopterin. By culturing unfused cells and hybridomas in HAT medium, only hybridomas that are resistant to aminopterin can be selectively retained and proliferated.

[0058] (f) Division into single cell clones (cloning) Hybridoma cloning can be performed using known methods such as the methylcellulose method, soft agarose method, and limiting dilution (see, for example, Barbara, BM and Stanley, MS: Selected Methods in Cellular Immunology, W.H. Freeman and Company, San Francisco (1980)). Among these methods, three-dimensional culture methods such as the methylcellulose method are particularly suitable. For example, hybridomas formed by cell fusion can be suspended and cultured in a methylcellulose medium such as ClonaCell-HY Selection Medium D (StemCell Technologies, #03804), and the formed hybridoma colonies can be recovered to obtain monoclonal hybridomas. Each recovered hybridoma colony is cultured, and hybridomas with stable antibody titers in the culture supernatant are selected as anti-HER3 monoclonal antibody-producing hybridoma strains.

[0059] (g) Preparation of monoclonal antibodies by hybridoma culture The hybridomas selected in this manner can be cultured to efficiently obtain monoclonal antibodies, but prior to culturing, it is desirable to screen for hybridomas that produce the desired monoclonal antibody. For this screening, a method known per se can be used. In the present invention, the antibody titer can be measured by, for example, the ELISA method described in the above item (b). The hybridomas obtained by the above method can be stored in a frozen state in liquid nitrogen or in a freezer at -80°C or below. Once the cloning is complete, the hybridoma is cultured in a normal medium instead of the HT medium. Large-scale culture is carried out by rotary culture using large culture bottles or spinner culture. Monoclonal antibodies that specifically bind to the protein of the present invention can be obtained from the supernatant of this large-scale culture by purification using methods well known to those skilled in the art, such as gel filtration. In addition, ascites containing large amounts of the monoclonal antibody of the present invention can be obtained by injecting the hybridoma into the abdominal cavity of a mouse of the same strain (e.g., the BALB / c described above) or a Nu / Nu mouse and allowing the hybridoma to proliferate. When administering intraperitoneally, a larger amount of ascites can be obtained by administering a mineral oil such as 2,6,10,14-tetramethylpentadecane (pristane) beforehand (3 to 7 days before). For example, an immunosuppressant was injected into the peritoneal cavity of a mouse of the same strain as the hybridoma to inactivate T cells, and then 10 6 ~10 7 Hybridoma clone cells (0.5 mL) suspended in serum-free medium are administered intraperitoneally. The abdominal cavity of the mouse is usually distended, and ascites fluid is collected from the mouse once the fluid has accumulated. This method yields monoclonal antibodies at concentrations approximately 100 times higher than those obtained in the culture medium. The monoclonal antibodies obtained by the above method can be purified, for example, by the method described in Weir, DM: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978). The monoclonal antibody thus obtained has high antigen specificity for HER3.

[0060] (h) Monoclonal antibody assay The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, examples of identification methods include the Ouchterlony method, the ELISA method, and the RIA method. Although the Ouchterlony method is simple, a concentration procedure is required when the concentration of the monoclonal antibody is low. On the other hand, when using the ELISA or RIA method, the culture supernatant is directly reacted with an antigen-adsorbing solid phase, and antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies, thereby making it possible to identify the isotype and subclass of the monoclonal antibody. As an even simpler method, a commercially available identification kit (for example, Mouse Typer Kit; manufactured by Bio-Rad) can also be used. Furthermore, protein can be quantified by the Folin-Lowry method and by calculation based on absorbance at 280 nm (1.4 (OD280) = 1 mg / mL of immunoglobulin). Furthermore, even when steps (a) to (h) of (2) are repeated to obtain a separate, independent monoclonal antibody, it is possible to obtain an antibody with cytotoxic activity equivalent to that of an anti-HER3 antibody. An example of such an antibody is an antibody that binds to the same epitope as the anti-HER3 antibody. If a newly produced monoclonal antibody binds to a partial peptide or partial three-dimensional structure to which the anti-HER3 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER3 antibody. Furthermore, by confirming that the monoclonal antibody competes with the binding of the anti-HER3 antibody to HER3 (i.e., that the monoclonal antibody inhibits the binding of the anti-HER3 antibody to HER3), it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER3 antibody, even if the specific sequence or structure of the epitope has not been determined. If it is confirmed that the epitope is the same, it is highly likely that the monoclonal antibody has antigen-binding ability or biological activity equivalent to that of the anti-HER3 antibody.

[0061] (3) Other antibodies The antibodies of the present invention include not only the monoclonal antibodies against HER3 described above, but also recombinant antibodies artificially modified for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods. Chimeric antibodies include antibodies in which the variable and constant regions are heterologous, such as chimeric antibodies in which the variable regions of a mouse- or rat-derived antibody are joined to the constant regions of a human-derived antibody (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) has been incorporated into a human-derived antibody (see Nature (1986) 321, pp. 522-525), and antibodies in which not only the CDR sequence but also some framework amino acid residues have been grafted onto a human antibody using CDR grafting (WO 90 / 07861). In this specification, "several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0062] In the present invention, it should be understood that the amino acid sequence of the binding protein of the present invention is not limited to the 20 common amino acids (Immunology-A Synthese s(2 nd Edition, ESGolub and DRGren, Eds. , Sinauer Associates, Sunderland, Mass. (1991) (incorporated herein by reference). For example, amino acids can include stereoisomers of the 20 common amino acids (e.g., D-amino acids), α,α-disubstituted amino acids, N-alkyl amino acids, unnatural amino acids such as lactic acid and other unconventional amino acids. Examples of unconventional amino acids that may also be suitable components for the binding proteins of the invention include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).

[0063] Furthermore, conservative amino acid substitutions are preferred for the amino acid substitutions herein. Conservative amino acid substitutions are substitutions that occur within amino acid groups that are related in their amino acid side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. Such amino acid substitutions are preferably made to the extent that they do not impair the properties of the substance having the original amino acid sequence. In the heavy and light chains of the antibody of the present invention, when the N-terminal amino acid is glutamic acid, it may be cyclized (constituted as pyroglutamine). In the present invention, such pyroglutamine is not distinguished from normal glutamine in terms of amino acid sequence. In addition, in the heavy and light chains of the antibody of the present invention, cysteine ​​may be cysteinylated. Such cysteinylated products are also included in the present invention. , and is not distinguishable from normal cysteine ​​in the amino acid sequence.

[0064] The antibody of the present invention further includes a human antibody that binds to HER3. An anti-HER3 human antibody refers to a human antibody that has only the gene sequence of an antibody derived from a human chromosome. An anti-HER3 human antibody can be produced by a method using a human antibody-producing mouse that has a human chromosome fragment containing the heavy and light chain genes of a human antibody (Tomizuka, K. et al., Na ture 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. Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et.al., Proc. Natl. Acad. Sci. USA (2000) 97, p. 722-727, etc. ) can be obtained.

[0065] Specifically, such human antibody-producing mice can be produced by generating knockout animals and transgenic animals as genetically modified animals in which the endogenous immunoglobulin heavy and light chain gene loci have been destroyed and instead the human immunoglobulin heavy and light chain gene loci have been introduced via a yeast artificial chromosome (YAC) vector or the like, and by crossbreeding these animals. Alternatively, eukaryotic cells can be transformed using recombinant DNA technology with cDNA encoding each of the heavy and light chains of such a human antibody, preferably with a vector containing the cDNA, and the transformed cells that produce the recombinant human monoclonal antibody can be cultured to obtain the antibody from the culture supernatant. Here, as the host, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes, and myeloma cells can be used. Production of human antibodies is described in detail in WO 2007 / 077028, the contents of which are incorporated herein by reference.

[0066] Methods for obtaining phage-display-derived human antibodies selected from a human antibody library are also known (see, for example, Wormstone, I. 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). For example, a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105-1116) can be used, in which the variable region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv), and phages that bind to the antigen are selected. By analyzing the genes of phages selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of an scFv that binds to an antigen has been determined, an expression vector containing that sequence can be constructed and introduced into an appropriate host for expression to obtain a human antibody (WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, WO 95 / 15388; Annu. Rev. Immunol (1994) 12, pp. 433-455; Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0067] One aspect of the present invention relates to proteins that bind to HER3. In some embodiments of the present invention, the isolated HER3 binding proteins of the present invention are those selected from the group consisting of (a) SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 152, 154, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 2 (b) CDHR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, or 230; and (c) a CDRH2 comprising the amino acid sequence set forth in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, or 230; and (d) a heavy chain amino acid sequence comprising a CDRH3 contained in the amino acid sequence set forth in SEQ ID NO: 4, 8, 12, 16, 20, a CDRL1 comprising the amino acid sequence set forth in 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, or 232;(e) a CDRL2 contained in the amino acid sequence set forth in SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, or 232; and (f) a CDRL2 contained in the amino acid sequence set forth in SEQ ID NO: a light chain amino acid sequence comprising a CDRL3 contained in the amino acid sequence set forth in SEQ ID NOs. 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, or 232;

[0068] The isolated HER3 binding protein of the present invention preferably comprises a heavy chain amino acid sequence comprising: (a) a CDRH1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 236, 251, 252, and 256; (b) a CDRH2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 258, 278, 280, and 282; and (c) a CDRH3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 283, 285, 309, 313, and 315; and (d) a CDRL1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 320, 334, 337, and 340; (e) a CDRL2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 343, 356, 351, and 344; and (f) a light chain amino acid sequence comprising a CDRL3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 360, 381, 385, and 387.

[0069] In another aspect of the invention, the isolated HER3 binding protein of the invention is selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, and 230, and / or a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 78, 80, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 146, 150, 154, 158 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228, and 232.

[0070] In yet another aspect of the invention, the isolated HER3 binding proteins of the invention are suitably selected from the group consisting of SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, 22 and 24, 26 and 28, 30 and 32, 36 and 38, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 60 and 58, 62 and 64, 66 and 68, 7 0 and 72, 74 and 76, 78 and 82, 80 and 82, 84 and 86, 88 and 90, 92 and 94, 96 and 98, 100 and 102, 104 and 106, 108 and 110, 112 and 114, 116 and 118, 122 and 124, 126 and 128, 130 and 132, 134 and 136, 138 and 140, 142 and 144, 146 and 148, 150 and 152, 154 and 156, 158 and 160, 162 and 164, 166 and 168, 170 and 172, 174 and 176, 178 and 180, 182 and 184, 186 and 188, 190 and 192, 194 and 196, 198 and 200, 202 and 204, 206 and 208, 210 and 212, 214 and 216, 218 and 220, 222 and 224, 226 and 228, or 230 and 232, or the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 34, 40, 60, 62, or 120 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 58 or 64. The isolated HER3 binding protein of the present invention more preferably comprises a heavy chain variable region amino acid sequence shown in SEQ ID NO: 42, 54, 70, 92 or 96, and a light chain variable region amino acid sequence shown in SEQ ID NO: 44, 56, 72, 94 or 98.

[0071] An antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 2 and 4 is designated "U1-39"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 6 and 8 is designated "U1-40"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 10 and 12 is designated "U1-38"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 14 and 16 is designated "U1-41"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 18 and 20 is designated "U1-42"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 22 and 24 is designated "U1-43"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 26 and 28 is designated "U1-44"; an antibody comprising the heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 30 and 32 is designated "U1-45"; The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 36 and 38 is designated "U1-45," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 36 and 38 is designated "U1-47," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 42 and 44 is designated "U1-49," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 46 and 48 is designated "U1-50," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 50 and 52 is designated "U1-51," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 54 and 56 is designated "U1-53," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 60 and 58 is designated "U1-55," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 62 and 64 is designated "U1-56," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 63 and 64 is designated "U1-57," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 64 and 66 is designated "U1-58," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 65 and 66 is designated "U1-59," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 66 and 68 is designated "U1-58"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 70 and 72 is designated "U1-59"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 74 and 76 is designated "U1-52"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 78 and 82 is designated "U1-61"; The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 80 and 82 is designated "U1-61.1." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 84 and 86 is designated "U1-62." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 88 and 90 is designated "U1-2." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 92 and 94 is designated "U1-7." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 96 and 98 is designated "U1-8." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 100 and 102 is designated "U1-10." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 104 and 106 is designated "U1-12." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 108 and 110 is designated "U1-13." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 112 and 114 is designated "U1-15." an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 116 and 118 is designated "U1-15"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 122 and 124 is designated "U1-20"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 126 and 128 is designated "U1-21"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 130 and 132 is designated "U1-22";The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 134 and 136 is designated "U1-23," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 138 and 140 is designated "U1-24," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 142 and 144 is designated "U1-25," the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 146 and 148 is designated "U1-26," and the antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 150 and 152 is designated "U1-27." The antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 154 and 156 is designated "U1-27," the antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 158 and 160 is designated "U1-31," the antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 162 and 164 is designated "U1-32," and the antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 166 and 168 is designated "U1-33." The antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 170 and 172 is designated "U1-36." The antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 174 and 176 is designated "U1-37." The antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 178 and 180 is designated "U1-34." The antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 182 and 184 is designated "U1-35." an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 186 and 188 is designated "U1-3"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 190 and 192 is designated "U1-4"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 194 and 196 is designated "U1-5"; an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 198 and 200 is designated "U1-6";The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 202 and 204 is designated "U1-8," SEQ ID NO: 206 and and 208 is designated "U1-11"; an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 210 and 212 is designated "U1-16"; an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 214 and 216 is designated "U1-17"; an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 218 and 220 is designated "U1-18"; an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 222 and 224 is designated "U1-33"; an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 226 and 228 is designated "U1-34"; An antibody comprising the variable region amino acid sequence is referred to as "U1-29," an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 230 and 232 is referred to as "U1-30," an antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 34 is referred to as "U1-46," an antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 40 is referred to as "U1-48," an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 60 and 58 is referred to as "U1-55.1," an antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 120 is referred to as "U1-19," and an antibody comprising the heavy chain variable region amino acid sequence and light chain variable region amino acid sequence shown in SEQ ID NOs: 62 and 64 is referred to as "U1-57.1." These antibodies are described in detail in the Examples. The isolated HER3-binding protein of the present invention more preferably comprises the heavy chain and light chain variable region amino acid sequences shown in SEQ ID NOs: 42 and 44, the heavy chain and light chain variable region amino acid sequences shown in SEQ ID NOs: 54 and 56, the heavy chain and light chain variable region amino acid sequences shown in SEQ ID NOs: 70 and 72, the heavy chain and light chain variable region amino acid sequences shown in SEQ ID NOs: 92 and 94, or the heavy chain and light chain variable region amino acid sequences shown in SEQ ID NOs: 96 and 98, and even more preferably, such a HER3-binding protein is the anti-HER3 antibody U1-49, U1-53, U1-59, U1-7, or U1-9.

[0072] [ka] JPEG2025072447000015.jpg 228139 JPEG2025072447000016.jpg 229147 JPEG2025072447000017.jpg 228141 JPEG2025072447000018.jpg 229140 JPEG2025072447000019.jpg 228140 JPEG2025072447000020.jpg 227143 JPEG2025072447000021.jpg 227144 JPEG2025072447000022.jpg 228140 JPEG2025072447000023.jpg 229142 JPEG2025072447000024.jpg 228140 JPEG2025072447000025.jpg 228146 JPEG2025072447000026.jpg 227145 JPEG2025072447000027.jpg 227147 JPEG2025072447000028.jpg 228149 JPEG2025072447000029.jpg 228142 JPEG2025072447000030.jpg 226145 JPEG2025072447000031.jpg 228150 JPEG2025072447000032.jpg 228143 JPEG2025072447000033.jpg 227147 JPEG2025072447000034.jpg 228150 JPEG2025072447000035.jpg 227148 JPEG2025072447000036.jpg 229149 JPEG2025072447000037.jpg 227146 JPEG2025072447000038.jpg 227152 JPEG2025072447000039.jpg 227146 JPEG2025072447000040.jpg 228151 JPEG2025072447000041.jpg 224142 JPEG2025072447000042.jpg 214162

[0073]

Chem.

[0074] If a newly produced monoclonal antibody binds to a partial peptide or partial three-dimensional structure bound by the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody, it can be determined that the antibody binds to the same epitope as the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody. Furthermore, by confirming that the antibody competes with the binding of the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody to HER3 (i.e., that the antibody interferes with the binding of the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody to HER3), it can be determined that the antibody binds to the same epitope as the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody, even if the specific sequence or structure of the epitope has not been determined. If it is confirmed that the epitope is the same, it is highly expected that the antibody will have biological activity equivalent to that of the U1-49, U1-53, U1-59, U1-7 or U1-9 antibody.

[0075] According to the present invention, the HER3 binding proteins of the present invention interact with at least one epitope in the extracellular portion of HER3. The epitope is preferably located within the amino-terminal domain, domain L1 (amino acids 19 to 184), the two cysteine-rich domains, domain S1 (amino acids 185 to 327) and domain S2 (amino acids 500 to 632), domain L2 (328 to 499) adjacent to the two cysteine-rich domains, or a combination of HER3 domains. The epitope may also be located within a combination of domains, such as, but not limited to, the epitope formed by portions of L1 and S1. Furthermore, the binding proteins of the present invention are further characterized in that their binding to HER3 reduces HER3-mediated signaling. According to the present invention, Thus, a reduction in HER3-mediated signaling can be caused, for example, by downregulating HER3, which at least partially eliminates HER3 molecules from the cell surface, or by stabilizing HER3 on the cell surface in a substantially inactive form (i.e., a form that exhibits lower signaling than the non-stabilized form). Alternatively, a reduction in HER3-mediated signaling can be caused by affecting (e.g., reducing or inhibiting) the binding of a ligand or another member of the HER family to HER3, the binding of GRB2 to HER-2, or the binding of GRB2 to SHC, by inhibiting receptor tyrosine phosphorylation, AKT phosphorylation, PYK2 tyrosine phosphorylation, or ERK2 phosphorylation, or by reducing tumor invasiveness. Alternatively, a reduction in HER3-mediated signaling can be caused by affecting (e.g., reducing or inhibiting) the formation of HER3-containing dimers with other HER family members. Among other examples, one example would be reducing or inhibiting the formation of the HER3-EGFR protein complex.

[0076] Furthermore, minor variations in the amino acid sequences set forth in any one of SEQ ID NOS: 1 to 232 are contemplated as encompassed by the present invention, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, and most preferably 99% of the sequence set forth in any one of SEQ ID NOS: 1 to 232. Variations can occur within framework regions (i.e., outside the CDRs), within the CDRs, or within the framework regions and CDRs. Preferred variations in the amino acid sequences set forth in SEQ ID NOS: 1 to 232, i.e., deletions, insertions, and / or substitutions of one to several amino acids, occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformational domains that occur in other binding proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. For example, Bowie et al., Science 253, 164 (1991) ;Proteins, Structures and Molecular Principles(Creighton, Ed., WHFreeman and Company, New York(1984));Introduction to Protein Structure(C. Branden and J. Tooze, eds., G Arland Publishing, New York, NY (1991); and Thornton et al., Nature 354:105 (1991), all of which are incorporated herein by reference. Thus, those skilled in the art will recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present invention. From among antibodies obtained by combining heavy and light chains with such variations in amino acid sequence, it is possible to select antibodies that are equivalent to or superior to the original antibody (parent antibody). The HER3-binding proteins, anti-HER3 antibodies, etc. of the present invention retain HER3-binding activity even if variations in amino acid sequence occur as described above. In the present invention, "homology" is synonymous with "identity." The homology between two amino acid sequences was determined using the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, The Blast algorithm can be determined by using the default parameters of Webb Miller and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402. m can also be accessed on the internet, for example, at www.ncbi.nlm.nih.gov / blast.

[0077] The chimeric, humanized, or human antibodies obtained by the above methods can be evaluated for their antigen-binding ability by known methods, and suitable antibodies can be selected. The anti-HER3 antibodies of the present invention include MEHD-7945A (or doligotuzumab), RG-7116, MM-111, MM-121 (or seribantumab), MM-141, LJM-716, huHER3-8, tri-specific anti-EGFR / ErbB3 zybody, GSK-2849330, REGN-1400, KTN-3379, AV-203, monospecific surrobody (ErbB3), lumretuzumab, MP-EV-20, ZW-9, and Dimercept TM , anti-Erb3 surfactant (SL-175 or SL-176), SYM-013, and variants, active fragments, modified forms, etc. thereof are also included.

[0078] Another example of an index used to compare antibody properties is antibody stability. Differential scanning calorimetry (DSC) is an instrument that can quickly and accurately measure the thermal denaturation midpoint (Tm), a good indicator of the relative structural stability of proteins. Differences in thermal stability can be compared by measuring Tm values ​​using DSC and comparing the values. It is known that the storage stability of antibodies correlates somewhat with their thermal stability (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273). Therefore, suitable antibodies can be selected using thermal stability as an index. Other indexes for antibody selection include high yield in appropriate host cells and low aggregation in aqueous solution. For example, the antibody with the highest yield does not necessarily have the highest thermal stability. Therefore, it is necessary to comprehensively evaluate the above-mentioned indexes to select the antibody most suitable for human administration.

[0079] The antibodies of the present invention also include modified antibodies. The term "modified antibodies" refers to antibodies of the present invention that have been chemically or biologically modified. Chemical modifications include attachment of a chemical moiety to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, and the like. Biological modifications include those that have undergone post-translational modifications (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, and methionine oxidation), and those that have undergone expression in prokaryotic host cells to add a methionine residue to the N-terminus. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens, among other purposes.

[0080] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the antibody of the present invention. Techniques for modulating antibody sugar chain modification are known, including, but not limited to, those described in International Publication Nos. 1999 / 54342, 2000 / 61739, and 2002 / 31140. The antibodies of the present invention also include antibodies in which the sugar chain modification has been modulated. When an antibody gene is isolated and then introduced into a suitable host to produce an antibody, a suitable combination of host and expression vector can be used. Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence and a gene encoding the light chain sequence of an antibody described herein. When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or they can be inserted into separate expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, examples of animal cells include mammalian cells, such as monkey COS cells (Gluzman, Y. Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), and dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220). When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis. Antibodies can be obtained by introducing the desired antibody gene into these cells by transformation and culturing the transformed cells in vitro. The yield of the antibody during this culture may vary depending on the antibody sequence, and antibodies with equivalent binding activity can be selected based on the yield as an indicator to determine which antibodies are easy to produce as pharmaceuticals. Therefore, the antibodies of the present invention also include antibodies obtained by a method for producing the antibody, which method comprises the steps of culturing the transformed host cells and recovering the desired antibody or a functional fragment of the antibody from the culture obtained in this step.

[0081] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134 (1995)). Similarly, 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 (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibodies of the present invention also include antibodies that have undergone such modifications and functional fragments of such antibodies, including deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and amidated deletions (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody of the present invention are not limited to the above types. The two heavy chains constituting the antibody of the present invention may be any one of heavy chains selected from the group consisting of full-length and the above-mentioned deletions, or a combination of any two of these. The quantitative ratio of each deletion may be affected by the type of mammalian cultured cells producing the antibody of the present invention and the culture conditions, but the main component of the antibody of the present invention can be an antibody in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains. The full-length antibody of the present invention (also simply referred to as "antibody" in the present invention) also includes such deletions and mixtures containing one or more of such deletions. Furthermore, the "antibody" of the present invention includes heavy or light chains in which, when the N-terminus is glutamine, it has been cyclized to pyroglutamine, and / or heavy or light chains in which some of the cysteines have been cysteinylated. Also included are those containing heavy or light chains.

[0082] Examples of the isotype of the anti-HER3 antibody of the present invention include, but are not limited to, IgA, IgD, IgE, IgG, or IgM, preferably IgG1, IgG2, IgG3, IgG4, IgM1, and IgM2, more preferably IgG or IgM, and most preferably IgG1, IgG2, or IgG4.

[0083] The biological activities of an antibody generally include antigen-binding activity, the activity of internalizing into cells expressing the antigen by binding to the antigen, the activity of neutralizing the activity of the antigen, the activity of enhancing the activity of the antigen, antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP). The function of the antibody of the present invention is the binding activity to HER3, preferably the activity of internalizing into HER3-expressing cells by binding to HER3. Furthermore, the antibody of the present invention has the cell-internalizing activity. In addition, it may also have ADCC activity, CDC activity and / or ADCP activity.

[0084] In certain respects, for example, with respect to generating antibodies against HER3 as therapeutic candidates, it may be desirable that the anti-HER3 antibodies of the invention be capable of fixing complement and participating in complement-dependent cytotoxicity (CDC). There are numerous antibody isotypes that are capable of fixing complement and participating in complement-dependent cytotoxicity (CDC), including, but not limited to, mouse IgM, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgM, human IgG1, human IgG3, and human IgA. It is understood that the antibody generated need not initially possess such an isotype; rather, the generated antibody can have any isotype, and that antibodies can be isotype-switched using conventional molecular biology techniques well known in the art by adding molecularly cloned V-region genes or cDNAs to molecularly cloned constant region genes or cDNAs in an appropriate expression vector, and then expressing the antibody in a host cell using techniques known in the art. Isotype-exchanged antibodies are molecularly engineered to have superior CDC compared to naturally occurring variants (Idusogie et al., J. Immunol., 166, 2571-2575), and can also have their Fc regions recombinantly expressed in host cells using techniques known in the art. Such techniques include, inter alia, direct recombinant techniques (see, e.g., U.S. Pat. No. 4,816,397) and cell-cell fusion techniques (e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418). In cell-cell fusion techniques, a myeloma or other cell line (e.g., CHO) carrying a heavy chain with any desired isotype is prepared, and another myeloma or other cell line (e.g., CHO) carrying a light chain is prepared. These cells are then fused, and a cell line expressing the intact antibody can be isolated. As an example, a human anti-HER3 IgG4 antibody with the desired binding to the HER3 antigen can be easily isotype-exchanged to create a human IgM, human IgG1, or human IgG3 isotype, while having the same variable region (which defines the antibody's specificity and some of its affinity). Such molecules may then be able to fix complement and participate in CDC.

[0085] Furthermore, it may be desirable for the anti-HER3 antibodies of the present invention to bind to Fc receptors on effector cells, such as monocytes and natural killer (NK) cells, and to participate in antibody-dependent cellular cytotoxicity (ADCC). There are numerous antibody isotypes that can do this, including, but not limited to, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, and human IgG3. It is understood that the antibody produced need not initially have such an isotype; rather, the produced antibody can have any isotype, and that antibody isotype exchange can be performed using conventional molecular biology techniques well known in the art by adding molecularly cloned V-region genes or cDNAs to molecularly cloned constant region genes or cDNAs in an appropriate expression vector, followed by expression of the antibody in a host cell using techniques known in the art. Isotype-exchanged antibodies can be molecularly engineered to have superior ADCC compared to naturally occurring variants (Shields et al., J. Biol. Chem., 276, 6591-6604), and can also have their Fc regions recombinantly expressed in host cells using techniques known in the art. Such techniques include, inter alia, direct recombinant techniques (see, e.g., U.S. Pat. No. 4,816,397) and cell-cell fusion techniques (see, e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418). In cell-cell fusion techniques, a myeloma or other cell line (e.g., CHO) carrying a heavy chain with any desired isotype is prepared, and another myeloma or other cell line (e.g., CHO) carrying a light chain is prepared. These cells can then be fused, and a cell line expressing the intact antibody can be isolated. As an example, a human anti-HER3 IgG4 antibody with the desired binding to the HER3 antigen can be made to have the same variable region (which defines the antibody specificity and some of the antibody's affinity) but a human IgG1 or human IgG3 isotype. Such molecules may then be able to bind to FcγR on effector cells and participate in ADCC.

[0086] The obtained antibody can be purified to homogeneity. Antibodies can be separated and purified using methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring, 1999). Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)). Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatographies can be carried out using liquid chromatographies such as HPLC and FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, columns using protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia). It is also possible to purify antibodies by using a carrier on which an antigen is immobilized, and taking advantage of their binding to the antigen.

[0087] [Anti-tumor compound] The antitumor compound conjugated to the anti-HER3 antibody-drug conjugate of the present invention will now be described. The antitumor compound used in the present invention is not particularly limited as long as it is a compound with antitumor effect and has a substituent or partial structure that can be bound to a linker structure. The antitumor compound exerts its antitumor effect when part or all of the linker is cleaved within tumor cells, liberating the antitumor compound portion. When the linker is cleaved at the binding site with the drug, the antitumor compound is released in its unmodified form, thereby exerting its inherent antitumor effect. The antitumor compound used in the present invention is a camptothecin derivative, exatecan ((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; the following formula:

[0088] [ka]

[0089] can be preferably used. Although this exatecan has excellent antitumor activity, it has not yet been commercially available as an antitumor drug. This compound can be easily obtained by known methods, and the amino group at position 1 can be preferably used as a binding site for a linker structure. In addition, exatecan may be released within tumor cells with part of the linker still bound, but even with this structure, it is an excellent compound that exhibits excellent antitumor effects. Since exatecan has a camptothecin structure, it is effective in acidic aqueous media (e.g., pH 3). It is known that in a basic aqueous medium (e.g., at about pH 10), the equilibrium is biased toward a structure in which the lactone ring is formed (closed ring isomer), whereas in a basic aqueous medium (e.g., at about pH 10), the equilibrium is biased toward a structure in which the lactone ring is opened (open ring isomer). Drug conjugates incorporating exatecan residues corresponding to such closed ring structures and open ring structures are expected to have equivalent antitumor effects, and it goes without saying that both forms are encompassed within the scope of the present invention.

[0090] Other antitumor compounds include, for example, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or its derivatives), taxol or its derivatives, other camptothecin or its derivatives (such as those described in Japanese Patent Application Laid-Open No. 6-87746), antitumor agents) and the like.

[0091] In antibody-drug conjugates, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-drug conjugates are produced by specifying reaction conditions, such as the amounts of raw materials and reagents used, so that a certain number of drugs are bound. However, unlike chemical reactions of small molecules, they are usually obtained as a mixture of drugs with different numbers of conjugated drugs. The number of drugs bound to one antibody molecule is specified and expressed as an average value, i.e., the average drug conjugation number. In the present invention, as a general rule, unless otherwise specified, i.e., except when referring to an antibody-drug conjugate with a specific drug conjugation number contained in a mixture of antibody-drug conjugates with different drug conjugation numbers, the average drug conjugation number is used. The number of exatecans bound to an antibody molecule can be controlled, and the average number of drugs bound to an antibody can be about 1 to 10, preferably 2 to 8, and more preferably 3 to 8. Those skilled in the art will be able to design a reaction for binding the required number of drugs to an antibody from the descriptions in the Examples of the present application, and will be able to obtain an antibody-drug conjugate in which the number of exatecans bound is controlled. The antibody-drug conjugate of the present invention is less likely to undergo aggregation, insolubility, fragmentation, etc., even when the number of drugs bound to one antibody molecule is large.

[0092] [Linker structure] In the anti-HER3 antibody-drug conjugate of the present invention, a linker structure that connects an antitumor compound to an anti-HER3 antibody is described. The linker has the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- or -L 1 -L 2 -L P - The antibody has the structure L 1 The end of (L 2 The antitumor compound binds to the -L a -(CH2)n 2 A carbonyl group in the -C(=O)- moiety or L P It binds at the C-terminus of n 1 represents an integer of 0 to 6, preferably an integer of 1 to 5, and more preferably an integer of 1 to 3.

[0093] 1.L 1 L 1 teeth, -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)- It is shown in the structure: where n 3 is an integer from 2 to 8, and "-(Succinimid-3-yl-N)-" is a group represented by the following formula:

[0094] [ka]

[0095] The structure is represented by the formula: Position 3 in this partial structure is the binding site for the anti-HER3 antibody. The binding to the antibody at position 3 is characterized by forming a thioether bond. The nitrogen atom at position 1 in this structural portion is bonded to the carbon atom of a methylene present in the linker containing this structure. That is, -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-L 2 - is a structure represented by the following formula (where "antibody-S-" is derived from an antibody):

[0096] [ka]

[0097] In the formula, n 3 is an integer from 2 to 8, preferably from 2 to 5.

[0098] L 1 Specific examples include: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)- The following can be mentioned:

[0099] 2.L 2 L 2 teeth, -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- The structure is shown by L 2 may not exist, in which case L 2 In particular, in the drug-linker structure of the present invention, L P may bind directly to the drug, in which case this L 2 is particularly preferably a single bond. 4 is an integer from 1 to 6, preferably from 2 to 4. 2 is the terminal amino group and L 1 and the carbonyl group at the opposite end of L P and combine.

[0100] L 2 Specific examples include: -NH-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)- The following can be mentioned:

[0101] 3.L P L P is a peptide residue consisting of 2 to 7 amino acids. In other words, it is composed of residues of an oligopeptide consisting of 2 to 7 amino acids linked by peptide bonds. P is N-terminally L 2 and at the C-terminus, the linker -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- part It binds to amino groups.

[0102] L P The amino acids constituting the amino acid are not particularly limited, but may be, for example, L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may also be used, and non-natural amino acids such as N-methylated amino acids may also be used. L PThe amino acid sequence of is not particularly limited, but examples of the constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), and aspartic acid (Asp; D). Among these, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid are preferred. The drug release pattern can be controlled by the type of amino acid. The number of amino acids may be 2 to 7.

[0103] L P As a concrete example, -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, -GGFGGGF- The above "(D-)D" means D-aspartic acid. Particularly preferred L-drug conjugates P Examples include -GGFG- and -DGGFG- peptide residues. Furthermore, in the drug-linker structure of the present invention, L P In some cases, the drug is directly bound to the L P An example of such a peptide is the pentapeptide residue -DGGFG-. This can be done.

[0104] 4.L a -(CH2)n 2 -C(=O)- La -(CH2)n 2 L in -C(=O)- a is the structure of -O- or a single bond. 2 is an integer of 0 to 5, preferably 0 to 3, and more preferably 0 or 1. L a -(CH2)n 2 Examples of -C(=O)- include the following structures: -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, -O-CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2CH2-C(=O)-. Among these, -O-CH2-C(=O)-, -O-CH2CH2-C(=O)- If so, L a is a single bond and n 2 It is preferred if is 0.

[0105] Linker -NH-(CH2)n 1 -L a -(CH2)n 2 Specific examples of the structure represented by -C(=O)- include: -NH-CH2-C(=O)-, -NH-CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-OC(=O)-, -NH-CH2CH2-O-CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-, The following can be mentioned:

[0106] Among these, more preferred are: -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-OC(=O)- is.

[0107] Linker -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- has a chain length of 4 to 7 atoms Those having a chain length of 5 or 6 atoms are preferred, more preferably those having a chain length of 5 or 6 atoms.

[0108] After the anti-HER3 antibody-drug conjugate of the present invention is transported into tumor cells, the linker moiety is cleaved to form NH2-(CH2)n 1 -L a -(CH2)n 2 It is believed that a drug derivative having a structure represented by -C(=O)-(NH-DX) is released to exert an antitumor effect. Examples of the antitumor derivatives that are released from the antibody-drug conjugate of the present invention to exert an antitumor effect include the aforementioned linker -NH-(CH2)n 1 -L a -(CH2)n 2 An antibody having a structural portion in which the terminal of the structure represented by -C(=O)- is an amino group Among the tumorigenic derivatives that may be mentioned are the following, which are particularly preferred: NH2-CH2CH2-C(=O)-(NH-DX), NH2-CH2CH2CH2-C(=O)-(NH-DX), NH2-CH2-O-CH2-C(=O)-(NH-DX), NH2-CH2CH2-O-CH2-C(=O)-(NH-DX). In the case of NH2-CH2-O-CH2-C(=O)-(NH-DX), the aminal structure in the molecule is unstable. Therefore, it further self-decomposes HO-CH2-C(=O)-(NH-DX) These compounds can also be suitably used as intermediates for producing the antibody-drug conjugates of the present invention. In addition, in the drug-linker structure of the present invention, L P may bind directly to the drug. P When the C-terminus of is glycine, the antitumor drug released is exatecan itself or a compound in which glycine is bound to the amino group of exatecan.

[0109] In the antibody-drug conjugate of the present invention in which the drug is exatecan, the drug-linker structure portion has the following structure: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX), or -L 1 -L 2 -L P -(NH-DX) The average number of these drug-linker structural moieties bound to one antibody may be 1 to 10, preferably 2 to 8, and more preferably 3 to 8. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Among these, the following are more preferred: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Furthermore, the following is preferable: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Particularly preferred are the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX).

[0110] In the antibody-drug conjugate of the present application, a preferred linker structure linking the anti-HER3 antibody and the drug can be constructed by linking the preferred structures shown for each linker moiety described above. The following structures can be suitably used as such linker structures. The left end of the structure is the binding site for the antibody, and the right end is the binding site for the drug. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-, -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-GGFG-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-DGGFG-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-。 Among these, the more preferred ones are as follows. -(N-Succinimid-3-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-DGGFG-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-。 Furthermore, preferably, the following can be mentioned. -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(N-Succinimid-�-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-. Particularly preferred are the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0111] [Manufacturing method] Next, a representative method for producing the antibody-drug conjugate of the present invention or a production intermediate thereof will be described. Hereinafter, compounds will be referred to by the numbers shown in each reaction scheme. That is, they will be referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will also be referred to in the same manner.

[0112] 1. Manufacturing method 1 The antibody-drug conjugate represented by formula (1), in which the antibody and the drug-linker structure are linked via a thioether, can be produced, for example, by the following method.

[0113] [ka]

[0114] [Wherein, AB represents an antibody having a sulfhydryl group, and L 1 'L 1 In the linker structure represented by the formula:

[0115] [ka]

[0116] (where the nitrogen atom is the binding site.) Specifically, -(Succinimid-3-yl-N)-(CH2)n 2 L represented by -C(=O)- 1 In the structure of the formula (I), the -(Succinimid-3-yl-N)- moiety is a maleimidyl group.

[0117] [ka]

[0118] This represents the group formed by removing one hydrogen atom from the amino group at position 1 of exatecan.

[0119] In the above reaction scheme, the compound of formula (1) can be interpreted as a structure in which one structural moiety from the drug to the linker terminal is bound to one antibody. However, this is a convenient description for the purpose of explanation, and in reality, multiple structural moieties are often bound to one antibody molecule. This situation also applies to the following description of the production method.

[0120] That is, the antibody-drug conjugate (1) can be produced by reacting the compound (2), which can be obtained by the method described below, with an antibody (3a) having a sulfhydryl group. Antibody (3a) having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). For example, Traut's reagent is reacted with the amino groups of an antibody; N-succinimidyl S-acetylthioalkanoates are reacted with the amino groups of an antibody, followed by hydroxylamine; N-succinimidyl 3-(pyridyldithio)propionate is reacted with the amino groups of an antibody, followed by a reducing agent; or a reducing agent such as dithiothreitol, 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is reacted with an antibody. and reducing disulfide bonds in the hinge region of the antibody to generate sulfhydryl groups; etc., but are not limited to these. Specifically, an antibody with partially or completely reduced hinge disulfides can be obtained by reacting the antibody with TCEP as a reducing agent in an amount of 0.3 to 3 molar equivalents per intra-antibody hinge disulfide in a buffer solution containing a chelating agent. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These can be used at a concentration of 1 mM to 20 mM. Examples of buffer solutions that can be used include sodium phosphate, sodium borate, and sodium acetate solutions. Specifically, an antibody (3a) with partially or completely reduced sulfhydryl groups can be obtained by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours. Here, a reaction is carried out to add a sulfhydryl group to the drug-linker moiety, thereby linking the drug-linker moiety via a thioether bond. Antibody-drug conjugates (1) can be produced using 2 to 20 molar equivalents of compound (2) per antibody (3a) bearing a sulfhydryl group, with 2 to 8 drugs bound per antibody. Specifically, a solution of compound (2) is added to a buffer solution containing antibody (3a) bearing a sulfhydryl group, followed by reaction. Examples of suitable buffer solutions include sodium acetate, sodium phosphate, and sodium borate. The pH during the reaction is 5 to 9, preferably around 7. Examples of suitable solvents for dissolving compound (2) include organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP). The organic solvent solution containing compound (2) can be added to a buffer solution containing antibody (3a) having a sulfhydryl group at 1 to 20% v / v and reacted. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by deactivating the reactivity of unreacted compound (2) with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC relative to the compound (2) used and incubating at room temperature for 10 to 30 minutes. The produced antibody-drug conjugate (1) was concentrated and buffered by the following common procedure. The antibody-drug conjugate (1) can be identified by performing antibody exchange, purification, antibody concentration, and measurement of the average number of drugs bound per antibody molecule.

[0121] Common Procedure A: Concentration of an Aqueous Solution of an Antibody or Antibody-Drug Conjugate The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (50,000 MWCO, Millipore Corporation) container and concentrated by centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) at 2000 to 3800 G for 5 to 20 minutes.

[0122] Common Procedure B: Antibody concentration measurement Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. The 280 nm extinction coefficient can be estimated from the amino acid sequence of the antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). The 280 nm extinction coefficient varies depending on the antibody. 80nm absorption coefficient (1.3mLmg -1 cm -1 ~1.8mLmg -1 cm -1 In the case of U1-59, 1.768 mL mg was used according to the amino acid sequence. -1 cm -1 The 280 nm extinction coefficient of was used as an estimate.

[0123] Common Procedure C: Antibody Buffer Exchange A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 matrix was equilibrated with phosphate buffer (10 mM, pH 6.0; referred to herein as PBS6.0 / EDTA) containing 137 mM sodium chloride and 5 mM ethylenediaminetetraacetic acid (EDTA) according to the manufacturer's instructions. 2.5 mL of antibody solution was loaded onto each column, and a 3.5 mL fraction was eluted with 3.5 mL of PBS6.0 / EDTA. This fraction was concentrated using the common procedure A, and the antibody concentration was measured using the common procedure B. The antibody concentration was then adjusted to 10 mg / mL using PBS6.0 / EDTA.

[0124] Common Procedure D: Purification of Antibody-Drug Conjugates A NAP-25 column was equilibrated with acetate buffer (10 mM, pH 5.5; herein referred to as ABS) containing sorbitol (5%). The antibody-drug conjugate reaction solution (approximately 2.5 mL) was loaded onto this NAP-25 column and eluted with the manufacturer's specified volume of buffer to obtain an antibody fraction. This fraction was loaded onto the NAP-25 column again, and the gel filtration purification procedure, in which the fraction was eluted with buffer, was repeated two or three times to obtain an antibody-drug conjugate from which unbound drug linkers and small molecular weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide) had been removed.

[0125] Common Procedure E: Measurement of antibody concentration and average number of drugs bound per antibody molecule in antibody-drug conjugates (1) The bound drug concentration in an antibody-drug conjugate can be calculated by measuring the UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths, 280 nm and 370 nm, and then performing the following calculation. Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in a system (additivity of absorbance), assuming that there is no change in the molar absorption coefficients of the antibody and drug before and after conjugation of the antibody and drug, the antibody concentration and drug concentration in the antibody-drug conjugate are expressed by the following relationship: A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Formula (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Formula (2) where A280 indicates the absorbance of the antibody-drug conjugate aqueous solution at 280 nm. , A 370 indicates the absorbance of the antibody-drug conjugate aqueous solution at 370 nm, and A A,280 indicates the absorbance of the antibody at 280 nm, and A A,370 is the absorbance of the antibody at 370 nm indicates A D,280 denotes the absorbance of the conjugate precursor at 280 nm, and A D,370 denotes the absorbance of the conjugate precursor at 370 nm, and ε A,280 is at 280 nm is the molar extinction coefficient of the antibody, and ε A,370 is the molar extinction coefficient of the antibody at 370 nm , ε D,280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D,370 is the molar extinction coefficient of the conjugate precursor at 370 nm, and C A antibody-drug conjugate indicates the antibody concentration in the adjugate, C D is the drug concentration in the antibody-drug conjugate Shows. where ε A,280 , ε A,370 , ε D,280 , ε D,370 is a value prepared in advance (a calculated estimated value or an actual measured value obtained from UV measurement of the compound). For example, ε A,280 is the antibody's amino acid sequence. From the amino acid sequence, the nucleotide sequence was calculated by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). It can be estimated that A,370 is usually zero. In the case of U1-59, the amino According to the acid sequence, ε A,280 The estimated value was 259400. D,280 and ε D,370 teeth The absorbance of a solution in which the conjugate precursor used is dissolved at a certain molar concentration is measured, and the absorbance is calculated according to the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). The A of the antibody-drug conjugate aqueous solution can be obtained by 280 and A 370 By measuring these values ​​and substituting them into equations (1) and (2) and solving the simultaneous equations, C A and C D Furthermore, C D C A The average number of drugs bound per antibody can be calculated by dividing by this. In the present invention, the above-described method for determining the average number of drugs bound per antibody is called the "UV method."

[0126] Common Procedure F: Measurement of the average number of drugs bound per antibody molecule in antibody-drug conjugates (2) The average number of drugs bound per antibody molecule in an antibody-drug conjugate can be determined by high-performance liquid chromatography (HPLC) analysis using the following method, in addition to the above-mentioned common procedure E. [F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). The mixture is incubated at 37° C. for 30 minutes to cleave the disulfide bond between the L chain and H chain of the antibody-drug conjugate. The resulting sample is used for HPLC analysis. [F-2.HPLC analysis] The HPLC analysis is carried out under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent T technologies) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Agilent T echnologies, P / N PL1912-1802) Column temperature: 80℃ Mobile phase A: 0.04% trifluoroacetic acid (TFA) in water Mobile phase B: Acetonitrile solution containing 0.04% TFA Gradient program: 29%-36% (0 min-12.5 min), 36%-42% (12.5 min-15 min), 42%-29% (15 min-15.1 min), 29%-29% (15.1 min-25 min) Sample injection volume: 15 μL [F-3. Data Analysis] [F-3-1] Drug-bound L chain (L chain with one drug bound: L1) and H chain (H chain with one drug bound: L2) to the L chain (L0) and H chain (H0) of an antibody without a drug bound to it. H chains with two drugs bound to them: H1, H chains with two drugs bound to them: H2, and H chains with three drugs bound to them: H3 The retention time increases in proportion to the number of molecules, resulting in increased hydrophobicity, so they are eluted in the order of L0, L1, H0, H1, H2, and H3. By comparing the retention times with L0 and H0, the detected peak can be assigned to one of L0, L1, H0, H1, H2, or H3. [F-3-2] Because the drug linker has UV absorption, the peak area value is corrected according to the molar extinction coefficients of the light chain, heavy chain, and drug linker according to the number of drug linkers bound, using the formula below.

[0127]

number

[0128]

number

[0129] Here, the molar extinction coefficients (280 nm) of the L chain and H chain of each antibody were calculated by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). Values ​​estimated from the amino acid sequence can be used. In the case of U1-59, the molar extinction coefficient of the L chain was estimated to be 34,690, and the molar extinction coefficient of the H chain was estimated to be 95,000, based on the amino acid sequence. Furthermore, the molar extinction coefficient (280 nm) of the drug linker was determined by reacting each drug linker with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether. [F-3-3] Calculate the peak area ratio (%) of each chain to the total corrected peak area according to the following formula.

[0130]

number

[0131] [F-3-4] Calculate the average number of drugs bound per antibody molecule in the antibody-drug conjugate according to the following formula. Average number of drug bonds = (L0 peak area ratio x 0 + L0 peak area ratio x 1 + H0 peak area ratio x 0 + H1 peak area ratio x 1 + H2 peak area ratio x 2 + H3 peak area ratio x 3) / 100 x 2

[0132] The intermediate compounds used in Production Method 1 are described below. The compound represented by formula (2) is represented by the following formula:

[0133] (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) or (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -(NH-DX) It is a compound represented by the formula: During the ceremony, n3 denotes the integers 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L a represents -O- or a single bond, (maleimid-N-yl)- is a group having the following formula: [ka]

[0134] a maleimidyl group (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by the formula: -(NH-DX) is a compound of the formula: [ka]

[0135] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in

[0136] L P The peptide residue L is preferably an amino acid residue selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and is used as a production intermediate. P Among these, peptide residues consisting of 4 or 5 amino acids are preferred as production intermediates. Pis a tetrapeptide residue of -GGFG- or a pentapeptide of -DGGFG- It is preferred as an intermediate, and more preferably -GGFG-.

[0137] Also, -NH-(CH2)n 1 -L a -(CH2)n 2 As for -, -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are preferred as production intermediates, and -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are more preferred. n 3 is preferably an integer of 2 to 8 as a production intermediate. L 2 is a single bond or -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- where n 4 is an integer of 2 to 4 is preferred as a production intermediate.

[0138] Furthermore, n 3 is an integer between 2 and 5, and L 2 is a single bond, and -NH-(CH2)n 1 -L a -(CH2)n 2 - is preferably -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. Among these, -NH-(CH2)n is more preferred. 1 -L a -(CH2)n 2- is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. 3 But integer Preferably, it is 2 or 5 of the above.

[0139] Also, n 3 is an integer between 2 and 5, and L 2 -NH-(CH2-CH2-O)n 4 -CH2-CH2-C(=O)-, n 4 is an integer from 2 to 4, and -NH-(CH2)n 1 -L a -(CH2)n 2 - is preferably -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. 4 is an integer of 2 or 4. Furthermore, -NH-(CH2)n 1 -L a Preferably, - is -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-.

[0140] Preferred examples of intermediates useful for producing the compounds of the present invention include the following: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0141] The anti-Her3 antibody-drug conjugate of the present invention can be produced by reacting a drug-linker compound selected from the above group of production intermediate compounds with an anti-Her3 antibody or a reactive derivative thereof to form a thioether bond at the disulfide bond present in the hinge region of the anti-Her3 antibody. In this case, it is preferable to use a reactive derivative of the anti-Her3 antibody, and particularly preferable to use a reactive derivative obtained by reducing the anti-Her3 antibody.

[0142] The following compounds are more preferred as intermediates in the production: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0143] Among the above intermediate compounds, there is also the compound of the following formula: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). The intermediate represented by the formula: is a more preferred compound. Particularly preferred is the formula: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX). It is a compound represented by the formula:

[0144] 2. Manufacturing method 2 The compound of formula (2), which is an intermediate used in the above production method, or a pharmacologically acceptable salt thereof can be produced, for example, by the following method.

[0145] [ka]

[0146] [In the formula, L 1 ' is L 1 The terminal of P is converted to a maleimidyl group. 1 , P 2 and P 3 indicates a protecting group.]

[0147] Carboxylic acid (5) can be converted to an activated ester, mixed acid anhydride, acid halide, or the like, and reacted with NH2-DX [exatecan; 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] (4) or a pharmacologically acceptable salt thereof in the presence of a base to produce compound (6). This reaction can be carried out using the same reagents and conditions as those normally used in peptide synthesis. Various activated esters are available, but they can be prepared by reacting carboxylic acid (5) with phenols such as p-nitrophenol, N-hydroxybenzotriazole, or N-hydroxysuccinimide using a condensing agent such as N,N'-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The activated ester can also be produced by the reaction of carboxylic acid (5) with pentafluorophenyl trifluoroacetate or the like; the reaction of carboxylic acid (5) with 1-benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphite; the reaction of carboxylic acid (5) with diethyl cyanophosphonate (salt-in method); the reaction of carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyl disulfide (Mukaiyama method); the reaction of carboxylic acid (5) with a triazine derivative such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); etc. The reaction can also be carried out by the acid halide method, which can be carried out by treating carboxylic acid (5) with an acid halide such as thionyl chloride or oxalyl chloride in the presence of a base. Compound (6) can be produced by reacting the activated ester, mixed acid anhydride, or acid halide of carboxylic acid (5) obtained as described above with compound (4) in the presence of a suitable base in an inert solvent at a reaction temperature of −78° C. to 150° C. The term “inert solvent” refers to a solvent that does not inhibit the intended reaction carried out in the reaction in which the solvent is used.

[0148] Specific examples of the base used in each of the above steps include sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, water, Examples of the organic base include carbonates, alkoxides, hydroxides, or hydrides of alkali metals or alkaline earth metals, such as sodium hydride and potassium hydride; organometallic bases typified by alkyllithiums such as n-butyllithium and dialkylaminolithiums such as lithium diisopropylamide; organometallic bases of bissilylamines, such as lithium bis(trimethylsilyl)amide; and organic bases such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diisopropylethylamine, and diazabicyclo[5.4.0]undec-7-ene (DBU).

[0149] Examples of inert solvents used in this reaction include halogenated hydrocarbon solvents such as dichloromethane, chloroform, and carbon tetrachloride; ether solvents such as tetrahydrofuran, 1,2-dimethoxyethane, and dioxane; aromatic hydrocarbon solvents such as benzene and toluene; and amide solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidin-2-one. In addition to these, sulfoxide solvents such as dimethyl sulfoxide and sulfolane; ketone solvents such as acetone and methyl ethyl ketone; and alcohol solvents such as methanol and ethanol can also be used in some cases. Furthermore, these solvents can also be used in combination.

[0150] Protecting group P of the terminal amino group of compound (6) 1Examples of the protecting group P include tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, benzyloxycarbonyl, and other amino-protecting groups commonly used in peptide synthesis. Other protecting groups for amino groups include alkanoyl groups such as acetyl; alkoxycarbonyl groups such as methoxycarbonyl and ethoxycarbonyl; arylmethoxycarbonyl groups such as para-methoxybenzyloxycarbonyl and para-(or ortho)nitrobenzyloxycarbonyl; arylmethyl groups such as benzyl and triphenylmethyl; aroyl groups such as benzoyl; and arylsulfonyl groups such as 2,4-dinitrobenzenesulfonyl and ortho-nitrobenzenesulfonyl. 1 may be selected depending on the properties of the compound that protects the amino group. The protecting group P of the terminal amino group of the obtained compound (6) 1 Compound (7) can be produced by deprotecting the protecting group. For this deprotection, reagents and conditions can be selected according to the protecting group. N-terminus to P 2 The peptide carboxylic acid (8) protected with P is converted to an active ester, mixed acid anhydride, etc., and then reacted with the resulting compound (7), to produce compound (9). The reaction conditions, reagents, bases, and inert solvents for forming the peptide bond between peptide carboxylic acid (8) and compound (7) may be appropriately selected from those described in the synthesis of compound (6). 2 The protecting group may be appropriately selected from those described above for compound (6), and may be selected depending on the properties of the compound protecting the amino group, etc. Alternatively, compound (9) can be produced by repeating the sequential reaction and deprotection of the amino acids or peptides constituting peptide carboxylic acid (8) to elongate them, as is commonly done in peptide synthesis. The protecting group P of the amino group of the obtained compound (9) 2 The compound (10) can be produced by deprotecting the protecting group. The reagents and conditions for this protecting group can be selected according to the protecting group. Carboxylic acid (11) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with the resulting compound (10) to produce compound (2). The reaction conditions and reagents for forming the peptide bond between carboxylic acid (11) and compound (10), as well as the base and inert solvent, may be appropriately selected from those described in the synthesis of compound (6).

[0151] Compound (9) can also be produced, for example, by the following method. N-terminus to P 2 The peptide carboxylic acid (8) protected by P is converted to an activated ester, mixed acid anhydride, etc., and the carboxyl group is converted to P in the presence of a base. 3 and reacting the compound (12) with a protected amine compound (13). Compound (13) can be produced by the following procedure. The reaction conditions, reagents, bases, and inert solvents for forming the peptide bond between peptide carboxylic acid (8) and compound (12) may be appropriately selected from those described in the synthesis of compound (6). The protecting group P of the amino group of compound (13) 2There are no particular limitations on the protecting group as long as it is a commonly used protecting group. Specific examples of protecting groups for hydroxyl groups include alkoxymethyl groups such as a methoxymethyl group; arylmethyl groups such as a benzyl group, 4-methoxybenzyl group, and triphenylmethyl group; alkanoyl groups such as an acetyl group; aroyl groups such as a benzoyl group; and silyl groups such as a tert-butyldiphenylsilyl group. Carboxy groups can be protected as esters with alkyl groups such as a methyl group, an ethyl group, and a tert-butyl group, or with arylmethyl groups such as an allyl group or a benzyl group. Examples of the amino group include alkyloxycarbonyl groups such as a tert-butyloxycarbonyl group, a methoxycarbonyl group, and an ethoxycarbonyl group; arylmethoxycarbonyl groups such as an allyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, a paramethoxybenzyloxycarbonyl group, and a para(or ortho)nitrobenzyloxycarbonyl group; alkanoyl groups such as an acetyl group; arylmethyl groups such as a benzyl group and a triphenylmethyl group; aroyl groups such as a benzoyl group; and arylsulfonyl groups such as a 2,4-dinitrobenzenesulfonyl group and an orthonitrobenzenesulfonyl group. Carboxy protecting group P 3 As the protecting group, any protecting group that is commonly used as a protecting group for a carboxy group in organic synthetic chemistry, particularly in peptide synthesis, may be used. Specifically, a methyl group, an ethyl group, an alkyl ester such as tert-butyl, an allyl ester, a benzyl ester, etc. may be used, and an appropriate protecting group may be selected from the above-mentioned protecting groups. In this case, it is preferable that the protecting group for the amino group and the protecting group for the carboxyl group can be removed by different methods or under different conditions. 2 is a tert-butyloxycarbonyl group, and P 3 A typical example is a combination in which each of the protecting groups is a benzyl group. The protecting groups may be selected from those described above depending on the properties of the compound protecting the amino group and the carboxy group, and when cleaving the protecting groups, reagents and conditions may be selected depending on the protecting groups. The protecting group P of the carboxy group of the obtained compound (13) 3The compound (14) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. The resulting compound (14) can be converted into an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) in the presence of a base to produce compound (9). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and inert solvents can be appropriately selected from those described for the synthesis of compound (6).

[0152] Compound (2) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (13) 2 The compound (15) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. Carboxylic acid derivative (11) can be converted to an activated ester, mixed acid anhydride, acid halide, or the like, and reacted with the resulting compound (15) in the presence of a base to produce compound (16). The reaction conditions, reagents, bases, and inert solvents for forming the amide bond between peptide carboxylic acid (11) and compound (15) can be appropriately selected from those described in the synthesis of compound (6). The protecting group of the carboxy group in the resulting compound (16) can be deprotected to produce compound (17). This deprotection can be carried out in the same manner as the deprotection of the carboxy group in the production of compound (14). Compound (17) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) in the presence of a base to produce compound (2). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis. The base and inert solvent may be appropriately selected from those described in the synthesis of compound (6).

[0153] 3. Manufacturing method 3 The intermediate compound of formula (2) can also be prepared by the following method.

[0154] [ka]

[0155] [In the formula, L 1 ' is L 1 The terminal of P is converted to a maleimidyl group. 4 indicates a protecting group]

[0156] Compound (11) is converted to an activated ester, a mixed acid anhydride, or the like, and the C-terminus is converted to P in the presence of a base. 4 Compound (19) can be produced by reacting the peptide carboxylic acid (18) protected with a compound (11). The reaction conditions, reagents, bases, and inert solvents for forming the peptide bond between the peptide carboxylic acid (18) and compound (11) can be appropriately selected from those described in the synthesis of compound (6). 4 is selected appropriately from the protecting groups mentioned above. The protecting group of the carboxy group of the resulting compound (19) is deprotected to produce compound (20). This deprotection can be carried out in the same manner as the deprotection of the carboxy group in the production of compound (14). The resulting compound (20) can be converted into an activated ester, a mixed acid anhydride, or the like, and reacted with compound (7) to produce compound (2). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and inert solvents can be appropriately selected from those described for the synthesis of compound (6).

[0157] 4. Manufacturing method 4 Among the intermediates (10) described in Production Method 2, n 1 =1, L a Compounds with =O The production method of (10b) is described in detail below. The compound represented by formula (10b), a salt thereof or a solvate thereof can be produced, for example, by the following method.

[0158] [ka]

[0159] [In the formula, L P represents the same as above, and L is an acyl group, such as an alkanoyl group, e.g., an acetyl group. X and Y are each an aroyl group such as an aryl group or a benzoyl group, or a hydrogen atom, and an oligopeptide consisting of 1 to 3 amino acids is represented by P 5 and P 7 represents the protecting group of the amino group, P 6 represents a protecting group for a carboxy group]

[0160] The compound represented by formula (21) can be prepared by the method described in JP-A-2002-60351 or the method described in the literature (J. Org. Chem., Vol. 51, p. 3196, 1986), or by applying the method. They can be produced by removing the protecting group or converting the functional group as necessary, or by treating the acid amide of an amino acid having a protected terminal amino group or an oligopeptide having a protected amino group with an aldehyde or ketone. Compound (23) can be produced by reacting compound (21) with compound (22) having a hydroxyl group in an inert solvent in the presence of an acid or a base under cooling to room temperature. Examples of acids that can be used here include inorganic acids such as hydrofluoric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid; organic acids such as acetic acid, citric acid, paratoluenesulfonic acid, and methanesulfonic acid; and Lewis acids such as tetrafluoroborate, zinc chloride, tin chloride, aluminum chloride, and iron chloride. Among these, sulfonic acids are preferred, with paratoluenesulfonic acid being particularly preferred. Furthermore, the base may be appropriately selected from the bases already mentioned, with particular preference given to alkali metal alkoxides such as potassium tert-butoxide, alkali metal or alkaline earth metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal hydrides such as sodium hydride and potassium hydride, organometallic bases typified by dialkylaminolithium such as lithium diisopropylamide, and organometallic bases of bissilylamine such as lithium bis(trimethylsilyl)amide. The solvent used in the reaction may be an ether solvent such as tetrahydrofuran or 1,4-dioxane, or an aromatic hydrocarbon solvent such as benzene or toluene. The above solvents may be mixed with water. Also, P 5 There are no particular limitations on the amino-protecting group shown in the formula (I) as long as it is a group that is normally used for protecting an amino group. Representative examples include the amino-protecting groups described in Production Method 2. 5 In such a case, the protecting group may be reintroduced by reacting the compound with an appropriate amino-protecting reagent, if necessary. Compound (24) can be prepared by removing the protecting group P of compound (23). 6 where P 6 Representative examples of the protecting group for the carboxyl group shown in the formula (23) are described in the production method 2, and can be appropriately selected from these. 5 and a protecting group P of the carboxy group 6 is preferably a protecting group that can be removed by different methods or conditions. For example, P 5 is a 9-fluorenylmethyloxycarbonyl group, and P6 A typical example is a combination in which the protecting group is a benzyl group. The protecting group can be selected depending on the properties of the compound that protects the amino group and the carboxy group. When removing these protecting groups, reagents and conditions may be selected according to the protecting group. Carboxylic acid (24) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) or a pharmacologically acceptable salt thereof in the presence of a base to produce compound (25). 5 The compound (26) can be prepared by the reaction of the compound (4) with the carboxylic acid (24) and the protecting group P 6 In the reaction for removing the compound, the same reagents and reaction conditions as those described in Production Method 2 may be used. Compound (26) is reacted with an amino acid having a protected terminal amino group or an oligopeptide (27) having a protected terminal amino group to produce compound (9b), and the protecting group P 7 Compound (10b) can be prepared by removing P 7 The amino-protecting group shown in is not particularly limited as long as it is a group normally used for protecting an amino group, and representative examples include the amino-protecting groups described in Production Method 2. For its removal, reagents and conditions may be selected according to the protecting group. In the reaction between compound (26) and compound (27), reaction reagents and conditions normally used in peptide synthesis may be applied. Compound (10b) produced by the above method can be converted into compound (1) of the present invention according to the above production method.

[0161] 5. Manufacturing method 5 The intermediate compound (2) can also be prepared by the following method.

[0162] [ka]

[0163] [In the formula, L1 ' is L 1 The terminal of L is converted to a maleimidyl group. P Ha-L p1 -L p2 -mosquito The structure is called P 3 , P 8 , P 9 , P 10 , P 11 , and P 12 indicates a protecting group.]

[0164] L P L p1 and L p2 Since it is formed by combining P The hydrophilic amino acid at the N-terminus of p1 Reason Since it will be p1 The N-terminus of the amino acid may be a hydrophilic amino acid. There may be a plurality of amino acids. p2 If you use a substance that contains hydrophilic amino acids, , depending on its position, L P L containing multiple hydrophilic amino acids at the N-terminus or at the N-terminus and other positions P can be manufactured.

[0165] N-terminus to P 2 The peptide or amino acid (28) protected by the formula (I) is converted into an active ester, a mixed acid anhydride, or the like, and reacted with the resulting compound (7), thereby producing compound (29). The reaction conditions, reagents, bases, and solvents for forming the amide bond between the peptide or amino acid (28) and compound (7) may be appropriately selected from those described in the synthesis of compound (6). 8 The protecting group may be appropriately selected from those described for compound (6) and may be selected depending on the properties of the compound, etc. Alternatively, compound (29) can be produced by repeating the reaction and deprotection of the amino acids or peptides constituting peptide or amino acid (28) in sequence, as is commonly used in peptide synthesis. The protecting group P of the amino group of the obtained compound (29)8 The compound (23) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the appropriate reagent and conditions depending on the protecting group. N-terminus to P 8 The carboxyl group, hydroxyl group, or amino group on the side chain is P 9 The amino acid or peptide (31) protected by the protecting group P is converted into an active ester, mixed acid anhydride, or the like, and reacted with the resulting compound (30), thereby producing compound (32). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the amino acid or peptide (31) and compound (30) may be appropriately selected from those described in the synthesis of compound (6). 8 and P 9 The protecting group P of the amino group may be appropriately selected from those described above for the protecting group of the amino group, carboxy group, or hydroxyl group of the compound (6). 9 and the protecting group P of the side chain functional group 10 It is necessary to be able to remove P by different methods or conditions. 9 9-Fluorenylmethyl is a methyloxycarbonyl group, and P 10 is a protecting group for the carboxy group, then tert-butyl Representative examples of the combination include a protecting group P of a functional group in a side chain, a protecting group for a hydroxyl group such as a methoxymethyl group, and a protecting group for an amino group such as a tert-butyloxycarbonyl group. 10 A protecting group that can be removed by subjecting it to acidic conditions is preferred. Preferably, but not limited to, the protecting group may be selected from those mentioned above depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected. Furthermore, when cleaving these protecting groups, reagents and conditions may be selected according to the protecting group. Incidentally, compound (32) can also be produced by sequentially reacting and deprotecting the constituent amino acids or peptides repeatedly to elongate them, as is commonly done in peptide synthesis. The protecting group P of the terminal amino group of the obtained compound (32) 9The compound (33) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. The carboxylic acid derivative (11) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with the resulting compound (33) to produce the compound (34). 1 ' is a compound having a structure in which the linker end has a maleimidyl group. The reaction conditions, reagents, bases and solvents for forming the peptide bond between the carboxylic acid derivative (11) and the compound (33) may be appropriately selected from those described in the synthesis of the compound (6). The protecting group P of the carboxyl group, hydroxyl group, or amino group in the amino acid side chain of the peptide portion of the obtained compound (34) 10 Compound (2) can be produced by deprotecting the The reagents and conditions may be selected depending on the protecting group.

[0166] Compound (29) can also be produced, for example, by the following method. N-terminus to P 8 The peptide or amino acid (28) protected by the 3 Compound (35) can be produced by reacting the compound (35) with an amine compound (12) protected with a protecting group P. The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the peptide or amino acid (28) and compound (12) can be appropriately selected from those described in the synthesis of compound (6). 8 The protecting group P of the carboxy group may be appropriately selected from those described above for the protecting group of compound (6). 3 As the protecting group, any protecting group that is commonly used as a protecting group for a carboxy group in organic synthetic chemistry, especially in peptide synthesis, may be used. Specifically, a protecting group may be appropriately selected from those described for the protecting group of compound (6), such as a methyl group, an ethyl group, an alkyl ester such as tert-butyl, an allyl ester, a benzyl ester, etc. In this case, the protecting group P of the amino group 8and a protecting group P of the carboxy group 3 It is necessary to be able to remove P by different methods or conditions. 8 is a tert-butyloxycarbonyl group, and P 3 A typical example is a combination in which each of the protecting groups is a benzyl group. The protecting groups may be selected from those described above depending on the properties of the compound protecting the amino group and the carboxy group, and when cleaving the protecting groups, reagents and conditions may be selected depending on the protecting groups. The protecting group P of the carboxy group of the obtained compound (35) 3 The compound (36) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagents and conditions according to the protecting group. The resulting compound (36) can be converted into an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) in the presence of a base to produce compound (29). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and solvents can be appropriately selected from those described in the synthesis of compound (6).

[0167] Compound (32) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (35) 8 The compound (37) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. Compound (38) can be produced by converting amino acid or peptide (31) into an activated ester, mixed acid anhydride, acid halide, or the like, and reacting the resulting compound (37) in the presence of a base. The reaction conditions, reagents, bases, and solvents for forming the amide bond between amino acid or peptide (31) and compound (37) can be appropriately selected from those described in the synthesis of compound (6). Here, the protecting group P of amino acid or peptide (31) can be used. 9 , P 10 transformation The protecting group P of compound (37) 3 Each of these must be removed by a different method or under different conditions. 9is a 9-fluorenylmethyloxycarbonyl group, and P 10 tert-butyloxy Carbonyl group, tert-butyl group, or methoxymethyl group, P 3 A typical example is a combination in which the protecting group P of the functional group in the side chain is a benzyl group. 10 The protecting group is preferably a protecting group that can be removed by subjecting it to acidic conditions, but is not limited thereto. The protecting group is not limited to any particular protecting group, and may be selected from those described above depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected, and when cleaving the protecting group, reagents and conditions may be selected according to the protecting group. The protecting group P of the carboxy group of the obtained compound (38) 3 The compound (39) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the appropriate reagent and conditions depending on the protecting group. Compound (39) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) in the presence of a base to produce compound (32). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and solvents can be appropriately selected from those described in the synthesis of compound (6).

[0168] Compound (34) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (38) 9 The compound (40) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. Carboxylic acid derivative (11) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and the resulting product is reacted with the resulting compound (40) in the presence of a base to produce compound (41). The reaction conditions, reagents, bases, and solvents for forming the amide bond between carboxylic acid derivative (11) and compound (40) may be appropriately selected from those described in the synthesis of compound (6). The protecting group P of the carboxy group of the obtained compound (41) 3The compound (42) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. Compound (42) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) in the presence of a base to produce compound (34). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and solvents can be appropriately selected from those described in the synthesis of compound (6).

[0169] Compound (34) can also be produced, for example, by the following method. The carboxylic acid derivative (11) is converted to an activated ester, a mixed acid anhydride, or an acid halide, and the carboxy group is converted to P in the presence of a base. 11 The carboxyl group, hydroxyl group or amino group on the side chain is P 10 Compound (44) can be produced by reacting the carboxylic acid derivative (11) with an amino acid or peptide (43) protected by the formula (44). The reaction conditions, reagents, bases, and solvents for forming the amide bond between the carboxylic acid derivative (11) and compound (43) can be appropriately selected from those described in the synthesis of compound (6). 10 and P 11 The protecting group P of the carboxyl group, hydroxyl group, or amino group of the compound (6) may be appropriately selected from those mentioned above. 11 and the protecting group P of the side chain functional group 10 It is necessary to be able to remove P by different methods or conditions. 11 is a benzyl group, and P 10 is a tert-butyl group or the like if it is a protecting group for a carboxy group, a methoxymethyl group or the like if it is a protecting group for a hydroxyl group, or a tert-butyloxycarbonyl group or the like if it is a protecting group for an amino group. 10 is subjected to acidic conditions Protecting groups that can be deprotected by cleaving the protecting group are preferred, but are not limited thereto, and may be selected from the above-mentioned groups depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected, and when cleaving the protecting group, reagents and conditions may be selected depending on the protecting group. The protecting group P of the carboxy group of the obtained compound (44) 11 By deprotecting the compound The deprotection can be carried out by selecting the appropriate reagents and conditions depending on the protecting group. Compound (45) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (30) in the presence of a base to produce compound (34). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and solvents can be appropriately selected from those described in the synthesis of compound (6). Alternatively, compound (45) may be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and the carboxyl group may be converted to P in the presence of a base. 12 and reacting with a protected amino acid or peptide (46) Compound (47) can be produced by this reaction. This reaction can be carried out by applying the reaction reagents and conditions normally used in peptide synthesis, and the reaction conditions, reagents, bases, and solvents can be appropriately selected from those described in the synthesis of compound (6). 10 and P 12 Examples of the protecting groups include those described above for the carboxyl, hydroxyl or amino groups of compound (6). However, in this case, the protecting group P 12 and the protecting group P of the side chain functional group 10 It is necessary to be able to remove P by different methods or conditions. 12 is a benzyl group, and P 10 is a protecting group for a carboxyl group, such as a tert-butyl group, For example, a protecting group for a methyl group is a methoxymethyl group, and for an amino group is a tert-butyloxy group. A typical example is a combination of a protecting group P of a functional group in a side chain, such as a hydroxyl group, a hydroxyl group, etc. 10 A protecting group that can be removed by subjecting it to acidic conditions is preferred. The protecting group may be selected from those listed above depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected, and when cleaving the protecting group, reagents and conditions may be selected according to the protecting group. Compound (47) can also be produced by sequentially reacting and deprotecting the constituent amino acids or peptides, and then elongating them. The protecting group P of the carboxy group of the obtained compound (47) 12 By deprotecting the compound (48) can be produced by selecting the reagents and conditions according to the protecting group. Compound (48) can be converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (7) in the presence of a base to produce compound (34). This reaction can be carried out using reaction reagents and conditions commonly used in peptide synthesis, and the reaction conditions, reagents, bases, and solvents can be appropriately selected from those described in the synthesis of compound (6). Compound (47) can also be produced, for example, by the following method. Amino acid or peptide (46) is converted to an activated ester, mixed acid anhydride, or acid halide, and the N-terminus is converted to P in the presence of a base. 9 The carboxyl group, hydroxyl group or amino group on the side chain is P 10 The peptide (49) can be produced by reacting the amino acid or peptide (46) with the amino acid or peptide (31) protected by the formula (32). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the amino acid or peptide (46) and the amino acid or peptide (31) can be appropriately selected from those described in the synthesis of compound (6). Here, the protecting group P of the carboxy group of the amino acid or peptide (46) is 12 and a protecting group P of the amino acid or peptide (31). 9 and P 10 As mentioned above, they must be removed by different methods or conditions. For example, P9 is a 9-fluorenylmethyloxycarbonyl group, and P 10 If is a protecting group for the carboxy group, ter t-butyl group, etc., a hydroxyl group protecting group is a methoxymethyl group, an amino group protecting group is a tert-butyloxycarbonyl group, etc., 12 is a benzyl group, etc. The following can be mentioned as a representative example. 10 is subjected to acidic conditions However, the protecting group is not limited thereto and may be selected from the above-mentioned groups depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected, and when cleaving the protecting group, reagents and conditions may be selected depending on the protecting group. The N-terminal protecting group P of the obtained peptide (49) 9 The peptide (50) can be produced by deprotecting the protecting group. The reagents and conditions can be selected according to the protecting group. Carboxylic acid derivative (11) can be converted to an activated ester, mixed acid anhydride, acid halide, or the like, and reacted with the resulting peptide (50) in the presence of a base to produce compound (47). The reaction conditions, reagents, bases, and solvents for forming the amide bond between carboxylic acid derivative (11) and peptide (50) can be appropriately selected from those described in the synthesis of compound (6).

[0170] 6. Manufacturing method 6 In the production intermediate (2), the linker is -L 1 -L 2 -L P -, and the L P A peptide residue in which the N-terminus is a hydrophilic amino acid, and in which the hydrophilic amino acid present at the N-terminus is a hydrophilic amino acid other than glycine, can also be produced by the method described below.

[0171] [ka]

[0172] [Wherein, L 1 'L 1 The terminal of L is converted to a maleimidyl group. P Ha-L p1 -L p2 - a structure consisting of P 8 , P 9 , P 10 , and P 12 indicates a protecting group.]

[0173] L P L p1 and L p2 Since it is formed by combining P The hydrophilic amino acid at the N-terminus of p1 Reason Since it will be p1 The N-terminus of the amino acid may be a hydrophilic amino acid. There may be a plurality of amino acids. p2 If you use a substance that contains hydrophilic amino acids, , depending on its position, L P L containing multiple hydrophilic amino acids at the N-terminus or at the N-terminus and other positions P can be manufactured. The N-terminus of the compound described in Production Method 5 is converted to P 8 The peptide or amino acid (28) protected by the protecting group P is converted into an active ester, a mixed acid anhydride, or the like, and reacted with the compound (4) or a salt thereof to produce the compound (51). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the peptide or amino acid (28) and the compound (4) may be appropriately selected from those described in the synthesis of the compound (6). 8 The protecting group may be appropriately selected from those described for compound (6) and may be selected depending on the properties of the compound protecting the amino group, etc. Alternatively, compound (51) can be produced by repeating the sequential reaction and deprotection of the amino acids or peptides constituting peptide or amino acid (28) to elongate the peptide or amino acid (28), as is commonly used in peptide synthesis. The protecting group P of the amino group of the obtained compound (51)8 The compound (52) can be produced by deprotecting the protecting group. The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. The N-terminus of the compound according to Production Method 4 is changed to P 9 The carboxyl group, hydroxyl group, or amino group on the side chain is P 10 The protected amino acid or peptide (31) is converted to an active ester, a mixed acid anhydride, or the like, The resulting compound (52) can be reacted to produce compound (53). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the amino acid or peptide (31) and compound (52) can be appropriately selected from those described in the synthesis of compound (6). 9 and P 10 The procedure is as described in Production Method 5. As is commonly used in synthesis, compound (53) can also be produced by sequentially reacting and deprotecting the constituent amino acids or peptides, and then elongating them. The protecting group P of the amino group of the obtained compound (53) 9 Compound (54) can be produced by deprotecting the protecting group. For this deprotection, reagents and conditions can be selected according to the protecting group. Carboxylic acid derivative (11) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and the resulting compound (54) is reacted to produce compound (55). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between carboxylic acid derivative (11) and compound (54) may be appropriately selected from those described in the synthesis of compound (6). The protecting group P of the carboxyl group, hydroxyl group, or amino group of the obtained compound (55) 10 Deprotection Compound (2) can be produced by deprotecting the protecting group. The reagents and conditions for this deprotection can be selected according to the protecting group.

[0174] Compound (53) can also be produced, for example, by the following method. The protecting group P of the carboxy group of peptide (49) described in Production Method 512 Deprotecting the The peptide (56) can be produced by the following procedure. For this deprotection, reagents and conditions can be selected according to the protecting group. The resulting peptide (56) can be converted into an active ester, a mixed acid anhydride, an acid halide, or the like, and reacted with compound (4) or a salt thereof to produce compound (53). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between compound (56) and compound (4) can be appropriately selected from those described in the synthesis of compound (6).

[0175] Compound (55) can also be prepared by the following method, for example. Compound (55) can be produced by converting compound (48) described in Production Method 5 into an activated ester, mixed acid anhydride, or the like, and reacting it with compound (4) in the presence of a base, or by converting amino acid or peptide (45) described in Production Method 5 into an activated ester, mixed acid anhydride, or the like, and reacting it with compound (52) described above in the presence of a base. The reaction conditions, reagents, bases, and solvents for forming the respective peptide bonds may be appropriately selected from those described in the synthesis of compound (6).

[0176] 7. Manufacturing method 7 Among the production intermediates represented by formula (2), the linker is -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-, and the L P When the N-terminus of the peptide residue is a hydrophilic amino acid, and the hydrophilic amino acid present at the N-terminus is a hydrophilic amino acid other than glycine, it can also be produced, for example, by the method described below.

[0177] [ka]

[0178] [In the formula, L 1 'L 1 The terminal of L is converted to a maleimidyl group. P Ha-L p1 -L p2 -mosquito The structure is called P 9 , P 13 indicates a protecting group.

[0179] The intermediate compound represented by formula (2) has a linker of -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- and -L 1 -L 2 -L P There are two variations of the structure indicated by -. The linker uses -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 The compound (2) having the structure represented by -C(=O)- can be prepared as follows. Compound (57) can be synthesized in the same manner as compound (32) described in Production Method 5, but unlike compound (32), the protecting group P 9 and the protecting group P of the side chain functional group 13 Different It is not necessary that the functional group of the side chain is a carboxyl group or a hydroxyl group, and the protecting group P of the amino group is 9 and a protecting group P of the carboxyl or hydroxyl group in the side chain 13 simultaneously For example, P 9 is a tert-butyloxycarbonyl group, and P 13 is a tert-butyl group, a trityl group, or P 3 is a benzyloxycarbonyl group, and P 13Representative examples include a combination in which R is a benzyl group or the like. The protecting group may be appropriately selected from those described for protecting groups of compound (6) depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected, and when cleaving the protecting group, reagents and conditions may be selected according to the protecting group. Compound (57) can be synthesized in the same manner as in Production Method 5, using a protected amino acid or peptide that satisfies the above properties. The protecting group P of compound (57) 9 , P 13 and (5) are deprotected sequentially or simultaneously to give compound (5) 1) can be produced by selecting the reagents and conditions according to the protecting group. Compound (58) is L P Although the functional group of the hydrophilic side chain of is not particularly protected, compound (2) can be produced by reacting compound (11) derived into an active ester, mixed acid anhydride, etc. in the presence of a base. The reaction conditions, reagents, bases, and solvents for forming each peptide bond may be appropriately selected from those described in the synthesis of compound (6). The linker uses -L 1 -L 2 -L P The compound (2) having the structure represented by - can be prepared as follows. Compound (59) can be synthesized in the same manner as compound (53) described in Production Method 6, but unlike compound (53), the protecting group P 3 and the protecting group P of the side chain functional group 8 It is not necessary that the protecting group P of the amino group can be removed by a different method or condition. 9 and a protecting group P of the carboxyl or hydroxyl group in the side chain 13 at the same time It can also be deprotected. For example, P 9 is a tert-butyloxycarbonyl group, and P 13 is a tert-butyl group, a trityl group, or P 3 is a benzyloxycarbonyl group, and P 13is a benzyl group, etc. The protecting group may be appropriately selected from those described for compound (6) depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected, and the reagents and conditions for cleaving the protecting group may be selected according to the protecting group. Compound (59) can be synthesized in the same manner as in Production Method 6, using a protected amino acid or peptide that satisfies the above properties. The protecting group P of compound (59) 9 , P 13 and (5) are deprotected sequentially or simultaneously to give compound (5) 3) can be produced by selecting the reagents and conditions according to the protecting group. Compound (60) is L P Although the functional group of the hydrophilic side chain of is not particularly protected, compound (2) can be produced by reacting compound (11) derived into an active ester, mixed acid anhydride, etc. in the presence of a base. The reaction conditions, reagents, bases, and solvents for forming each peptide bond may be appropriately selected from those described in the synthesis of compound (6).

[0180] 8. Manufacturing method 8 Among the compounds (43) shown in Production Method 5, the linker-L P -ga, -L p1 When the structure is -Gly-Gly-Phe-Gly-, it can also be produced by the following method.

[0181] [ka]

[0182] [In the formula, P 9 , P 10 indicates a protecting group.

[0183] The amino acid or peptide (31) described in Production Method 5 can be converted to an activated ester, mixed acid anhydride, or acid halide, and then reacted with glycylglycyl-L-phenylalanyl-N-[(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]glycinamide (the free form of the pharmaceutical compound described in WO 1997 / 46260) (61) or a salt thereof in the presence of a base to produce compound (62). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the amino acid or peptide (31) and compound (61) can be appropriately selected from those described in the synthesis of compound (6). The N-terminal protecting group P 3 , a protecting group P of the side chain functional group 10 As for the manufacturing method 5, In addition, the protecting group P of the functional group of the side chain 10 It is not necessary to have an amino acid sequence protected only at the N-terminus. The reaction can be carried out using a monoacid or peptide (31) to give compound (62).

[0184] 9. Manufacturing method 9 Among the compounds represented by formula (2), the linker is -L 1 -L 2 -L P -, and the L P When the C-terminus of the oligopeptide is an oligopeptide consisting of two or more glycines, which is bound to a drug, and the N-terminus of the peptide residue is a hydrophilic amino acid, the peptide residue is glycine, it can also be produced, for example, by the method described below.

[0185] [ka]

[0186] [In the formula, L 1 'L 1 The terminal of L is converted to a maleimidyl group.P is L p1 -L p2 from The structure is P 12 and P 14 indicates a protecting group.

[0187] L P L p1 and L p2 Since it is formed by combining P The number of glycines that make up the C-terminus of L P The number of C-terminal glycines contained in the enzyme and the number of repetitions of using these glycines during the reaction can be taken into consideration when designing the enzyme. Peptide (63) is an oligopeptide whose C-terminus is two or more glycines, and when the N-terminus of the peptide residue is a hydrophilic amino acid, the peptide residue is glycine, and the N-terminus is P 14 Peptide (63) is a peptide synthesis As is commonly used in the synthesis of hydroxybenzoates, the constituent amino acids or peptides can be synthesized by sequentially repeating condensation reactions and deprotection. Compound (64) can be produced by converting peptide (63) into an activated ester, mixed acid anhydride, or the like, and reacting it with compound (4) or a salt thereof. The reaction conditions, reagents, bases, and solvents for forming the peptide bond between peptide (63) and compound (4) can be appropriately selected from those described in the synthesis of compound (6). Protecting group P 14 is the compound (6) The composition may be appropriately selected from those described above. Compound (64) also has an N-terminus converted to P 14 The protected amino acid or peptide (65) was activated. The compound (52) can also be prepared by converting the compound (52) into a protective ester, mixed acid anhydride, or the like, and reacting the resulting compound with the compound (52) described in Preparation Method 6. The reaction conditions, reagents, bases, and solvents for forming the peptide bond between the amino acid or peptide (65) and the compound (52) may be appropriately selected from those described in the synthesis of the compound (6). 14 The same as described in the synthesis of compound (6) It is sufficient to select and use it appropriately from the above. The protecting group P of the amino group of the obtained compound (64) 14 By deprotecting the compound (6 6) can be produced by selecting the reagents and conditions according to the protecting group. Carboxylic acid derivative (11) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and the resulting compound (66) is reacted to produce compound (2). The reaction conditions, reagents, bases, and solvents for forming the amide bond between carboxylic acid derivative (11) and compound (66) may be appropriately selected from those described in the synthesis of compound (6). Compound (2) can also be produced by the following method. Compound (67) is L p1 The N-terminal glycine of 2 and the compound ( The amino acid or peptide (4) described in Production Method 5 can be synthesized in the same manner as in Production Method 45. Compound (68) can be produced by converting compound (6) into an active ester, a mixed acid anhydride, an acid halide, or the like, and reacting it with compound (67). Here, the amino acid or peptide (46) is glycine or an oligopeptide having two or more glycines at its C-terminus, and the C-terminus is P 12 Protected by: Amino acid or peptide The reaction conditions, reagents, bases and solvents for forming the amide bond between (46) and compound (67) may be appropriately selected from those described in the synthesis of compound (6). Compound (68) can be prepared by converting compound (11) into an active ester, a mixed acid anhydride, or the like, and converting the C-terminus to P 12 It can also be prepared by reacting the protected peptide (69) with Here, peptide (69) is an oligopeptide whose C-terminus is composed of two or more glycines, and when the N-terminus is a hydrophilic amino acid, the peptide residue is glycine. Peptide (69) can be synthesized by repeating the condensation reaction and deprotection of the constituent amino acids or peptides in sequence, as is commonly used in peptide synthesis. The reaction conditions, reagents, bases, and solvents for forming the peptide bond between peptide (69) and compound (11) may be appropriately selected from those described in the synthesis of compound (6). Protecting group P 12 is deprotected under acidic conditions. Protective groups that can be used are preferred, but are not limited thereto, and may be appropriately selected from those described in the synthesis of compound (6). The protecting group P of the carboxy group of the obtained compound (68) 12 By deprotecting the compound The deprotection can be carried out by selecting the reagents and conditions according to the protecting group. Compound (70) can be converted into an activated ester, a mixed acid anhydride, or the like, and reacted with compound (4) or a salt thereof to produce compound (2). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between compound (70) and compound (4) can be appropriately selected from those described in the synthesis of compound (6). Alternatively, compound (2) can be produced by the following method. Compound (52) described in Production Method 6 can be converted to an activated ester, a mixed acid anhydride, or the like, and reacted with compound (67) in the presence of a base to produce compound (2). The reaction conditions, reagents, bases, and solvents for forming a peptide bond between compound (67) and compound (52) may be appropriately selected from those described in the synthesis of compound (6).

[0188] The intermediate compounds in Production Methods 1 to 9 are all salts and / or hydrates. It is also possible.

[0189] The antibody-drug conjugates of the present invention may become hydrated by absorbing moisture or by adsorbing water when left in the air or when subjected to a purification procedure such as recrystallization, and such compounds or salts containing water are also encompassed by the present invention. The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugate of the present invention may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or carbon-14 ( 14 C) and the like. In addition, the compound of the present invention can be used in combination with, for example, tritium ( 3 H), iodine-125( 125 I), carbon-14( 14 C), copper-64( 64 Cu), Ruthenium-89( 89 Zr), Iodine-124( 124 I), Fluorine-18( 18 F), Indium-111( 111 I), carbon-11( 11 C) or Iodine-131( 131 I) Radiolabeled with a radioisotope such as Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo diagnostic imaging agents. All isotopic variants of the antibody-drug conjugates of the invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0190] [Pharmaceuticals] The anti-HER3 antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells and can therefore be used as a pharmaceutical, particularly as a therapeutic and / or preventive agent for cancer. Cut. In other words, the anti-HER3 antibody-drug conjugates of the present invention can be selected and used as drugs for chemotherapy, a major cancer treatment, and as a result, can slow the growth of cancer cells, suppress their proliferation, and even destroy them. This can relieve cancer patients from cancer-related symptoms, improve their quality of life, and achieve therapeutic effects while preserving their lives. 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. In addition to being used alone in such drug therapies, it 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. In addition to the therapeutic uses described above, the anti-HER3 antibody-drug conjugate of the present invention can be expected to suppress the growth of, and even destroy, micrometastatic cancer cells. In particular, when HER3 expression is confirmed in primary cancer cells, administration of the anti-HER3 antibody-drug conjugate of the present invention can be expected to suppress or prevent cancer metastasis. For example, it can be expected to suppress and destroy cancer cells present in body fluids during the metastatic process, as well as suppress and destroy microscopic cancer cells immediately after implantation in any tissue. Therefore, it can be expected to suppress and prevent cancer metastasis, especially after surgical removal of cancer. The anti-HER3 antibody-drug conjugate of the present invention can be administered to patients as a systemic therapy, and can also be administered locally to cancer tissues to be expected to have a therapeutic effect. The antibody-drug conjugate (1) has excellent antitumor activity, safety, and physical properties, and has demonstrated anti-breast cancer, anti-lung cancer, and anti-melanoma effects in vitro. The antibody-drug conjugate (2) has excellent antitumor activity, safety, and physical properties, and has shown anti-breast cancer, anti-lung cancer, anti-colon cancer, and anti-melanoma effects in vitro, and U It showed stronger anti-breast cancer and anti-melanoma effects than 1-59. The antibody-drug conjugate (3) has excellent antitumor activity, safety, and physical properties, and exhibits anti-breast cancer, anti-lung cancer, anti-ovarian cancer, anti-colon cancer, and anti-melanoma effects in vitro, and exhibits stronger anti-breast cancer, anti-lung cancer, anti-colon cancer, anti-gastric cancer, and anti-melanoma effects than U1-59 in vivo. It showed anti-melanoma activity. The antibody-drug conjugate (4) has excellent antitumor activity, safety, and physical properties, and has demonstrated anti-breast cancer activity in vitro. The antibody-drug conjugate (5) has excellent antitumor activity, safety, and physical properties, and has demonstrated anti-breast cancer, anti-lung cancer, and anti-melanoma effects in vitro. The antibody-drug conjugate (6) has excellent antitumor activity, safety, and physical properties, and exhibits anti-breast cancer, anti-lung cancer, and anti-melanoma effects in vitro, and is more potent than U1-59 in vivo. It showed anti-breast cancer activity. The antibody-drug conjugate (7) has excellent antitumor activity, safety, and physical properties, and has demonstrated anti-breast cancer, anti-lung cancer, and anti-melanoma effects in vitro. The antibody-drug conjugate (8) has excellent antitumor activity, safety, and physical properties, and exhibits anti-breast cancer, anti-lung cancer, and anti-melanoma effects in vitro, and is more potent than U1-59 in vivo. It showed anti-breast cancer activity. The antibody-drug conjugate (9) has excellent antitumor activity, safety, and physical properties, and has demonstrated anti-breast cancer, anti-lung cancer, anti-ovarian cancer, anti-colon cancer, and anti-melanoma effects in vitro. The antibody-drug conjugate (10) has excellent antitumor activity, safety, and physical properties. It has anti-breast cancer, anti-lung cancer, anti-colon cancer and anti-melanoma effects in vitro and anti-melanoma effects in vivo. It showed stronger anti-breast cancer, anti-lung cancer, anti-colon cancer, anti-gastric cancer, and anti-melanoma effects than U1-59. The antibody-drug conjugate (11) has excellent antitumor activity, safety, and physical properties. It showed anti-breast cancer activity in vitro. The antibody-drug conjugate (12) has excellent antitumor activity, safety, and physical properties. It has shown anti-breast cancer, anti-lung cancer, anti-ovarian cancer, anti-colon cancer and anti-melanoma effects in vitro. The antibody-drug conjugate (13) has excellent antitumor activity, safety, and physical properties. It has anti-breast cancer, anti-lung cancer, anti-colon cancer and anti-melanoma effects in vitro and anti-melanoma effects in vivo. It exhibited stronger anti-breast cancer (including triple-negative breast cancer) activity, anti-lung cancer activity, anti-colon cancer activity, anti-gastric cancer activity, anti-pancreatic cancer activity, and anti-melanoma activity than U1-59. The antibody-drug conjugate (14) has excellent antitumor activity, safety, and physical properties. It showed anti-breast cancer activity in vitro. The antibody-drug conjugate (16a) has excellent antitumor activity, safety, and physical properties, and has been shown to have anti-breast cancer (including luminal breast cancer and triple-negative breast cancer) and anti-melanoma effects in vivo. It has shown anti-ovarian cancer, anti-bladder cancer, anti-lung cancer, anti-head and neck cancer, and anti-gastric cancer activity, either alone or in combination with trastuzumab, gefitinib, cetuximab, panitumumab, or pertuzumab.

[0191] Examples of cancers to which the anti-HER3 antibody-drug conjugates of the present invention can be applied include lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, metastatic breast cancer, luminal breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, and penile cancer. In particular, triple-negative breast cancer (which does not express HER2, estrogen receptor, or progesterone receptor) is currently the only treatment available, and is said to have a poor prognosis. Although there have been few reports on HER3 expression in triple-negative breast cancer, if HER3 expression is observed in patients with triple-negative breast cancer, the anti-HER3 antibody-drug conjugate of the present invention can be used as a therapeutic and / or preventive agent. However, the cancer cells to be treated are not limited to these, as long as they express a protein that can be recognized by the antibody in the antibody-drug conjugate. The anti-HER3 antibody-drug conjugate of the present invention targets cancer cells that express the HER3 protein, which can be recognized by the antibody in the antibody-drug conjugate. As used herein, "cancer expressing HER3 protein" refers to cancers containing cells that have HER3 protein on their cell surface, or cancers that secrete HER3 protein into the blood. HER3 protein is overexpressed in various human tumors, and immunohistochemical staining (IHC) is used to evaluate HER3 protein overexpression in tumor (primary and metastatic) specimens, fluorescent in situ hybridization (FISH) is used to evaluate HER3 gene amplification, and blood The evaluation can be performed using methods commonly used in the field, such as enzyme-linked immunosorbent assay (ELISA) for evaluating overexpression of HER3 protein in a specimen. Furthermore, the anti-HER3 antibody-drug conjugate of the present invention exerts an anti-tumor effect by the anti-HER3 antibody recognizing and further internalizing the HER3 protein expressed on the surface of cancer cells. Therefore, the therapeutic targets of the anti-HER3 antibody-drug conjugate of the present invention are not limited to "cancers expressing HER3 protein," and can also be, for example, leukemia, malignant lymphoma, plasma cell carcinoma, myeloma, or sarcoma.

[0192] The anti-HER3 antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0193] Substances used in pharmaceutical compositions containing the anti-HER3 antibody-drug conjugates of the present invention can be appropriately selected and applied in terms of dosage and administration concentration from formulation additives and other substances commonly used in this field.

[0194] The anti-HER3 antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical composition typically contains one or more pharmaceutical carriers (e.g., sterile liquids). 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 and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients can be selected from those known in the art. The 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.

[0195] Various delivery systems are known and can be used to administer the anti-HER3 antibody-drug conjugates of the present invention. Introduction methods 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, the ligand-drug conjugate is administered by infusion. Parenteral administration is a preferred route of administration.

[0196] In a representative embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the medicament 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 medicament is in a form to be administered by infusion, 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.

[0197] The pharmaceutical composition of the present invention may contain only the anti-HER3 antibody-drug conjugate of the present application as an active ingredient, or it may contain the anti-HER3 antibody-drug conjugate and at least one other drug (e.g., a cancer therapeutic agent). The anti-HER3 antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, thereby enhancing the anti-cancer effect. The other drug, such as an anti-cancer drug, used for such a purpose may be administered, for example, followed by administration of a pharmaceutical composition containing the anti-HER3 antibody-drug conjugate of the present invention as an active ingredient, or after administration of a pharmaceutical composition containing the anti-HER3 antibody-drug conjugate as an active ingredient, or may be administered to an individual simultaneously with the antibody-drug conjugate, separately (individually), or consecutively, or at different administration intervals. In the present invention, the "pharmaceutical composition comprising an antibody-drug conjugate and another drug" encompasses both cases where the antibody-drug conjugate and the other drug are administered simultaneously as a single formulation, and cases where the antibody-drug conjugate and the other drug are administered separately as separate formulations simultaneously, consecutively, or at different administration intervals. Such cancer treatment agents include 5-FU, trastuzumab, trastuzumab emtansine (T-DM1), cetuximab, gefitinib, panitumumab, pertuzumab, abraxane, erlotinib, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), paclitaxel, docetaxel, and pemetrex. Examples of anti-cancer drugs include ed, sorafenib, vinblastin, vinorelbine, vemurafenib, or the drugs described in International Publication No. 2003 / 038043, as well as LH-RH analogs (e.g., leuprorelin, goserelin), estramustine phosphate, estrogen antagonists (e.g., tamoxifen, raloxifene), and aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), but are not limited thereto as long as they have anti-tumor activity. These cancer therapeutic agents can be classified based on their targets into anti-FGER agents (e.g., cetuximab, gefitinib, panitumumab), anti-HER2 agents (e.g., trastuzumab, T-DM1, pertuzumab), anti-HER3 agents (e.g., patritumab, MM-121, MM-111), and anti-VEGF agents (e.g., infliximab, adalimumab). They can be further classified into anti-EGFR antibodies such as cetuximab and panitumumab, anti-HER2 antibodies such as trastuzumab and pertuzumab, anti-HER3 antibodies such as patritumab, MM-121 and MM-111, and anti-VEGF antibodies such as infliximab and adalimumab. The anti-HER3 antibody-drug conjugate of the present invention exhibits excellent therapeutic effects when i) administered in combination with an anti-HER2 agent or anti-HER2 antibody in the treatment of gastric cancer, breast cancer, triple-negative breast cancer, etc., or ii) administered in combination with an anti-EGFR agent or anti-EGFR antibody in the treatment of lung cancer, head and neck cancer, gastric cancer, breast cancer, triple-negative breast cancer, etc. The other drug may be one or more, and the other drug may be an anticancer drug or a drug for reducing side effects caused by the concomitant drug.

[0198] In the present invention, a "pharmaceutical composition comprising an anti-HER3 antibody-drug conjugate and another drug" is synonymous with a "pharmaceutical composition in which an anti-HER3 antibody-drug conjugate and another drug are administered in combination." In the present invention, the phrase "administered in combination" of an anti-HER3 antibody-drug conjugate and another drug means that the anti-HER3 antibody-drug conjugate and another drug are taken up into the body of the recipient over a certain period of time. The anti-HER3 antibody-drug conjugate and another drug may be administered as a single formulation, or each may be formulated separately and administered separately. When formulated separately, the timing of their administration is not particularly limited, and they may be administered simultaneously, at different times with an interval, or on different days. When the anti-HER3 antibody-drug conjugate and another drug are administered at different times or days, the order of their administration is not particularly limited. Typically, each formulation is administered according to its respective administration method, and the administrations may be the same or different. Furthermore, when each is formulated separately, the administration method (administration route) of each formulation may be the same or different. Furthermore, the anti-HER3 antibody-drug conjugate and the other drug do not need to be present in the body at the same time, as long as they are taken into the body within a certain period (e.g., one month, preferably one week, more preferably several days, and even more preferably one day), and the active ingredient of one may have disappeared from the body when the other active ingredient is administered. Examples of dosage forms for a "pharmaceutical composition administered in combination with an anti-HER3 antibody-drug conjugate and another drug" include: 1) administration of a single formulation containing the anti-HER3 antibody-drug conjugate and the other drug; 2) simultaneous administration of two formulations obtained by separately formulating the anti-HER3 antibody-drug conjugate and the other drug via the same administration route; 3) administration of two formulations obtained by separately formulating the anti-HER3 antibody-drug conjugate and the other drug via the same administration route, with a time lag; 4) simultaneous administration of two formulations obtained by separately formulating the anti-HER3 antibody-drug conjugate and the other drug via different administration routes; and 5) administration of two formulations obtained by separately formulating the anti-HER3 antibody-drug conjugate and the other drug via different administration routes, with a time lag. The dosage, administration interval, administration form, formulation, etc. of a "pharmaceutical composition administered in combination with an anti-HER3 antibody-drug conjugate and another drug" are similar to, but not limited to, those of the pharmaceutical composition containing the anti-HER3 antibody-drug conjugate of the present invention. When such a pharmaceutical composition is formulated into two different formulations, it may be a kit containing them. In the present invention, the "combination" of an anti-HER3 antibody-drug conjugate and another drug means that the anti-HER3 antibody-drug conjugate and the other drug are "administered in combination."

[0199] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as lyophilized preparations, they may contain appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0200] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the anti-HER3 antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its therapeutic effect at a smaller dose, as the affinity of the antibody-drug conjugate for the antigen, i.e., the dissociation constant (Kd value) for the antigen, of the antibody-drug conjugate increases (the lower the Kd value). Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, a dose of about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days. [Example]

[0201] The present invention will be specifically described by the following examples, but the present invention is not limited thereto. Furthermore, the examples should not be construed as limiting in any sense. Furthermore, reagents, solvents, and starting materials not specifically described herein are readily available from commercial sources.

[0202] Reference Example 1 Preparation of U1-59 U1-59 was prepared based on the method described in WO 2007 / 077028.

[0203] Example 1 Antibody-drug conjugate (1) [ka]

[0204] Step 1: tert-butyl (4-{[(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}-4-oxobutyl)carbamate 4-(tert-Butoxycarbonylamino)butanoic acid (0.237 g, 1.13 mmol) was dissolved in dichloromethane (10 mL), N-hydroxysuccinimide (0.130 g, 1.13 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.216 g, 1.13 mmol) were added, and the mixture was stirred for 1 hour. The reaction solution was added dropwise to a solution of exatecan mesylate (0.500 g, 0.94 mmol) and triethylamine (0.157 mL, 1.13 mmol) in N,N-dimethylformamide (10 mL) and stirred at room temperature for one day. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 8:2 (v / v)] to give the title compound (0.595 g, quantitative yield). 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.31(9H,s),1.58(1H,t,J=7.2Hz),1.66(2H,t,J=7.2Hz),1.89-1.82(2H,m),2.12-2.21(3H, m),2.39(3H,s),2.92(2H,t,J=6.5Hz),3.17(2H,s),5.16(1H,d,J=19.2 Hz),5.24(1H,d,J=18.8Hz),5.42(2H,s),5.59-5.55(1H,m),6.53(1H,s ),6.78(1H,t,J=6.3Hz),7.30(1H,s),7.79(1H,d,J=11.0Hz),8.40(1H,d,J=8.6Hz). MS(APCI)m / z:621(M+H) + .

[0205] Step 2: 4-amino-N-[(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]butanamide trifluoroacetate The compound obtained in Step 1 above (0.388 g, 0.61 mmol) was dissolved in dichloromethane (9 mL). Trifluoroacetic acid (9 mL) was added and the mixture was stirred for 4 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) partition organic layer] to obtain the title compound (0.343 g, quantitative). 1 H-NMR(400MHz,DMSO-d6)0.87(3H,t,J=7.2Hz),1.79-1.92(4H,m),2.10-2.17(2H ,m),2.27(2H,t,J=7.0Hz),2.40(3H,s),2.80-2.86(2H,m),3.15-3.20(2H,m),5.1 5(1H,d,J=18.8Hz),5.26(1H,d,J=18.8Hz),5.42(2H,s),5.54-5.61(1H,m),6.55( 1H,s),7.32(1H,s),7.72(3H,brs),7.82(1H,d,J=11.0Hz),8.54(1H,d,J=8.6Hz). MS(APCI)m / z:521(M+H) + .

[0206] Step 3: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(4-{[(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}-4-oxobutyl)glycinamide N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (0.081 g, 0.19 mmol) was dissolved in dichloromethane (3 mL), and N-hydroxysuccinimide (0.021 g, 0.19 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.036 g, 0.19 mmol) were added and stirred for 3.5 hours. The reaction solution was added dropwise to a solution of the compound obtained in Step 2 (0.080 g, 0.15 mmol) in N,N-dimethylformamide (1.5 mL) and stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 8:2 (v / v)] to obtain the title compound (0.106 g, 73%). 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.36(9H,s),1.71(2H,m),1.86(2H,t,J=7.8Hz),2.15-2.19(4H,m),2.40(3H,s),2.77(1H,dd ,J=12.7,8.8Hz),3.02(1H,dd,J=14.1,4.7Hz),3.08-3.11(2H,m),3.16 -3.19(2H,m),3.54(2H,d,J=5.9Hz),3.57-3.77(4H,m),4.46-4.48(1H,m ),5.16(1H,d,J=19.2Hz),5.25(1H,d,J=18.8Hz),5.42(2H,s),5.55-5. 60(1H,m),6.53(1H,s),7.00(1H,t,J=6.3Hz),7.17-7.26(5H,m),7.31( 1H,s),7.71(1H,t,J=5.7Hz),7.80(1H,d,J=11.0Hz),7.92(1H,t,J=5.7Hz),8.15(1H,d,J=8.2Hz),8.27(1H,t,J=5.5Hz),8.46(1H,d,J=8.2Hz). MS(APCI)m / z:939(M+H) + .

[0207] Step 4: Glycylglycyl-L-phenylalanyl-N-(4-{[(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}-4-oxobutyl)glycinamide trifluoroacetate The compound obtained in step 3 above (1.97 g, 2.10 mmol) was dissolved in dichloromethane (7 mL). Trifluoroacetic acid (7 mL) was added and the mixture was stirred for 1 hour. The solvent was evaporated under reduced pressure, and toluene was added for azeotropy. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) partition organic layer] to obtain the title compound (1.97 g, 99%). 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.71-1.73(2H,m),1.82-1.90(2H,m),2.12-2.20(4H,m),2.40(3H,s),2.75(1 H,dd,J=13.7,9.4Hz),3.03-3.09(3H,m),3.18-3.19(2H,m),3.58-3.60(2H,m),3.64(1H,d,J=5.9Hz),3.69(1H,d,J=5.9Hz),3.7 2(1H,d,J=5.5Hz),3.87(1H,dd,J=16.8,5.9Hz),4.50-4.56(1H,m),5.16(1H,d,J=19.2Hz),5.25(1H,d,J=18.8Hz),5.42(2H,s) ,5.55-5.60(1H,m),7.17-7.27(5H,m),7.32(1H,s),7.78-7.81(2H,m),7.95-7.97(3H,m),8.33-8.35(2H,m),8.48-8.51(2H,m). MS(APCI)m / z:839(M+H) + .

[0208] Step 5: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(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}-4-oxobutyl)glycinamide To a solution of the compound obtained in step 4 (337 mg, 0.353 mmol) in N,N-dimethylformamide (1.2 mL), triethylamine (44.3 mL, 0.318 mmol) and N-succinimidyl 6-maleimidohexanoate (119.7 mg, 0.388 mmol) were added and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 5:1 (v / v)] to obtain the title compound (278.0 mg, 76%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.3Hz),1.12-1.22(2H,m),1.40-1.51(4H,m),1.66-1.76(2H,m),1.80-1.91(2H,m),2.05-2.21(6H,m) ,2.39(3H,s),2.79(1H,dd,J=14.0,9.8Hz),2.98-3.21(5H,m),3.55-3. 77(8H,m),4.41-4.48(1H,m),5.15(1H,d,J=18.9Hz),5.24(1H,d,J=18.9 Hz),5.40(1H,d,J=17.1Hz),5.44(1H,d,J=17.1Hz),5.54-5.60(1H,m), 6.53(1H,s),6.99(2H,s),7.20-7.27(5H,m),7.30(1H,s),7.70(1H,t,J= 5.5Hz), 7.80(1H,d,J=11.0Hz),8.03(1H,t,J=5.8Hz),8.08(1H,t,J=5.5Hz),8.14(1H,d,J=7.9Hz),8.25(1H,t,J=6.1Hz),8.46(1H,d,J=8.5Hz). MS(APCI)m / z:1032(M+H) + .

[0209] Step 6: Antibody-drug conjugate (1) Antibody reduction: U1-59 prepared in Reference Example 1 was prepared using the common procedures B and C described in Production Method 1, except that the medium was replaced with PBS 6.0 / EDTA, resulting in an antibody concentration of 10 mg / mL. This solution (1.00 mL) was placed in a 2.0 mL polypropylene tube, and a 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0307 mL; 4.6 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.050 mL) were added. After confirming that the pH of this solution was within 7.4 ± 0.1, it was incubated at 37°C for 1 hour to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: After incubating the above solution in a water bath at 22°C for 10 minutes, dimethyl sulfoxide (Sigma-Aldrich Co., LLC; 0.0586 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound obtained in step 5 (0.0615 mL; 9.2 equivalents per antibody molecule) were added and incubated in a water bath at 22°C for 40 minutes to conjugate the drug linker to the antibody. Next, 100 mM NAC (Sigma-Aldrich Co., LLC) aqueous solution (0.0123 mL) was added and the mixture was further stirred at room temperature for 20 minutes using a tube rotator (MTR-103, AS ONE Corporation) to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) described in Production Method 1 to obtain 6 mL of a solution containing the title antibody-drug conjugate. Characterization: Common procedure E (molar extinction coefficient of drug linker, ε D,280 =7280ε D,370 =23400) to obtain the following characteristic values. Antibody concentration: 1.29 mg / mL, antibody yield: 7.74 mg (77%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 4.9.

[0210] Example 2 Antibody-drug conjugate (2) [ka]

[0211] Step 1: Antibody-drug conjugate (2) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1 using U1-59 prepared in Reference Example 1 and the compound obtained in Step 5 of Example 1. Antibody concentration: 12.0 mg / mL, antibody yield: 226.8 mg (91%), common procedure E The average number of drugs bound per antibody molecule (n) measured was 4.9.

[0212] Example 3 Antibody-drug conjugate (3) [ka]

[0213] Step 1: Antibody-drug conjugate (3) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1 using U1-59 prepared in Reference Example 1 and the compound obtained in Step 5 of Example 1. Antibody concentration: 16.9 mg / mL, antibody yield: 219.7 mg (88%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 4.9.

[0214] Example 4 Antibody-drug conjugate (4) [ka]

[0215] Step 1: Antibody-drug conjugate (4) Antibody reduction: U1-59 prepared in Reference Example 1 was prepared using common procedures B and C described in Production Method 1, except that the medium was replaced with PBS6.0 / EDTA, resulting in an antibody concentration of 10 mg / mL. This solution (1.00 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0187 mL; 2.8 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0170 mL) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 1 hour to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: A dimethyl sulfoxide solution (0.0314 mL; 4.7 equivalents per antibody molecule) containing 10 mM of the compound obtained in step 5 above was added to the above solution at room temperature, and the mixture was incubated at 15°C for 1 hour to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0123 mL; 18.4 equivalents per antibody molecule) was added, and the mixture was further incubated at room temperature for 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) described in Production Method 1 to obtain 6 mL of a solution containing the title antibody-drug conjugate, and then the solution was concentrated using common procedure A. Characterization: Common procedure E (molar extinction coefficient of drug linker, ε D,280 =5000, ε D,370 = 19000) to obtain the following characteristic values. Antibody concentration: 1.02 mg / mL, antibody yield: 6.1 mg (61%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 2.9; common procedure F (molar extinction coefficient of the drug linker: ε D,280 per antibody molecule measured using a 5000 mAb Average number of drug bonds (n): 3.2.

[0216] Example 5 Antibody-drug conjugate (5) [ka]

[0217] Step 1: tert-butyl (5S,14S)-5-benzyl-1-{[(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}-14-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazahexadecan-16-oate Under ice cooling, glycylglycyl-L-phenylalanyl-N-[(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]glycinamide (a pharmaceutical compound described in WO 1997 / 46260 in its free form; 0.250 g, 0.332 mmol), N-hydroxysuccinimide (57.2 mg, 0.497 mmol), and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-aspartic acid 4 To a solution of 10 mL of N,N-dimethylformamide (10.0 mL) of tert-butyl ether (0.205 g, 0.497 mmol), N,N'-dicyclohexylcarbodiimide (0.123 g, 0.497 mmol) was added and the mixture was stirred at room temperature for 2 days. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 9:1 (v / v)] to obtain the title compound (0.278 g, 73%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.1Hz),1.35(9H,s),1.79-1.90(2 H,m),2.03-2.25(2H,m),2.40(3H,s),2.40-2.51(2H,m),2.64-2.82(2H,m),2 .98(1H,dd,J=13.7,4.6Hz),3.16(2H,brs),3.55(1H,dd,J=16.7,5.7Hz),3.6 3-3.80(4H,m),4.16-4.34(3H,m),4.36-4.50(2H,m),5.23(2H,s),5.37(1H,d ,J=16.5Hz),5.43(1H,d,J=16.5Hz),5.51-5.62(1H,m),6.52(1H,s),7.10-7 .25(5H,m),7.26-7.33(3H,m),7.39(2H,t,J=7.3Hz),7.65-7.72(3H,m),7.80 (1H,d,J=11.0Hz),7.86(2H,d,J=7.3Hz),7.98(1H,t,J=5.5Hz),8.07(1H,d,J =7.8Hz),8.15(1H,t,J=5.5Hz),8.31(1H,t,J=5.5Hz),8.41(1H,d,J=8.7Hz). MS(ESI)m / z:1147(M+H) + .

[0218] Step 2: tert-butyl (5S,14S)-14-amino-5-benzyl-1-{[(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}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazahexadecan-16-oate To a solution of the compound (0.279 g, 0.242 mmol) obtained in Step 1 above in N,N-dimethylformamide (2.00 mL) was added piperidine (0.240 mL, 2.42 mmol), and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 2:1 (v / v)] to obtain the title compound (0.265 g, quantitative) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.2Hz),1.39(9H,s),1.81-1.94(1H,m),2.07-2.28(2H,m),2.37(1H,dd,J=15.8,8.0Hz),2. 43(3H,s),2.60(1H,dd,J=15.8,4.9Hz),2.75-2.82(1H,m),3.00(1H,dd,J=13.9,4.5Hz),3.16-3.25(2H,m),3.50-3.61(2H,m),3.65-3 .81(5H,m),4.40-4.51(1H,m),5.27(2H,dd,J=24.1,19.0Hz),5.43(2H,dd,J=21.3,16.2Hz),5.56-5.65(1H,m),6.55(1H,s),7.15-7. 28(5H,m),7.33(1H,s),7.83(1H,d,J=11.0Hz),8.04(1H,t,J=5.7Hz),8.09(1H,d,J=8.2Hz),8.26-8.39(2H,m),8.44(1H,d,J=8.2Hz).

[0219] Step 3: tert-butyl (5S,14S)-5-benzyl-14-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-1-{[(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]quinoline-1- yl]amino}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazahexadecan-16-oate To a solution of the compound (0.100 g, 0.108 mmol) obtained in step 2 above in N,N-dimethylformamide (2 mL), N-succinimidyl 6-maleimidohexanoate (40 mg, 0.130 mmol) was added and stirred at room temperature for 2 days. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 9:1 (v / v)] to obtain the title compound (80 mg, 66%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.2Hz),1.13-1.23(2H,m),1.37(9H,s),1.42-1.54(4H,m),1.80- 1.96(2H,m),2.08-2.25(4H,m),2.35-3.76(15H,m),2.43(3H,s),4.39-4.49(1H,m),4.55-4.67(1H,m),5.21 -5.34(2H,m),5.43(2H,dd,J=21.1,16.4Hz),5.56-5.64(1H,m),6.55(1H,s),7.01(2H,d,J=0.8Hz),7.16-7. 26(5H,m),7.33(1H,s),7.83(1H,d,J=11.3Hz),8.04-8.18(3H,m),8.30-8.37(1H,m),8.43(1H,d,J=8.6Hz). MS(ESI)m / z:1118(M+H) + .

[0220] Step 4: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-α-aspartylglycylglycyl-L-phenylalanyl-N-[(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]glycinamide Trifluoroacetic acid (4.00 mL) was added to the compound obtained in Step 3 (70.0 mg, 62.6 μmol) under ice cooling, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to obtain the title compound (55.0 mg, 83%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.4Hz),1.14-1.24(2H,m),1.41 -1.53(4H,m),1.79-1.95(2H,m),2.08-2.28(4H,m),2.37-2.60(2H,m),2. 42(3H,s),2.63-2.82(2H,m),2.99(1H,dd,J=14.1,5.1Hz),3.12-3.25(2H ,m),3.29-3.44(1H,m),3.52-3.80(6H,m),4.38-4.48(1H,m),4.56(1H,dd ,J=13.7,7.4Hz),5.27(2H,dd,J=24.3,18.8Hz),5.43(2H,dd,J=21.5,16. 4Hz),5.57-5.62(1H,m),6.55(1H,s),7.01(2H,s),7.15-7.26(5H,m),7.3 3(1H,s),7.82(1H,d,J=11.0Hz),7.98(1H,brs),8.08(1H,d,J=6.7Hz),8. 15(1H,d,J=7.8Hz),8.34(1H,brs),8.44(1H,d,J=8.6Hz),12.26(1H,brs). MS(ESI)m / z:1062(M+H) + .

[0221] Step 5: Antibody-drug conjugate (5) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1, using U1-59 prepared in Reference Example 1 and the compound obtained in Step 4 above. Antibody concentration: 1.36 mg / mL, antibody yield: 8.16 mg (82%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =7620, ε D,370 The average number of drugs bound per antibody molecule (n) measured using a ELISA (using a ELISA kit with a ELISA kit): 5.0.

[0222] Example 6 Antibody-drug conjugate (6) [ka]

[0223] Step 1: Antibody-drug conjugate (6) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1 using U1-59 prepared in Reference Example 1 and the compound obtained in Step 4 of Example 5. Antibody concentration: 11.5 mg / mL, antibody yield: 224.2 mg (90%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =7620, ε D,370 The average number of drugs bound per antibody molecule (n) measured using a ELISA (using a ELISA kit with a ELISA kit): 4.6.

[0224] Example 7 Antibody-drug conjugate (7) [ka]

[0225] Step 1: tert-butyl (3S,12S)-12-benzyl-21-{[(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}-3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4,7,10,13,16,21-hexaoxo-5,8,11,14,17-pentaazahenicosan-1-noate (2S)-4-tert-butoxy-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxobutanoic acid (0.625 g, 1.52 mmol) was dissolved in dichloromethane (10.0 mL), and N-hydroxysuccinimide (0.175 g, 1.52 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.291 g, 1.52 mmol) were added and stirred for 1 hour. The reaction solution was stirred at 200°C for 1 hour. The compound obtained in Step 4 of Example 1 (1.00 g, 1.01 mmol) was added to the N,N-dimethylaminopropyl The resulting mixture was added dropwise to a chloroformamide solution (10.0 mL) and stirred at room temperature for 20 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform, The mixture was purified with a mixture of chloroform and methanol (8:2 (v / v)) to give the title compound (0.873 g, 70%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.4Hz),1.37(9H,s),1.68-1.78(2H,m),1.81-1.93(2H,m),2.10-2.23(4H,m),2.41(3H,s) ,2.68-2.85(3H,m),2.99-3.22(5H,m),3.58-3.81(6H,m),4.19-4.36(3H,m),4.38-4.52(2H,m),5.17(1H,d,J=19.2Hz),5.25(1H,d,J =19.2Hz),5.43(2H,s),5.54-5.62(1H,m),6.55(1H,s),7.15-7.34(8H,m),7.41(2H,t,J=7.2Hz),7.66-7.75(4H,m),7.81(1H,d,J=1 1.0Hz),7.88(2H,d,J=7.4Hz),8.01-8.06(1H,m),8.14(1H,d,J=8.2Hz),8.17-8.22(1H,m),8.25-8.30(1H,m),8.47(1H,d,J=8.6Hz). MS(APCI)m / z:1232(M+H) + .

[0226] Step 2: tert-butyl (3S,12S)-12-benzyl-3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-21-{[(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}-4,7,10,13,16,21-hexaoxo-5,8,11,14,17-pentaazahenicosan-1-noate The compound obtained in Step 1 above (0.800 g, 0.649 mmol) was dissolved in N,N-dimethylformamide (3.00 mL), piperidine (0.643 mL, 6.49 mmol) was added, and the mixture was stirred for 1 hour. The solvent was evaporated to dryness under reduced pressure, and the resulting residue was dissolved in N,N-dimethylformamide (10 mL). N-Succinimidyl 6-maleimidohexanoate (0.300 g, 0.974 mmol) was added, and the mixture was stirred for 20 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 8:2 (v / v)] to obtain the title compound (0.224 g, 29%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.6Hz),1.15-1.22(2H,m),1.35(9H ,s),1.44-1.47(4H,m),1.71-1.73(2H,m),1.80-1.91(2H,m),2.08(2H,t,J=7 .6Hz),2.13-2.20(4H,m),2.40(3H,s),2.67(1H,dt,J=11.1,4.8Hz),2.78(1H ,dd,J=13.6,9.4Hz),2.99-3.17(6H,m),3.31-3.36(2H,m),3.57-3.76(6H,m) ,4.45-4.47(1H,m),4.57-4.60(1H,m),5.16(1H,d,J=18.7Hz),5.25(1H,d,J= 18.7Hz),5.42(2H,s),5.55-5.60(1H,m),6.53(1H,s),6.99(2H,s),7.15-7.2 7(5H,m),7.31(1H,s),7.70(1H,t,J=5.4Hz),7.80(1H,d,J=10.9Hz),7.99(1H ,t,J=5.7Hz),8.09-8.12(3H,m),8.25(1H,t,J=6.0Hz),8.45(1H,d,J=9.1Hz). MS(APCI)m / z:1203(M+H) + .

[0227] Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-α-aspartylglycylglycyl-L-phenylalanyl-N-(4-{[(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}-4-oxobutyl)glycinamide The compound obtained in Step 2 above (0.224 g, 0.186 mmol) was reacted in the same manner as in Step 2 of Example 1 to give the title compound (21.2 mg, 10%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.13-1.21(2H,m),1.42-1.45(6H,m),1.70-1.72(2H,m),1.85-1.88(2H,m),2. 06-2.20(6H,m),2.39(3H,s),2.63-2.67(1H,m),2.78-2.81(1H,m),3.04-3.12(6H,m),3.63-3.70(6H,m),4.46-4.52(2H,m),5.1 6(1H,d,J=19.2Hz),5.25(1H,d,J=18.8Hz),5.42(2H,s),5.55-5.58(1H,m),6.53(1H,s),6.99(2H,s),7.18-7.23(6H,m),7.30(1 H,s),7.71(1H,t,J=5.5Hz),7.79(1H,d,J=10.9Hz),7.99-8.02(1H,m),8.10-8.11(3H,m),8.27-8.30(1H,m),8.47-8.50(1H,m). MS(APCI)m / z:1147(M+H) + .

[0228] Step 4: Antibody-drug conjugate (7) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1, using U1-59 prepared in Reference Example 1 and the compound obtained in Step 3 above. Antibody concentration: 1.39 mg / mL, antibody yield: 8.34 mg (83%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =7670, ε D,370 The average number of drugs bound per antibody molecule (n) measured using a ELISA (using a ELISA kit with a ELISA kit): 4.7.

[0229] Example 8 Antibody-drug conjugate (8) [ka]

[0230] Step 1: Antibody-drug conjugate (8) Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 3 of Example 7, The title antibody-drug conjugate was obtained by the same method as in step 6. Antibody concentration: 11.2 mg / mL, antibody yield: 228.5 mg (91%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =7670, ε D,370 The average number of drugs bound per antibody molecule (n) measured using a ELISA (using a ELISA kit with a ELISA kit): 4.7.

[0231] Example 9 Antibody-drug conjugate (9)

[0232] [ka]

[0233] Step 1: N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl-L-phenylalanyl-N-(4-{[(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}-4-oxobutyl)glycinamide The compound obtained in Step 4 of Example 1 (100 mg, 0.119 mmol) was reacted in the same manner as in Step 5 of Example 1 using N-succinimidyl 3-(2-(2-(3-maleimidopropanamido)ethoxy)ethoxy)propanoate (50.7 mg, 0.119 mmol) instead of N-succinimidyl 6-maleimidohexanoate to obtain the title compound (66.5 mg, 48%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.85(3H,t,J=7.4Hz),1.65-1.74(2H,m),1.77-1.90(2H,m),2.07-2.19(4H,m),2.30(2H,t,J=7.2Hz),2.33-2.36(2 H,m),2.38(3H,s),2.76(1H,dd,J=13.7,9.8Hz),2.96-3.18(9H,m),3.4 2-3.44(4H,m),3.53-3.76(10H,m),4.43(1H,td,J=8.6,4.7Hz),5.14(1 H,d,J=18.8Hz),5.23(1H,d,J=18.8Hz),5.38(1H,d,J=17.2Hz),5.42(1H,d,J=17.2H z),5.52-5.58(1H,m),6.52(1H,s),6.98(2H,s),7.12-7.17(1H,m),7.18-7.25(4H,m) ,7.29(1H,s),7.69(1H,t,J=5.5Hz),7.78(1H,d,J=11.3Hz),7.98-8.03(2H,m),8.11 (1H,d,J=7.8Hz),8.16(1H,t,J=5.7Hz),8.23(1H,t,J=5.9Hz),8.44(1H,d,J=9.0Hz). MS(APCI)m / z:1149(M+H) + .

[0234] Step 2: Antibody-drug conjugate (9) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1, using U1-59 prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 2.08 mg / mL, antibody yield: 18.7 mg (94%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =4964, ε D,370 The average number of drugs bound per antibody molecule (n) measured using ELISA kit (using ELISA kit No. 18982): 5.6.

[0235] Example 10 Antibody-drug conjugate (10) [ka]

[0236] Step 1: Antibody-drug conjugate (10) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1 using U1-59 prepared in Reference Example 1 and the compound obtained in Step 1 of Example 9. Antibody concentration: 19.7 mg / mL, antibody yield: 236.4 mg (95%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =4964, ε D,370 Average number of drugs bound per antibody molecule (n): 6.2; common procedure F (molar extinction coefficient of the drug linker, ε D,280 Antibodies measured using 4964 Average number of drugs bound per molecule (n): 6.4.

[0237] Example 11 Antibody-drug conjugate (11)

[0238] [ka]

[0239] Step 1: Antibody-drug conjugate (11) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 4, using U1-59 prepared in Reference Example 1 and the compound obtained in Step 1 of Example 9. Antibody concentration: 0.88 mg / mL, antibody yield: 5.28 mg (53%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =4964, ε D,370 Average number of drugs bound per antibody molecule (n): 3.0; common procedure F (molar extinction coefficient of the drug linker, ε D,280 Antibody measured using 4964 Average number of drug conjugates per molecule (n): 3.3.

[0240] Example 12 Antibody-drug conjugate (12) [ka]

[0241] Step 1: ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl acetate Pyridine (1.16 mL, 14.7 mmol) and lead tetraacetate (6.84 g, 14.7 mmol) were added to a mixture of N-9-fluorenylmethoxycarbonylglycylglycine (4.33 g, 12.2 mmol), tetrahydrofuran (120 mL), and toluene (40.0 mL), and the mixture was heated to reflux for 5 hours. After the reaction mixture was cooled to room temperature, insoluble materials were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate and washed with water and saturated brine. The organic layer was dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate = 9:1 (v / v) to ethyl acetate] to obtain the title compound (3.00 g, 67%) as a colorless solid. 1 H-NMR(400MHz,CDCl3)δ:2.07(3H,s),3.90(2H,d,J=5.1Hz),4.23(1H,t,J=7.0Hz),4.46(2H,d,J=6.6Hz),5.26(2H,d,J=7.0Hz),5.32(1H,brs) ,6.96(1H,brs),7.32(2H,t,J=7.3Hz),7.41(2H,t,J=7.3Hz),7.59(2H,d,J=7.3Hz),7.77(2H,d,J=7.3Hz).

[0242] Step 2: Benzyl [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate To a solution of the compound obtained in Step 1 (3.68 g, 10.0 mmol) and benzyl glycolate (4.99 g, 30.0 mmol) in tetrahydrofuran (40.0 mL), potassium tert-butoxide (2.24 g, 20.0 mmol) was added at 0°C and stirred at room temperature for 15 minutes. Ethyl acetate and water were added to the reaction solution at 0°C, followed by extraction with ethyl acetate and chloroform. The resulting organic layer was dried over sodium sulfate and filtered. The solvent was evaporated under reduced pressure, and the resulting residue was dissolved in dioxane (40.0 mL) and water (10.0 mL). Sodium bicarbonate (1.01 g, 12.0 mmol) and 9-fluorenylmethyl chloroformate (2.59 g, 10.0 mmol) were added, followed by stirring at room temperature for 2 hours. Water was added to the reaction solution, which was then extracted with ethyl acetate. The resulting organic layer was dried over sodium sulfate and filtered. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v / v) to 0:100] to obtain the title compound (1.88 g, 40%) as a colorless oil. 1 H-NMR(400MHz,CDCl3)δ:3.84(2H,d,J=5.5Hz),4.24(3H,t,J=6.5Hz),4.49(2H,d,J=6.7Hz),4.88(2H,d,J=6.7Hz),5.15-5.2 7(1H,m),5.19(2H,s),6.74(1H,brs),7.31-7.39(7H,m),7.43(2H,t,J=7.4Hz),7.61(2H,d,J=7.4Hz),7.79(2H,d,J=7.4Hz).

[0243] Step 3: [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid The compound obtained in Step 2 (1.88 g, 3.96 mmol) was dissolved in ethanol (40 mL) and ethyl acetate (20 mL). Palladium-carbon catalyst (376 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. Insoluble matter was removed by filtration through Celite, and the solvent in the filtrate was evaporated under reduced pressure to give the title compound (1.52 g, quantitative) as a colorless solid. 1H-NMR(400MHz,DMSO-d6)δ:3.62(2H,d,J=6.3Hz),3.97(2H,s),4.18-4.32(3H,m),4.60(2H,d,J=6.7Hz),7. 29-7.46(4H,m),7.58(1H,t,J=5.9Hz),7.72(2H,d,J=7.4Hz),7.90(2H,d,J=7.4Hz),8.71(1H,t,J=6.5Hz).

[0244] Step 4: 9H-Fluoren-9-ylmethyl (2-{[(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]amino}-2-oxoethyl)carbamate To a solution of exatecan mesylate (0.283 g, 0.533 mmol), N-hydroxysuccinimide (61.4 mg, 0.533 mmol), and the compound obtained in step 3 (0.205 g, 0.533 mmol) in N,N-dimethylformamide (10 mL) was added N,N-diisopropylethylamine (92.9 μL, 0.533 mmol) and N,N'-dicyclohexylcarbodiimide (0.143 g, 0.693 mmol) under ice cooling, and the mixture was stirred at room temperature for 3 days. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) partition organic layer] to obtain the title compound (0.352 g, 82%) as a pale brown solid. Obtained as a solid. 1H-NMR(400MHz,DMSO-d6)δ:0.81(3H,t,J=7.4Hz),1.73-1.87(2H,m),2.06-2.20(2H,m),2.34(3H,s),3.01-3.2 3(2H,m),3.58(2H,d,J=6.7Hz),3.98(2H,s),4.13-4.25(3H,m),4.60(2H,d,J=6.7Hz),5.09-5.22(2H,m),5.32- 5.42(2H,m),5.50-5.59(1H,m),6.49(1H,s),7.24-7.30(3H,m),7.36(2H,t,J=7.4Hz),7.53(1H,t,J=6.3Hz),7. 66(2H,d,J=7.4Hz),7.75(1H,d,J=11.0Hz),7.84(2H,d,J=7.4Hz),8.47(1H,d,J=8.6Hz),8.77(1H,t,J=6.7Hz). MS(ESI)m / z:802(M+H) + .

[0245] Step 5: 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 To a solution of the compound obtained in Step 4 (0.881 g, 1.10 mmol) in N,N-dimethylformamide (11.0 mL), piperidine (1.1 mL) was added and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to give a mixture containing the title compound. This mixture was used in the next reaction without further purification.

[0246] Step 6: N-[(9H-fluoren-9-ylmethoxy)carbonyl]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 To a solution of the mixture obtained in Step 5 (0.439 mmol), N-hydroxysuccinimide (0.101 g, 0.878 mmol), and N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (the compound described in JP 2002-60351 A; 0.440 g, 0.878 mmol) in N,N-dimethylformamide (50 mL) was added N,N'-dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) under ice cooling, and the mixture was stirred at room temperature for 4 days. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 9:1 (v / v)] to give the title compound (0.269 g, 58%) as a pale orange solid. MS(ESI)m / z:1063(M+H) + .

[0247] Step 7: 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 To a solution of the compound obtained in Step 6 (0.269 g, 0.253 mmol) in N,N-dimethylformamide (4.00 mL), piperidine (0.251 mL, 2.53 mmol) was added and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to give a mixture containing the title compound. This mixture was used in the next reaction without further purification.

[0248] Step 8: 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 To a solution of the compound obtained in Step 7 (0.253 mmol) in N,N-dimethylformamide (10 mL), N-succinimidyl 6-maleimidohexanoate (0.156 g, 0.506 mmol) was added and stirred at room temperature for 3 days. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 9:1 (v / v)] to obtain the title compound (0.100 g, 38%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.83(3H,t,J=7.2Hz),1.09-1.21(2H,m),1.33-1.47(4H,m),1.75-1.90(2H,m),2.00-2.23(4H,m),2.36(3 H,s),2.69-2.81(1H,m),2.94-3.03(1H,m),3.06-3.22(2H,m),3.23-3.74(6H,m),3.98(2H,s),4.39-4.50(1H,m),4.60(2H,d,J=6.7H z),5.17(2H,s),5.39(2H,s),5.53-5.61(1H,m),6.50(1H,s),6.96(2H,s),7.11-7.24(5H,m),7.28(1H,s),7.75(1H,d,J=11.0Hz),7. 97(1H,t,J=5.7Hz),8.03(1H,t,J=5.9Hz),8.09(1H,d,J=7.8Hz),8.27(1H,t,J=6.5Hz),8.48(1H,d,J=9.0Hz),8.60(1H,t,J=6.5Hz). MS(ESI)m / z:1034(M+H) + .

[0249] Step 9: Antibody-drug conjugate (12) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1, using U1-59 prepared in Reference Example 1 and the compound obtained in Step 8 above. Antibody concentration: 2.11 mg / mL, antibody yield: 19.0 mg (95%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =5178, ε D,370 The average number of drugs bound per antibody molecule (n) measured using ELISA (using ELISA kit = 20217): 4.9.

[0250] Example 13 Antibody-drug conjugate (13) [ka]

[0251] Step 1: Antibody-drug conjugate (13) The title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 1 using U1-59 prepared in Reference Example 1 and the compound obtained in Step 8 of Example 12. Antibody concentration: 22.2 mg / mL, antibody yield: 244.2 mg (98%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =5178, ε D,370 Average number of drugs bound per antibody molecule (n): 6.2; common procedure F (molar extinction coefficient of the drug linker, ε D,280 Antibodies measured using 5178 Average number of drugs bound per molecule (n): 7.0.

[0252] Example 14 Antibody-drug conjugate (14) [ka]

[0253] Step 1: Antibody-drug conjugate (14) Antibody reduction: U1-59 prepared in Reference Example 1 was prepared using common procedures B and C described in Production Method 1, except that the medium was replaced with PBS6.0 / EDTA, resulting in an antibody concentration of 10 mg / mL. This solution (1.00 mL) was placed in a 2.0 mL polypropylene tube, and a 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0160 mL; 2.4 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.; 0.0150 mL) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 1 hour to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: After incubating the above solution in a 15°C water bath for 10 minutes, dimethyl sulfoxide (Sigma-Aldrich Co. LLC; 0.0209 mL) and a dimethyl sulfoxide solution containing 10 mM of the compound obtained in Step 8 of Example 12 (0.0315 mL; 5.0 equivalents per antibody molecule) were added, and the mixture was incubated in a 15°C water bath for 60 minutes to conjugate the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0050 mL) was added, and the mixture was further stirred at room temperature for 20 minutes using a tube rotator (MTR-103, AS ONE Corporation) to terminate the drug linker reaction. The following property values ​​were obtained by the same purification procedure and property evaluation as in Step 6 of Example 1. Antibody concentration: 1.46 mg / mL, antibody yield: 8.76 mg (88%), common procedure E ( The molar extinction coefficient of the lag linker is ε D,280 =5178, ε D,370 Average number of drugs bound per antibody molecule (n): 2.5; common procedure F (molar extinction coefficient of the drug linker, ε D,280 Antibody measured using 5178 Average number of drug conjugates per molecule (n): 2.9.

[0254] Example 15 Antibody-drug conjugate (15) [ka]

[0255] Step 1: Antibody-drug conjugate (15) Antibody reduction: U1-59 prepared in Reference Example 1 was adjusted to an antibody concentration of 10 mg / mL using common procedures B and C described in Production Method 1, except that the medium was replaced with PBS 6.0 / EDTA. This solution (100 mL) was placed in a 250 mL polycarbonate Erlenmeyer flask. The mixture was placed in a container and stirred magnetically at room temperature. 1M dipotassium hydrogen phosphate (1.70 mL) was added, followed by 10 mM TCEP (4.010 mL; 6.0 equivalents per antibody molecule). After confirming that the pH of the solution was within 7.0 ± 0.1, stirring was stopped and the mixture was incubated at 37°C for 1 hour to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: After cooling the above solution to 15°C, a DMSO solution (6.684 mL; 10.0 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 8 of Example 12 was slowly added dropwise under stirring. The mixture was stirred at 15°C for the first 30 minutes, and then incubated for the next hour with stirring stopped to allow the drug linker to bind to the antibody. Next, a 100 mM NAC aqueous solution (0.862 mL; 12.9 equivalents per antibody molecule) was added under stirring, and the mixture was further incubated at room temperature for 20 minutes to quench the reactivity of any unreacted drug linker. Purification: While stirring, 20% aqueous acetic acid (approximately 0.6 mL) and ABS (100 mL) were slowly added to the above solution, and the pH of the solution was adjusted to 5.5 ± 0.1. This solution was microfiltered (Millipore Co. Millex-HV filter, 0.45 μm, PVDF membrane) to remove cloudy material. This solution was then filtered using an ultrafiltration membrane (Merck Co., Pellicon XL Cassette, Biomax 50 KDa), a tube pump (Cole-Parmer, USA, Masterflex Pump model 77521-40, pump head model 7518-00), and tubing (Cole-Parmer, USA, Masterflex Tuner). Ultrafiltration purification was performed using an ultrafiltration apparatus consisting of a filter L / S16. Specifically, ultrafiltration purification was performed while ABS was added dropwise to the reaction solution as a purification buffer (total 1600 mL). This removed unbound drug linkers and other low-molecular-weight reagents, and the buffer was replaced with ABS, followed by concentration. The resulting purified solution was microfiltered (0.22 μm (Millipore Co. Millex-GV filter, PVDF membrane) to obtain 37.5 mL of a solution containing the title antibody-drug conjugate. Antibody concentration: 26.5 mg / mL, antibody yield: 993.0 mg (90%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =5178, ε D,370 Average number of drugs bound per antibody molecule (n): 6.3; common procedure F (molar extinction coefficient of drug linker, ε D,280 Antibodies measured using 5178 Average number of drugs bound per molecule (n): 7.3.

[0256] Example 16a Antibody-drug conjugate (16a) [ka]

[0257] Step 1: Antibody-drug conjugate (16a) Antibody reduction: U1-59 prepared in Reference Example 1 was adjusted to an antibody concentration of 10 mg / mL using common procedures B and C described in Production Method 1, except that the medium was replaced with PBS 6.0 / EDTA. This solution (15 mL) was placed in a 50 mL polyethylene terephthalate container. The mixture was placed in a 1000-well plate and stirred with a magnetic stirrer at room temperature. 1M dipotassium hydrogen phosphate (0.255 mL) was added, followed by 10 mM TCEP (0.601 mL; 6.0 equivalents per antibody molecule). After confirming that the pH of this solution was within 7.0±0.1, the mixture was incubated at 37°C for 2 hours to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: After cooling the above solution to 15°C, a DMSO solution (1.002 mL; 10.0 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 8 of Example 12 was slowly added dropwise under stirring. The mixture was stirred at 15°C for 30 minutes to allow the drug linker to bind to the antibody. Next, a 100 mM NAC aqueous solution (0.129 mL; 12.9 equivalents per antibody molecule) was added under stirring, and the mixture was further incubated at room temperature for 20 minutes to terminate the reactivity of the unreacted drug linker. Purification procedures and characterization were carried out in the same manner as in Step 6 of Example 1. The following characteristic values ​​were obtained by the evaluation. Antibody concentration: 2.36 mg / mL, antibody yield: 140 mg (59.5 mL) (94%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =5178, ε D,370 Average number of drugs bound per antibody molecule (n): 6.4; common procedure F (molar extinction coefficient of drug linker, ε D,280 (using =5178) The average number of drugs bound per antibody molecule (n) was determined to be 7.7.

[0258] Example 16b Antibody-drug conjugate (16b) [ka]

[0259] Antibody reduction: U1-59 prepared in Reference Example 1 was adjusted to an antibody concentration of 10 mg / mL using common procedures B and C described in Production Method 1, except that the medium was replaced with PBS 6.0 / EDTA. This solution (900 mL) was placed in a 2000 mL polycarbonate Erlenmeyer flask. The mixture was placed in a sco container, and under magnetic stirring at room temperature, 1 M dipotassium hydrogen phosphate aqueous solution (15.3 mL) was added, followed by 10 mM TCEP aqueous solution (36.1 mL; 6.0 equivalents per antibody molecule). After confirming that the pH of this solution was within 7.0 ± 0.1, stirring was stopped and the mixture was incubated at 37°C for 2 hours to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: After cooling the above solution to 15°C, a DMSO solution (60.16 mL; 10.0 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 8 of Example 12 was slowly added dropwise under stirring. The mixture was stirred at 15°C for 30 minutes to allow the drug linker to bind to the antibody. Next, a 100 mM NAC aqueous solution (7.76 mL; 12.9 equivalents per antibody molecule) was added under stirring, and the mixture was further incubated at room temperature for 20 minutes to terminate the reactivity of any unreacted drug linker. Purification: While stirring, 20% aqueous acetic acid (approximately 5 mL) and ABS (1000 mL) were slowly added to the above solution, and the pH of the solution was adjusted to 5.5±0.1. This solution was microfiltered (Millipore Co. Stericup, 0.45 μm, PVDF membrane) to remove cloudy matter. This solution was then filtered through an ultrafiltration membrane (Merck Co., Pellicon 2 mini casette, Ultracel 30 KDa, 0.1 m). 2 ), tube pump (USA Palmer Masterflex Pump model 7528-20, pump head Ultrafiltration purification was performed using an ultrafiltration apparatus consisting of a filter (model 77800-62) and tubing (Masterflex Tubes L / S24 and 25, Cole-Parmer, USA). Specifically, ultrafiltration purification was performed while ABS was added dropwise to the reaction solution as a purification buffer (total of 16 L). Unbound drug linkers and other low-molecular-weight reagents were removed, and the buffer was replaced with ABS. The reaction solution was then concentrated to obtain approximately 500 mL of a solution containing the title antibody-drug conjugate. Antibody concentration: 19.66 mg / mL, antibody yield: 9830 mg (109%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =5178, ε D,370 The average number of drugs bound per antibody molecule (n) measured using ELISA (using ELISA kit = 20217): 6.5.

[0260] Example 16c Antibody-drug conjugate (16c) [ka]

[0261] The title antibody-drug conjugate was obtained in the same manner as in Example 16b using U1-59 prepared in Reference Example 1 and the compound obtained in Step 8 of Example 12. Antibody concentration: 16.21 mg / mL, antibody yield: 9726 mg (600 mL, 108%), common procedure E (molar extinction coefficient of drug linker, ε D,280 =5178, ε D,370 The average number of drugs bound per antibody molecule (n) measured using ELISA (using ELISA kit = 20217): 6.5.

[0262] Example 16d Antibody-drug conjugate (16d) [ka]

[0263] The antibody-drug con...

Claims

[Claim 1] The invention described herein.

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