Anti-her2 antibody-drug conjugate

Abstract

To provide an antitumor drug which is excellent in terms of antitumor effect and safety and has an excellent therapeutic effect.SOLUTION: Provided is an antibody-drug conjugate in which an antitumor compound represented by the following formula and an anti-HER2 antibody are conjugated via a linker having a structure represented by the following formula: -L1-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O)-, wherein the anti-HER2 antibody is bound to a terminal of L1, and the antitumor compound is bound to a carbonyl group of -(CH2)n2-C(=O)- moiety with a nitrogen atom of an amino group at position 1 as a bonding site.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an antibody-drug conjugate useful as an antitumor drug, in which an anti-HER2 antibody and an antitumor drug are linked via a linker structure moiety. [Background technology]

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

[0003] Antitumor small molecule compounds that inhibit topoisomerase I and exert antitumor effects. Among them, camptothecin derivatives of the following formula are known.

[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 in clinical practice, this compound does not require enzymatic activation to exhibit its antitumor effect. In addition, it has a stronger topoisomerase activity than SN-38, the active ingredient of irinotecan, and topotecan, which is also used in clinical practice. The enzyme I inhibitory activity was observed, and the compound showed stronger cytotoxic activity against various cancer cells in vitro. In particular, it was observed against cancer cells that exhibit resistance to SN-38 and other drugs due to the expression of P-glycoprotein. Furthermore, a strong antitumor effect was observed in a mouse model in which a human tumor was subcutaneously transplanted, but although clinical trials were conducted, it has not yet been marketed (see Non-Patent Documents 5 to 10). It was unclear whether exatecan would function effectively as an ADC.

[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 maintains high blood retention, and furthermore, by utilizing the increased permeability of tumor neovasculature and tumor tissue retention, it passively enhances targeting to tumor sites. DE-310 is activated by enzymatic cleavage of the peptide spacer. The active ingredient, exatecan, and exatecan with glycine bound to the amino group are released continuously, resulting in improved pharmacokinetics. In non-clinical studies of various tumor models, DE-310 demonstrated greater efficacy than exatecan monotherapy, despite the total amount of exatecan contained therein being reduced compared to exatecan monotherapy. Clinical trials of DE-310 have demonstrated efficacy in some cases, and it has been reported that the active ingredient accumulates in tumors rather than in normal tissues. Meanwhile, it has also been reported that the accumulation of DE-310 and the active ingredient in tumors is not significantly different from that in normal tissues, and that 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] HER2 is one of the representative growth factor receptor-type oncogene products identified as the human epidermal growth factor receptor type 2-associated oncogene, and is a transmembrane receptor protein with a tyrosine kinase domain and a molecular weight of 185 kDa (Non-Patent Document 15). The DNA sequence and amino acid sequence of HER2 are published in public databases and can be referenced by accession numbers such as M11730 (Genbank) and NP_004439.2 (NCBI). HER2(neu,ErbB-2) is EGFR(epidermal growth factor receptor: HER2 is a member of the epidermal growth factor receptor (EGFR) family, and is known to play an important role in cell proliferation, differentiation, and survival in both normal and cancer cells by forming homodimers or heterodimers with other EGFR receptors, HER1 (EGFR, ErbB-1), HER3 (ErbB-3), and HER4 (ErbB-4) (Non-Patent Documents 16-18), which activates HER2 through autophosphorylation of intracellular tyrosine residues (Non-Patent Documents 19 and 20). HER2 is overexpressed in various types of cancer, including breast cancer, gastric cancer, and ovarian cancer (Non-Patent Documents 21-26), and has been reported to be a negative prognostic factor in breast cancer (Non-Patent Documents 27 and 28).

[0009] Trastuzumab, also known as recombinant humanized anti-HER2 monoclonal antibody (huMAb4D5-8, rhuMAb HER2, Herceptin®), is a humanized version (Patent Document 6) of the murine anti-HER2 antibody 4D5 (Non-Patent Document 29, Patent Document 5). Trastuzumab specifically binds to the extracellular domain IV of HER2 and exerts its anticancer effects by inducing antibody-dependent cellular cytotoxicity (ADCC) and inhibiting signal transduction from HER2 (Non-Patent Documents 30, 31). Because trastuzumab is highly effective against tumors overexpressing HER2 (Non-Patent Document 32), it was launched in the United States in 1999 and in Japan in 2001 as a therapeutic agent for patients with metastatic breast cancer overexpressing HER2. While the therapeutic efficacy of trastuzumab in breast cancer has been well documented (Non-Patent Document 33), responders to trastuzumab are HER2-overexpressing breast cancer patients who have received a wide range of conventional anticancer treatments. Approximately 15% of breast cancer patients present with thrombocytopenia, and approximately 85% of patients in this group are treated with trastuzumab. They either do not respond or only respond poorly.

[0010] Therefore, there is a recognized need for therapeutic agents that target diseases associated with HER2 expression for patients suffering from HER2-overexpressing tumors or disorders associated with HER2 expression who do not respond or respond poorly to trastuzumab. Examples of therapeutic agents include T-DM1 (trastuzumab emtansine, Kadcyla (registered trademark); Non-Patent Document 34), in which an antitumor drug is linked to trastuzumab via a linker structure, and HER2-targeting drugs that target the extracellular domain II of HER2. Pertuzumab (Perjeta®; Non-Patent Document 35, Patent Document 7) has been developed, which is designed to inhibit telodimer formation. However, its response, potency, and range of indications are still insufficient, and there remains an unmet need for treatment targeting HER2. [Prior art documents] [Patent documents]

[0011] [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] International Publication No. 2000 / 25825 [Patent Document 5] U.S. Patent No. 5,677,171 [Patent Document 6] U.S. Patent No. 5,821,337 [Patent Document 7] International Publication No. 01 / 00244 [Non-patent literature]

[0012] [Non-Patent Document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-patent document 2] Alley,SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [Non-licensed document 3] DamleN. K. Expert Opin. Biol. Ther. (2004) 4, 1445-1452.

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[0013] 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]

[0014] The inventors considered that, because anti-HER2 antibodies are antibodies that can target tumor cells, i.e., antibodies that have the properties of being able to recognize tumor cells, being able to bind to tumor cells, being able to be internalized into tumor cells, being cytotoxic to tumor cells, or having cytocidal activity against tumor cells, etc., by converting the antitumor compound exatecan into an antibody-drug conjugate in which it is bound to the antibody via a linker structure moiety, it would be possible to more reliably transport the antitumor compound to tumor cells and enable the antitumor effect of the compound to be exerted specifically on tumor cells, thereby ensuring the antitumor effect and enhancing the cytocidal effect of the anti-HER2 antibody, and furthermore, to reduce the dosage of the antitumor compound compared to when the compound is administered alone, i.e., by these means, it would be possible to mitigate the effect of the antitumor compound on normal cells and thereby achieve 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-HER2 antibody and exatecan are linked via this linker. They also found that this conjugate exhibits excellent antitumor effects, thereby completing the present invention.

[0015] That is, the present invention is [1] The following formula [ka] and an anti-HER2 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- 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-HER2 antibody via a linker having the structure shown in

[0016] Here, the anti-HER2 antibody is L 1 The antitumor compound binds to the amino acid at the 1-position. The nitrogen atom of the -(CH2)n group is used as the bonding site. 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 4 represents —CH2CH2—C(═O)— or a single bond, where n 4indicates an integer between 1 and 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: The anti-HER2 antibody is bound to the 3-position of this structure, and the nitrogen atom at the 1-position is bound to the methylene group in the linker structure containing this.

[0017] Furthermore, the present invention also relates to the following: [2]L P The peptide residues are phenylalanine, glycine, valine, lysine, and citrulline. The antibody-drug conjugate according to [1], wherein the peptide residue is composed of an amino acid selected from the group consisting of phenylalanine, serine, glutamic acid, and aspartic acid. [3]L P is a peptide residue selected from the group consisting of: -Drug conjugates: -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 four amino acids [1] or [2] Antibody-drug conjugates. [5]L Pis a tetrapeptide residue -GGFG- according to any one of [1] to [4]. Antibody-drug conjugates.

[0018] [6]n 3 is an integer from 2 to 5, and L 2 The antibody-drug conjugate according to any one of [1] to [5], wherein is a single bond. [7]n 3 is an integer from 2 to 5, and L 2 -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4 The antibody-drug conjugate according to any one of [1] to [5], wherein —NH—(CH2)n 1 -L a -(CH2)n 2 The antibody-drug conjugate according to any one of [1] to [7], wherein —C(═O)— is a partial structure having a chain length of 4 to 7 atoms. [9]-NH-(CH2)n 1 -L a -(CH2)n 2 The antibody-drug conjugate according to any one of [1] to [7], wherein —C(═O)— is a partial structure having a chain length of 5 or 6 atoms.

[10] -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)-, -NH-CH2CH2-O-CH2-C(=O)-, or The antibody-drug conjugate according to any one of [1] to [9], wherein —NH—CH2CH2—OC(═O)— gate.

[11] -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 [1] to [9], wherein

[0019]

[12] -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 [9], 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)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-OC(=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).

[0020] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the following formula: The anti-HER2 antibody is bound to the 3-position of this structure, and the nitrogen atom at the 1-position is bound 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-.

[0021]

[13] -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 [9], 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).

[0022] Here, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- are as defined above.

[0023]

[14] The following formula [ka] and an anti-HER2 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-HER2 antibody via a linker having the structure shown in Here, the anti-HER2 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 4 represents —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: The anti-HER2 antibody is bound to the 3-position of this structure, and the nitrogen atom at the 1-position is bound to the methylene group in the linker structure containing this.

[0024]

[15] 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 ais 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 n 1 is 2 and n 2 is 1 and n 3 is 5 and L 2 is a single bond, and L a The antibody-drug conjugate according to

[14] , wherein

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

[14] or

[15] , wherein

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

[14] or

[15] , wherein

[18] -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

[14] to

[17] , wherein

[0025]

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

[14] to

[18] , 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)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-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),

[0026] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the following formula: The anti-HER2 antibody is bound to the 3-position of this structure, and the nitrogen atom at the 1-position is bound 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-.

[0027]

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

[14] to

[18] , 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.

[0028]

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

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

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

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

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

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

[0029]

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

[23] , a salt thereof, or a hydrate of the same.

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

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

[26] The antitumor and / or anticancer agent according to

[25] , for use in lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell carcinoma, myeloma, or sarcoma.

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

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

[28] The pharmaceutical composition according to

[27] , for use in treating lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasma cell carcinoma, myeloma, or sarcoma.

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

[23] , a salt thereof, or a hydrate of the same.

[0030]

[30] 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) with an anti-HER2 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.

[0031] 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 are phenylalanine, glycine, valine, lysine, citrulline, serine, and glutamine. represents a peptide residue consisting of 2 to 7 amino acids selected from the group consisting of carboxylic acids, carboxylic acids, and aspartic acids; 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 compound of the 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

[0032]

[31] The method of producing according to

[30] , wherein the method of binding the drug-linker moiety to the anti-HER2 antibody is a method of converting the antibody into a reactive derivative by reduction treatment.

[0033]

[32] The method of producing according to

[30] or

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

[33] The method of producing according to

[30] or

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

[34] The method of producing according to

[30] or

[31] , wherein the average number of the selected drug-linker structures bound per antibody is in the range of 3 to 8. An antibody-drug conjugate obtained by any one of the production methods described in

[35]

[30] to

[34] .

[0034]

[36] An antibody-drug conjugate obtained by treating an anti-HER2 antibody under reducing conditions and then reacting it with a compound selected from the following group to form a thioether bond at the sulfide bond in the hinge region of the antibody: (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)-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)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-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-O-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), and (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX).

[0035] Here, (maleimid-N-yl)- is a compound 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- indicates the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[0036]

[37] An antibody-drug conjugate obtained by treating an anti-HER2 antibody under reducing conditions and then reacting it with a compound selected from the following group to form a thioether bond at the sulfide 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), and (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.

[0037]

[38] The antibody-drug conjugate according to

[36] or

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

[39] The antibody-drug conjugate according to

[36] or

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

[40] The antibody-drug conjugate according to

[36] or

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

[0038] Anti-HER2 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]

[0039] [Figure 1] 1 shows the amino acid sequence of the humanized anti-HER2 monoclonal antibody heavy chain (SEQ ID NO: 1). [Figure 2] 1 shows the amino acid sequence of the humanized anti-HER2 monoclonal antibody light chain (SEQ ID NO: 2). [Figure 3] Figure 1 shows the antitumor effect of antibody-drug conjugate (27) or trastuzumab on nude mice subcutaneously implanted with human breast cancer cell line KPL-4 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents the tumor volume. [Figure 4]

[0039] Figure 1 shows the antitumor effect of antibody-drug conjugates (8), (28), or trastuzumab emtansine on nude mice subcutaneously implanted with human gastric cancer cell line NCI-N87 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 5] This figure shows the antitumor effects of antibody-drug conjugates (8), (29), (30), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with the human breast cancer cell line JIMT-1. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 6] This figure shows the antitumor effect of antibody-drug conjugate (31), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human pancreatic cancer cell line Capan-1 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 7] Figure 1 shows the antitumor effect of antibody-drug conjugate (50) on nude mice subcutaneously implanted with human gastric cancer cell line NCI-N87 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents the tumor volume. [Figure 8]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human breast cancer cell line ST225 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 9]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human breast cancer cell line ST910 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 10]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human colon cancer cell line CTG-0401 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 11]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human non-small cell lung cancer cell line CTG-0860 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 12]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human cholangiocarcinoma cell line CTG-0927 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 13]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human esophageal cancer cell line CTG-0137 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. [Figure 14]

[0039] Figure 1 shows the antitumor effects of antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine on nude mice subcutaneously implanted with human ovarian cancer cell line SK-OV-3 cells. In the figure, the horizontal axis represents the number of days after cell implantation, and the vertical axis represents tumor volume. DETAILED DESCRIPTION OF THE INVENTION

[0040] 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.

[0041] The anti-HER2 antibody-drug conjugate of the present invention is an antitumor drug in which an antitumor compound is bound to an anti-HER2 antibody via a linker structure moiety, and will be described in detail below.

[0042] [antibody] The anti-HER2 antibody used in the anti-HER2 antibody-drug conjugate of the present invention may be derived from any species, but preferably includes human, rat, mouse, and rabbit. When the antibody is derived from a species other than human, it is preferably chimerized or humanized using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Anti-HER2 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 the antibody to tumor cells can be confirmed using flow cytometry. The uptake of antibodies into cells can be assessed using two methods: (1) an assay in which the antibody taken up into cells is visualized by fluorescence microscopy using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761); and (2) an assay in which the antibody taken up into cells is visualized by fluorescence microscopy using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody. (3) an assay that measures the amount of fluorescence taken up into cells (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (4) a Mab-ZAP assay that uses an immunotoxin that binds to a therapeutic antibody, which, when taken up into cells, releases the toxin and suppresses cell proliferation. (BioTechniques 28:162-165, January 2000) A recombinant conjugate protein of the catalytic domain of diphtheria toxin and protein G can also be used as an immunotoxin. The antitumor activity of antibodies can be confirmed in vitro by measuring their inhibitory activity against cell proliferation. For example, cancer cell lines that overexpress the target protein of the antibody can be cultured, and the antibody can be added to the culture system at various concentrations to measure the inhibitory activity against focus formation, colony formation, and spheroid growth. Antitumor activity can be confirmed by administering the antibody to nude mice transplanted with the cell line and measuring changes in cancer 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.

[0043] Anti-HER2 antibodies can be obtained by known means. For example, they 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, antibodies may be prepared 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)). Furthermore, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells, and a monoclonal antibody can be obtained. 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.

[0044] There are no particular limitations on the anti-HER2 antibodies that can be used in the present invention, but for example, those having the following properties are desirable. (1) An anti-HER2 antibody characterized by the following properties: (a) Binds specifically to HER2. (b) having the activity of being internalized into HER2-expressing cells by binding to HER2. (2) The antibody according to (1) above, which binds to the extracellular domain of HER2. (3) The antibody according to (1) or (2) above, which is a monoclonal antibody. (4) Antibody-dependent cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity The antibody according to any one of (1) to (3) above, having the following properties: (5) The antibody according to any one of (1) to (4) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized 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 consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2. (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.

[0045] The anti-HER2 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," "protein," and "protein" are used interchangeably. As used herein, the term "cells" includes cells within an animal body and cultured cells. As used herein, the term "HER2" is used interchangeably with HER2 protein. As used herein, the anti-HER2 antibody is not particularly limited, and examples thereof include pertuzumab (WO 01 / 00245) and trastuzumab (U.S. Pat. No. 5,821,337), with trastuzumab being preferred. However, the anti-HER2 antibody is not limited to this, as long as it specifically binds to HER2, more preferably has the activity of being internalized into HER2-expressing cells upon binding to HER2. As used herein, "trastuzumab" is also referred to as HERCEPTIN (registered trademark), huMAb4D5-8, or rhuMAb4D5-8, and is a humanized antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1 (Figure 1) and a light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 2 (Figure 2). As used herein, the term "specifically binds" means binding that is not nonspecific adsorption. The dissociation constant (hereinafter referred to as "KD") can be used as a criterion for determining whether binding is specific. The KD value of a suitable antibody for the HER2 protein is 1 x 10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less, or 1 x 10 -6 M or less; more preferably 5 x 10 -7 M or less, 2×10 -7 M or less, or 1 x 10 -7 M or less; even more preferably 5 x 10 -8 M or less, 2×10 -8 M or less, or 1 x 10 -8 M or less; optimally 5 × 10 -9 M or less, 2×10 -9 M or less, or 1 x 10 -9 The binding between the HER2 protein and the antibody can be measured using known methods such as the Surface Plasmon Resonance method, the ELISA method, and the RIA method. As used herein, "CDR" refers to complementarity determining region (CDR). It is known that the heavy and light chains of an antibody molecule each contain three CDRs. CDRs, also known as hypervariable domains, are located within 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 chain polypeptide chains. As used herein, with regard to antibody CDRs, the heavy chain CDRs are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and the light chain CDRs are referred to as CDRL1, CDRL2, and CDRL3 from the amino-terminal end 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" refers to hybridization under stringent conditions using a commercially available hybridization solution, ExpressHyb Hybridization Solution. Hybridization was performed at 68°C in a DNA-immobilized filter in the presence of 0.7-1.0 M NaCl, followed by hybridization in 0.1-2x SSC solution (1x SSC is 150 mM The term "hybridization" refers to hybridization under conditions that can be identified by washing at 68°C using a buffer consisting of 100 mM NaCl and 15 mM sodium citrate, or equivalent conditions.

[0046] 1.HER2 HER2 is a representative growth factor receptor-type oncogene product identified as the human epidermal growth factor receptor type 2-associated oncogene. It is a transmembrane receptor protein with a molecular weight of 185 kDa and a tyrosine kinase domain. It is a member of the EGFR family consisting of HER1 (EGFR, ErbB-1), HER2 (neu, ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). HER2 is known to play an important role in cell proliferation, differentiation, and survival in both normal and tumor cells by activating itself through autophosphorylation of intracellular tyrosine residues upon homodimerization or heterodimerization with other EGFRs, HER1, HER3, or HER4. The HER2 protein used in the present invention can be directly purified from HER2-expressing cells of humans or non-human mammals (rats, mice, etc.), or can be prepared and used as a cell membrane fraction of such cells. HER2 can also be synthesized in vitro or produced in host cells by genetic engineering. Specifically, genetic engineering involves incorporating HER2 cDNA into an expression vector and then synthesizing the protein in a solution containing enzymes, substrates, and energy sources necessary for transcription and translation, or by transforming other prokaryotic or eukaryotic host cells to express HER2, thereby obtaining the protein. Furthermore, the genetically engineered HER2-expressing cells or cell lines expressing HER2 can also be used as the HER2 protein. The DNA sequence and amino acid sequence of HER2 are published in public databases and can be referenced by accession numbers such as M11730 (Genbank) and NP_004439.2 (NCBI). In addition, there are also proteins that have the same biological activity as the above-mentioned HER2 protein, which are composed of an amino acid sequence in which one or several amino acids are substituted, deleted and / or added. Included in HER2. The human HER2 protein consists of an N-terminal signal sequence consisting of 22 amino acid residues, an extracellular domain consisting of 630 amino acid residues, a transmembrane domain consisting of 23 amino acid residues, and an intracellular domain consisting of 580 amino acid residues.

[0047] 2. Preparation of anti-HER2 antibodies The antibodies against HER2 of the present invention can be obtained, for example, by immunizing an animal with HER2 or any polypeptide selected from the amino acid sequence of HER2, and collecting and purifying the antibodies produced in vivo, according to methods commonly used in this field. The species of HER2 that serves as the antigen is not limited to humans; animals can also be immunized with HER2 derived from non-human animals such as mice and rats, rat p185neu, etc. In this case, antibodies that can be used to treat human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous HER2 with human HER2. Alternatively, a hybridoma can be established by fusing antibody-producing cells that produce antibodies against HER2 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, HER2, is expressed in host cells through genetic manipulation of the HER2 gene. This can be obtained by Specifically, a vector capable of expressing the HER2 gene is prepared, introduced into host cells to express the gene, and the expressed HER2 is then purified. Alternatively, the above-described genetically engineered HER2-expressing cells or cell lines expressing HER2 can be used as the HER2 protein. Anti-HER2 antibodies can be obtained by known means. Specific methods for obtaining antibodies against HER2 are described below.

[0048] (1) Antigen preparation Antigens for producing anti-HER2 antibodies include HER2 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. HER2 can be used after direct purification from human tumor tissues or tumor cells, or it can be obtained by synthesizing HER2 in vitro or by producing it in host cells by genetic engineering. Specifically, in genetic engineering, the antigen can be obtained by inserting HER2 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 HER2. It is also possible to obtain an antigen as a secreted protein by expressing a fusion protein linking the extracellular domain of HER2, a membrane protein, with the constant domain of an antibody in an appropriate host-vector system. HER2 cDNA can be obtained, for example, by the so-called PCR method, which involves performing a polymerase chain reaction (PCR; see Saiki, RK, et al., Science (1988) 239, pp. 487-489) using a cDNA library expressing HER2 cDNA as a template and primers that specifically amplify HER2 cDNA. 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 commonly used vertebrate cells include, but are not limited to, 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 (1980) 77, pp. 4126-4220). 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 is Proteins can be separated and purified by various known separation procedures that utilize their physical and chemical properties. 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 also be used as antigens. Alternatively, cell lines expressing HER2 can also be used as antigens. Examples of such cell lines include human breast cancer lines SK-BR-3, BT-474, KPL-4, or JIMT-1, human gastric cancer line NCI-N87, and human ovarian cancer line SK-OV-3, but are not limited to these cell lines as long as they express HER2.

[0049] (2) Production of anti-HER2 monoclonal antibodies An example of an antibody that specifically binds to HER2 is a monoclonal antibody that specifically binds to HER2, 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 biopolymers to be used as antigens or preparation of antigen-expressing cells (b) a step of 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) Examining the physiological activity and binding specificity of the monoclonal antibody thus produced, or testing 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.

[0050] (a) Antigen purification As the antigen, HER2 or a part thereof prepared by the method described above can be used. In addition, membrane fractions prepared from HER2-expressing recombinant cells, or HER2-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, HER2-expressing cell lines can also be used as antigens.

[0051] (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 used as an immunogen to immunize laboratory animals. Another method involves immunizing laboratory animals with the cells as immunogens. Any animal used in known hybridoma production methods can be used as the laboratory animal without any problems. Specific examples include mice, rats, goats, sheep, cattle, and horses. However, from the viewpoint of the 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 and sales companies 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 HER2 or its recombinants, known methods can be used, as described in detail in, for example, Weir, D.M., Handbook of Experimental Immunology Vol. I.II.III., Blackwell Scientific Publications, Oxford (1987); Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, Charles C. Thomas Publisher, Springfield, Illinois (1964). 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. One to ten days, preferably two to five days, and more preferably two to three days after the booster immunization, spleen cells or lymphocytes containing antibody-producing cells are aseptically removed from the immunized animal. At this time, the antibody titer is measured, 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 can be, for example, an RIA method or an ELISA method, but is not limited to these methods. The ELISA method can be carried out by 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 (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.

[0052] (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 cultured 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 is added), Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle's Medium (hereinafter referred to as "Dulbecco's The cells are subcultured in Modified Eagle Medium (hereinafter referred to as "DMEM"), but 3 to 4 days before cell fusion, they are subcultured in normal medium (e.g., ASF104 medium (Ajinomoto Co., Inc.) containing 10% FCS) and then subcultured at 2 × 10 7 Ensure that the above number of cells is maintained.

[0053] (d) Cell fusion Fusion of antibody-producing cells with myeloma cells can be carried out appropriately according to known methods (e.g., Weir, D.M., Handbook of Experimental Immunology Vol. I.II.III., Blackwell Scientific Publications, Oxford (1987); Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, Charles C. Thomas Publisher, Springfield, Illinois (1964)), under conditions that do not excessively reduce cell viability. Examples of such methods include 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.

[0054] (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.

[0055] (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 HER2 monoclonal antibody-producing hybridoma strains.

[0056] (g) Preparation of monoclonal antibodies by hybridoma culture The hybridomas selected in this manner can be cultured to efficiently obtain monoclonal antibodies, but it is desirable to screen for hybridomas that produce the desired monoclonal antibody prior to culturing. 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-tetramethyl pentadecane (pristane) beforehand (3 to 7 days before). For example, an immunosuppressant was injected into the abdominal 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 ascites fluid is then collected from the mouse once the abdomen has become distended and 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 to HER2. The monoclonal antibody of the present invention is not particularly limited, but an example thereof is the mouse monoclonal antibody 4D5 (ATCC CRL 10463).

[0057] (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 if steps (a) to (h) in (2) are repeated to obtain a separate, independently obtained monoclonal antibody, it is possible to obtain an antibody with cytotoxic activity equivalent to that of the anti-HER2 antibody obtained in step (g). An example of such an antibody is an antibody that binds to the same epitope as the anti-HER2 antibody obtained in step (g). If a newly produced monoclonal antibody binds to a partial peptide or partial three-dimensional structure to which the anti-HER2 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope. Furthermore, by confirming that the monoclonal antibody competes with the binding of the anti-HER2 antibody to HER2 (i.e., that the monoclonal antibody interferes with the binding of the anti-HER2 antibody to HER2), it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER2 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-HER2 antibody.

[0058] (3) Other antibodies The antibodies of the present invention include not only the monoclonal antibodies against HER2 as described above, but also genetically engineered antibodies that have been 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 a human-derived constant region (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). Chimeric antibodies of the present invention are not particularly limited, and include the chimeric antibody 4D5, which contains the heavy chain constant region of human IgG1 or IgG2. 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), antibodies in which not only the CDR sequence but also some framework amino acid residues have been transplanted into a human antibody by CDR grafting (WO 90 / 07861), and antibodies produced by gene conversion mutagenesis. Examples include antibodies that have been humanized using a humanized mutagenesis strategy (U.S. Pat. No. 5,821,337).

[0059] 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.

[0060] 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.

[0061] By combining sequences that are highly homologous to the above heavy chain and light chain amino acid sequences, it is possible to select antibodies with biological activity equivalent to each of the above antibodies. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added to the heavy chain or light chain amino acid sequence, it is also possible to select antibodies with biological activity equivalent to each of the above antibodies. Note that, in this specification, "homology" is used interchangeably with "identity."

[0062] Homology between two amino acid sequences can be determined using the default parameters of the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, 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). The Blast algorithm can also be accessed online at www.ncbi.nlm.nih.gov / blast.

[0063] Further examples of the antibody of the present invention include a human antibody that binds to HER2. An anti-HER2 human antibody refers to a human antibody that has only the gene sequence of an antibody derived from a human chromosome. Anti-HER2 human antibodies can be obtained by a method using human antibody-producing mice carrying human chromosomal fragments containing the heavy and light chain genes of human antibodies (see, for example, Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727).

[0064] 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.

[0065] 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).

[0066] Another example of an index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is an instrument that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good index of the relative structural stability of proteins. By measuring the Tm value and comparing the values, differences in thermal stability can be compared. It is known that the storage stability of an antibody correlates to a certain extent with its thermal stability (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and suitable antibodies can be selected using thermal stability as an indicator. Other indicators for selecting antibodies include high yield in appropriate host cells and low aggregation in aqueous solution. For example, an antibody with the highest yield does not necessarily exhibit the highest thermal stability, so it is necessary to comprehensively evaluate the above-mentioned indicators to select an antibody that is most suitable for administration to humans.

[0067] 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.

[0068] 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. 99 / 54342, 00 / 61739, and 02 / 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. Particularly, 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.

[0069] In addition, the carboxyl-terminal lysine residue of the heavy chain of the antibody produced in mammalian cultured cells is deleted. It is known that deletion of two amino acid residues, glycine and lysine, from the carboxyl terminus of the heavy chain results in a deletion and amidation of the proline residue at the carboxyl terminus (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibodies of the present invention also include antibodies and functional fragments of such antibodies that have been modified, including deletions in which one or two amino acids have been 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 has been amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletions in the carboxyl terminus of the heavy chain 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 type of heavy chain selected from the group consisting of full-length and the above-mentioned deletion variants, or a combination of any two types. Although the quantitative ratio of each deletion variant may be affected by the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions, 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.

[0070] The isotype of the antibody of the present invention can be, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG2.

[0071] 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 antigen activity, the activity of enhancing antigen activity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). The biological activity of the antibody of the present invention is the activity of binding to HER2, and preferably the activity of internalizing into HER2-expressing cells by binding to HER2. Furthermore, the antibody of the present invention may have ADCC, CDC, and / or ADCP activity in addition to the cell-internalizing activity. stomach.

[0072] 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 chromatography such as HPLC or 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 Co., Ltd.). Examples include companies such as 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.

[0073] [Anti-tumor compound] The antitumor compound conjugated to the anti-HER2 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:

[0074] [ka]

[0075] 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 structures are encompassed within the scope of the present invention.

[0076] 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.

[0077] In antibody-drug conjugates, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. The production of antibody-drug conjugates is carried out by specifying the reaction conditions, such as the amounts of raw materials and reagents used, so that the number of drugs bound is constant. However, unlike chemical reactions of small molecules, the resulting mixture contains a variable number of drugs bound to it. The number of drugs bound to one antibody molecule is usually specified and expressed as an average value, i.e., the average drug binding number. In the present invention, the average drug binding number is used in principle unless otherwise specified, i.e., except when referring to an antibody-drug conjugate having a specific drug binding number contained in a mixture of antibody-drug conjugates having different drug binding numbers. 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.

[0078] [Linker structure] The linker structure that connects the antitumor compound to the anti-HER2 antibody in the anti-HER2 antibody-drug conjugate of the present invention is described below. The linker has the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- The antibody has the structure L 1 The end of (L 2 The antitumor compound binds to the -L a -(CH2)n 2 It bonds through the carbonyl group of the -C(=O)- moiety. n 1 represents an integer of 0 to 6, preferably an integer of 1 to 5, and more preferably 1 to 3.

[0079] 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 compound represented by the following formula:

[0080] [ka]

[0081] The structure is represented by the formula: Position 3 in this partial structure is the binding site for the anti-HER2 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):

[0082] [ka]

[0083] In the formula, n 3 is an integer from 2 to 8, preferably from 2 to 5.

[0084] 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:

[0085] 2.L 2 L2 teeth, -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- The structure is shown by L 2 may not exist, in which case L 2 is 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.

[0086] 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:

[0087] 3.L P L P is a peptide residue consisting of 2 to 7 amino acids. It is composed of oligopeptide residues consisting of amino acids linked by peptide bonds. P is N L at the end 2 and at the C-terminus, the linker -NH-(CH2)n 1 -La -(CH2)n 2 It binds to the amino group of the -C(=O)- moiety. Here, "peptide residue" or "oligopeptide residue" indicates a divalent group derived from a peptide consisting of two or more amino acid residues, with the N-terminus and C-terminus being the binding sites.

[0088] L P The amino acids constituting the amino acid are not particularly limited, but may be, for example, L- or D-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 P The amino acid sequence of the present invention is not particularly limited, but may include, for example, phenylalanine ... Examples of amino acids that can be used 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. L-amino acids having a sequence of amino acids arbitrarily selected from these amino acids may overlap. P Just build it. The drug release pattern can be controlled by the type of amino acid. The number of amino acids can range from 2 to 7.

[0089] 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 For example, the tetrapeptide residue -GGFG- is used. It is possible.

[0090] 4.L a -(CH2)n 2 -C(=O)- L a -(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)-, -OC(=O)-. Among these, -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, -OC(=O)-, If so, L a is a single bond and n 2 It is preferred if is 0.

[0091] 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-O-CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-, -NH-CH2-OC(=O)-, -NH-CH2CH2-OC(=O)-, -NH-CH2CH2CH2-OC(=O)-, -NH-CH2CH2CH2CH2-OC(=O)-, The following can be mentioned:

[0092] Among these, more preferred are: -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-O-CH2-C(=O)- is.

[0093] Linker -NH-(CH2)n 1 -L a -(CH2)n 2 The -C(=O)- preferably has a chain length of 4 to 7 atoms, more preferably 5 or 6 atoms.

[0094] After the anti-HER2 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 Drug derivatives with the structure -C(=O)-(NH-DX) The antitumor derivatives that are released from the antibody-drug conjugates of the present invention to exhibit antitumor effects include the -NH-(CH2)n linker described above. 1 -L a -(CH2)n 2 Examples of antitumor derivatives include those having a structural moiety in which the terminal of the structure represented by -C(=O)- is an amino group, and particularly preferred are the following: NH2-CH2CH2-C(=O)-(NH-DX), NH2-CH2CH2CH2-C(=O)-(NH-DX), NH2-CH2-O-CH2-C(=O)-(NH-DX), NH2-CHCH2-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.

[0095] In the antibody-drug conjugate of the present invention in which the drug is exatecan, the drug-linker structure moiety [-L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(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)-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)-GGFG-NH-CH2CH2-OC(=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). Among these, the more preferred ones are as follows. -(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)-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)-GGFG-NH-CH2CH2-O-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), -(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). 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).

[0096] In the antibody-drug conjugates of the present invention, a preferred linker structure linking the anti-HER2 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)-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)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. Among these, the more preferred ones are as follows. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(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)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-OC(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. Furthermore, the following can be preferably mentioned: -(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)-.

[0097] [Manufacturing method] Next, a representative method for producing the antibody-drug conjugate of the present invention or a production intermediate thereof will be described. In the following, the compound numbers shown in each reaction scheme will be used to indicate the compounds. That is, they will be referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will be described in the same manner.

[0098] 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.

[0099] [ka]

[0100] [Wherein, AB represents an antibody having a sulfhydryl group, and L 1 'L 1 In the linker structure represented by the formula:

[0101] [ka]

[0102] where the nitrogen atom is the binding site. 1 Of the -(Succinimid-3-yl-N)-(CH2)n 3 It represents a group in which the -(Succinimid-3-yl-N)- moiety in -C(=O)- is a maleimidyl group. Also, -(NH-DX) represents the following formula:

[0103] [ka]

[0104] This represents the group formed by removing one hydrogen atom from the amino group at position 1 of exatecan.

[0105] 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, but 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.

[0106] 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. The antibody (3a) having a sulfhydryl group 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 can be reacted with the amino group of the antibody; N-succinimide S-acetylthioalkanoates are reacted with the amino groups of antibodies, followed by hydroxylamine; N-succinimidyl 3-(pyridyldithio)propionate is reacted with the amino groups, followed by a reducing agent; dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and other reducing agents are reacted with antibodies. 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-pyridone (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) can be concentrated, buffer exchanged, purified, and its antibody concentration measured, and the average number of drugs bound per antibody molecule measured, by the following common procedures, and the antibody-drug conjugate (1) can be identified.

[0107] 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 Co.) container and concentrated by centrifugation (2000 to 3800 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

[0108] 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 (1.3 mL mg) differs for each antibody. -1 cm -1 ~1.8mLmg -1 cm -1 ) was used.

[0109] Common Procedure C-1: Buffer Exchange of Antibody A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 support was equilibrated with phosphate buffer (10 mM, pH 6.0; herein referred to 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. Common Procedure C-2: Buffer Exchange of Antibodies A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 matrix was equilibrated with phosphate buffer (50 mM, pH 6.5; referred to herein as PBS6.5 / EDTA) containing 50 mM sodium chloride and 2 mM EDTA according to the manufacturer's instructions. 2.5 mL of antibody solution was loaded onto each NAP-25 column, and a 3.5 mL fraction was eluted with 3.5 mL of PBS6.5 / EDTA. This fraction was concentrated using common procedure A, and the antibody concentration was measured using common procedure B. The antibody concentration was then adjusted to 20 mg / mL using PBS6.5 / EDTA.

[0110] Common Procedure D: Purification of Antibody-Drug Conjugates A NAP-25 column was equilibrated with either a commercially available phosphate buffer (PBS7.4, Cat. No. 10010-023, Invitrogen), a sodium phosphate buffer (10 mM, pH 6.0; herein referred to as PBS6.0) containing 137 mM sodium chloride, or an acetate buffer (10 mM, pH 5.5; herein referred to as ABS) containing 5% sorbitol. The antibody-drug conjugate reaction solution (approximately 1.5 mL) was loaded onto the NAP-25 column, and the antibody fraction was collected by elution with the amount of buffer specified by the manufacturer. This fraction was again loaded onto the NAP-25 column and eluted with buffer. This gel filtration purification procedure was repeated two or three times to obtain an antibody-drug conjugate free of unbound drug linkers and low molecular weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide).

[0111] 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 the 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 relational equations: A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A formula( I) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 CA Formula (II) where A 280 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 indicates the absorbance of the antibody at 370 nm, and 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 denotes the molar extinction coefficient of the antibody at 280 nm, and ε A,370 denotes the molar extinction coefficient of the antibody at 370 nm, and 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 indicates the antibody concentration in the antibody-drug conjugate, and C D indicates the drug concentration in the antibody-drug conjugate. 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 can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A,370 is usually zero. In the examples, the molar extinction coefficient of trastuzumab is ε A,280 = 215400 (calculated estimate) and ε A,370 =0 was used. D,280 and ε D,370The molar extinction coefficient (ε) of the drug linker in the examples is calculated by measuring the absorbance of a solution containing the conjugate precursor dissolved at a certain molar concentration, according to the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). Unless otherwise specified, the molar extinction coefficient (ε) of the drug linker in the examples is calculated by the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). D,280 = 5000 (actual average value), ε D,370 = 19000 (actually measured average value). 280 and A 370 By measuring these values ​​and substituting them into equations (I) and (II) 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.

[0112] 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% (1 2.5-15 minutes), 42%-29% (15 minutes-15.1 minutes), 29%-29% (15.1 minutes-25 minutes) Sample injection volume: 15 μL [F-3. Data Analysis] [F-3-1] In contrast to the L chain (L0) and H chain (H0) of an antibody that do not have any drugs bound to them, the L chain (L chain with one drug bound: L1) and H chain (H chain with one drug bound: H1, H chain with two drugs bound: H2, H chain with three drugs bound: H3) with drugs bound to them have increased hydrophobicity and longer retention times in proportion to the number of drugs bound to them, and are therefore eluted in the order of L0, L1, H0, H1, H2, and H3. By comparing the retention times with those of L0 and H0, the detected peak can be assigned to either 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.

[0113]

number

[0114]

number

[0115] The molar extinction coefficients (280 nm) of the L and H chains of each antibody can be estimated from the amino acid sequences of the L and H chains of each antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). In the case of trastuzumab, the molar extinction coefficient of the L chain was estimated to be 26,150, and the molar extinction coefficient of the H chain was estimated to be 81,290, based on the amino acid sequence. 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.

[0116]

number

[0117] [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 bindings = (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

[0118] The intermediate compounds used in Production Method 1 are described below. The compound represented by formula (2) in Production Method 1 is represented by the following 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) It is a compound represented by the formula: During the ceremony, n 3 denotes the integers 2 to 8, L 2is -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 are phenylalanine, glycine, valine, lysine, citrulline, serine, and glutamine. represents a peptide residue consisting of 2 to 7 amino acids selected from the group consisting of carboxylic acids, carboxylic acids, and aspartic acids; 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 compound of the formula

[0119] [ka]

[0120] a maleimidyl group (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by the formula: -(NH-DX) is represented by the following formula:

[0121] [ka]

[0122] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in

[0123] L 2 is a single bond or -NH-(CH2CH2-O)n 4 n when -CH2CH2-C(=O)- 4 is an integer The compounds 2 to 4 are preferred as production intermediates. L P The peptide residues are phenylalanine, glycine, valine, lysine, citric acid, Compounds that are peptide residues consisting of amino acids selected from phosphorus, serine, glutamic acid, and aspartic acid are preferred as production intermediates. P 4 A compound that is a peptide residue composed of the amino acid L is preferred as a production intermediate. P is a tetrapeptide residue of -GGFG-, which is preferred as a production intermediate. .

[0124] Also, -NH-(CH2)n 1 -L a -(CH2)n 2 - is a compound selected from the group consisting of -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, and -NH-CH2CH2-O-CH2-. Compounds of the formula -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2 are preferred as intermediates in the production.

[0125] Furthermore, the compound represented by formula (2) is 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 -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. It is preferred as an intermediate. More preferred is -NH-(CH2)n 1 -L a -(CH2)n 2 - is -NH-CH2CH2- , -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. to, n3 is an integer of 2 or 5 is preferred.

[0126] In addition, the compound represented by formula (2) is 3 is an integer between 2 and 5, and L 2 -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4 is an integer from 2 to 4, and -NH-(CH2)n 1 -L a -(CH2)n 2 Compounds in which - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are preferred as production intermediates. 4 is an integer of 2 or 4. Furthermore, -NH-(CH2)n 1 -L a -(CH2)n 2 - is -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. .

[0127] 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)-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)。

[0128] The anti-HER2 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-HER2 antibody or a reactive derivative thereof to form a thioether bond at the disulfide bond present in the hinge region of the anti-HER2 antibody. In this case, it is preferable to use a reactive derivative of an anti-HER2 antibody, and particularly a reactive derivative obtained by reducing an anti-HER2 antibody.

[0129] 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)-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-CH2-CH2-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).

[0130] 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), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), The compound represented by the formula: is more preferred.

[0131] To ensure a sufficient amount of conjugate, multiple conjugates prepared under similar conditions with similar average drug contents (e.g., ±1) can be mixed to create a new lot. In this case, the average drug contents will fall within the range of the average drug contents before mixing.

[0132] 2. Manufacturing method 2 The compound of formula (2), which is an intermediate used in the above production method, and a pharmacologically acceptable salt thereof can be produced, for example, by the following method.

[0133] [ka]

[0134] [In the formula, L 1 ' indicates a terminal maleimidyl group, and P 1 , P 2 , and P 3 indicates a protecting group.]

[0135] The carboxylic acid (5) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and reacted with NH2-DX (4) or a pharmacologically acceptable salt thereof in the presence of a base to give the compound NH2-DX (4) can be used to produce 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). This reaction can be carried out using the same reaction reagents and conditions as those typically used in peptide synthesis. Various activated esters are available, including those 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. Activated esters can also be prepared by reacting carboxylic acid (5) with pentafluorophenyl trifluoroacetate; carboxylic acid (5) with 1-benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphite; carboxylic acid (5) with diethyl cyanophosphonate (salt-in method); or carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyl. It can also be produced by reaction with a disulfide (Mukaiyama method), 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 involves treating carboxylic acid (5) with an acid halide such as thionyl chloride or oxalyl chloride in the presence of a base. The activated ester, mixed acid anhydride, or acid halide of carboxylic acid (5) obtained as described above can be reacted with compound (4) in the presence of a suitable base in an inert solvent at a reaction temperature of −78° C. to 150° C. to produce compound (6). 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.

[0136] Specific examples of the base used in each of the above steps include carbonates, alkoxides, hydroxides, and hydrides of alkali metals or alkaline earth metals, such as sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, 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 tertiary amines or nitrogen-containing heterocyclic compounds, such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diisopropylethylamine, and diazabicyclo[5.4.0]undec-7-ene (DBU).

[0137] 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.

[0138] Protecting group P of the terminal amino group of compound (6) 1Examples include tert-butyloxycarbonyl Protecting groups for amino groups commonly used in peptide synthesis, such as a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, or the like, can be used. Other protecting groups for amino groups include alkanoyl groups such as an acetyl group; alkoxycarbonyl groups such as a methoxycarbonyl group and an ethoxycarbonyl group; arylmethoxycarbonyl groups such as a para-methoxybenzyloxycarbonyl group and a para-(or ortho)nitrobenzyloxycarbonyl 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. Protecting group P 1 is a compound that protects the amino group The thickness may be selected depending on the properties of the material. The protecting group P of the terminal amino group of the obtained compound (6) 1 By deprotecting the compound The deprotection can be carried out by selecting the reagent and conditions according to the protecting group. N-terminus to P 2 The protected peptide carboxylic acid (8) can be converted to an activated ester, mixed acid anhydride, etc. Compound (9) can be produced by derivatizing the compound (7) and reacting it with the resulting compound (7). The reaction conditions and reagents for forming the peptide bond between peptide carboxylic acid (8) and compound (7), as well as the base and inert solvent, can be appropriately selected from those described in the synthesis of compound (6). 2 is appropriately selected from the protecting groups described for compound (6). The amino acid may be selected depending on the properties of the compound protecting the amino group. As is commonly used in peptide synthesis, compound (9) can also be produced by sequentially repeating the reaction and deprotection of the amino acids or peptides that make up peptide carboxylic acid (8) to elongate it. The protecting group P of the amino group of the obtained compound (9) 2 By deprotecting the compound (1 0) can be produced. For this deprotection, reagents and conditions 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, reagents, bases, and inert solvents for forming the peptide bond between carboxylic acid (11) and compound (10) can be appropriately selected from those described in the synthesis of compound (6).

[0139] Compound (9) can also be produced, for example, by the following method. N-terminus to P 2 The protected peptide carboxylic acid (8) can be converted to an activated ester, mixed acid anhydride, etc. In the presence of a base, the carboxyl group is converted to P 3 and reacting with the amine compound (12) protected by Compound (13) can be produced by the above 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) 2 As the protecting group, any commonly used protecting group can be used. There are no restrictions. Specific examples of the protecting group for a hydroxyl group include alkoxymethyl groups such as a methoxymethyl group, arylmethyl groups such as a benzyl group, a 4-methoxybenzyl group, and a 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. A carboxyl group can be protected as an ester with an alkyl group such as a methyl group, an ethyl group, and a tert-butyl group, an allyl group, or an arylmethyl group such as 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 a result, he has been involved in organic synthesis, especially in peptide synthesis. A protecting group that is commonly used as a protecting group for a carboxy group may be used, specifically, a methyl group, an ethyl group, an alkyl ester such as tert-butyl, an allyl ester, a benzyl ester, etc., and the protecting group may be appropriately 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 is obtained by deprotecting The compound (14) can be produced by deprotection using a suitable reagent and conditions depending on 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 in the synthesis of compound (6). Just use it.

[0140] Compound (2) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (13) 2 Compound (15) was obtained by deprotecting For this deprotection, reagents and conditions may 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 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, and the reaction conditions, reagents, bases, and inert solvents can be appropriately selected from those described for the synthesis of compound (6).

[0141] 3. Manufacturing method 3 The intermediate compound of formula (2) can also be prepared by the following method.

[0142] [ka]

[0143] [In the formula, L 1 ' is L with the structure in which the terminal is converted to a maleimidyl group. 1 and P 4 indicates a protecting group]

[0144] 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 was mentioned earlier The protecting group may be appropriately selected and used. The protecting group of the carboxy group of the obtained 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).

[0145] 4. Manufacturing method 4 Among the intermediates (10) described in Production Method 2, n 1 =1, L a =O compound (1 The compound represented by formula (10b), a salt thereof or a solvate thereof can be produced, for example, by the following method.

[0146] [ka]

[0147] [In the formula, L P represents the same as above, L represents an acyl group, which is an alkanoyl group such as an acetyl group or an aroyl group such as a benzoyl group, or represents a hydrogen atom, and X and Y represent an oligopeptide consisting of 1 to 3 amino acids, P 5 and P 7 represents the protecting group of the amino group, P 6 represents a protecting group for a carboxy group]

[0148] 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, particularly paratoluenesulfonic acid, are preferred. Furthermore, the base can be appropriately selected from the bases already mentioned. In particular, alkali metal alkoxides such as potassium tert-butoxide; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal or alkaline earth 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 are preferred. 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 The amino-protecting groups exemplified by the following are generally used for protecting amino groups: There are no particular limitations as long as the protecting group is a protective group for the amino group. Representative examples include the protecting groups for the amino group described in Production Method 2. 5 The protecting group of the amino group is cleaved as shown in In this case, a protecting group may be introduced again by reacting the compound with an appropriate amino group-protecting reagent, if necessary. Compound (24) can be prepared by removing the protecting group P of compound (23). 6 Manufactured by removing Here, P 6 Examples of the protecting group for the carboxy group include the following in Production Method 2: Representative examples are listed, and an appropriate selection can be made therefrom. In compound (23), the protecting group P of the amino group 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 9-Fluorenylmethyloxycarbonyl is a phenyl group, and P 6 is a benzyl group. The protecting groups can be selected depending on the properties of the compound that protects the amino and carboxy groups, and when removing the protecting groups, reagents and conditions can be selected depending on the protecting groups. 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 By removing the compound (26) can be produced by the reaction of compound (4) with carboxylic acid (24) and the addition of a protecting group P 6 In the reaction for removing , the same reagents and reaction conditions as those described in Production Method 2 can be used. good. 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.

[0149] 5. Manufacturing method 5 Among the intermediates (2) described in Production Method 2, n 1 = 1, n 2 =1, L a =O compounds The production method of (2) will be described in detail below: The compound represented by formula (2), its salt or solvate thereof can be produced, for example, by the following method.

[0150] [ka]

[0151] [In the formula, L1’ , L 2 , L P represents the same as above, Z represents an oligopeptide consisting of 1 to 3 amino acids, P 8 represents the protecting group of the amino group, P 9 represents a protecting group for a carboxy group.]

[0152] Protecting group P of amino acid or oligopeptide (28) whose terminal amino and carboxy groups are protected 8 The compound (29) is obtained by removing the amine (29). Compound (30) can be produced by reacting compound (11) with P 8 Shown as The protecting group for the amino group to be used is not particularly limited as long as it is a group that is usually used for protecting an amino group, and representative examples include the protecting groups for the amino group described in Production Method 2. 8 When removing the protecting group, the reagents and conditions can be selected according to the protecting group. In the reaction of compound (29) with carboxylic acid (11), the same reagents and reaction conditions as those described in Production Method 2 may be used. Protecting group P of compound (30) 9 Compound (31) is prepared by removing The intermediate (2b) can be produced by reacting the carboxylic acid (31) with the compound (26). 8 The typical protecting groups for the carboxyl group shown in the following formula are In addition to the above, the deprotection reaction may use the same reagents and reaction conditions as those described in Production Method 2. In addition, the reaction between compound (26) and carboxylic acid (31) may use the same reaction reagents and conditions as those usually used in peptide synthesis. Compound (2b) produced by the above method can be converted into compound (1) of the present invention according to the above production method.

[0153] 6. Manufacturing method 6 Among the intermediates (17) described in Production Method 2, n 1 = 1, n 2 =1, La =O compound The compound represented by formula (17b), a salt thereof or a solvate thereof can also be produced, for example, by the following method.

[0154] [ka]

[0155] [In the formula, L 1’ , L 2 , L P , X, Y, P 5 , P 6 , and P 7 indicates the same as above.]

[0156] The amino group-protecting group P of the compound (23) in which the terminal amino group and the terminal carboxy group are protected 5 The compound (32) is prepared by deprotecting the amine (32), and the resulting amine (32) is reacted with an oligopeptide (27) in which the terminal amino group or amino group is protected to prepare the compound (33). 5 The protecting group for the amino group shown in There are no particular limitations on the group as long as it is used to protect the amino group, and representative examples include the amino-protecting groups described in Production Method 2. 5 Even when removing The reagents and conditions should be selected according to the protecting group. 6 The carboxyl group shown as and the protecting group P 7 As a protecting group for the amino group shown in the following formula (1), a typical example is Examples of the protecting groups for the carboxyl group and amino group described in Method 2 are as follows. In compound (33), the protecting group P 6 and the amino group protecting group P 7 is preferably a protecting group that can be removed by the same method or conditions. For example, P 6 is a benzyl ester group, and P 7is a benzyloxycarbonyl group. Compound (34) is a compound obtained by removing the protecting group P of the carboxy group of compound (33). 6 and the amino group protecting group P 7 The carboxy protecting group P 6 and amino groups Protecting group P 7 Compound (37) can also be prepared by sequentially removing each of the Kiru et al., P 6 and P 7 are protecting groups that can be removed by the same method or conditions, compound (34) can be conveniently prepared by removing both in one step. Compound (17b) can be produced by reacting the obtained compound (34) with compound (11). The reaction of compound (34) with compound (11) can be carried out using the same reagents and reaction conditions as those described in Production Method 2.

[0157] The anti-HER2 antibody-drug conjugate of the present invention may become a hydrate by absorbing moisture or by adsorbing water when left in the air or when subjected to recrystallization or purification procedures, and such water-containing compounds or salts 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 atomic isotopes in unnatural proportions. 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), or carbon-14 ( 14The radiolabeled compounds may be radiolabeled with radioisotopes such as HCl, ... , are included within the scope of the present invention.

[0158] [Pharmaceuticals] The anti-HER2 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-HER2 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-HER2 antibody-drug conjugate of the present invention can also be expected to have an effect of suppressing the growth of, and even destroying, micrometastatic cancer cells. In particular, when HER2 expression is confirmed in primary cancer cells, administration of the anti-HER2 antibody-drug conjugate of the present invention can be expected to have an effect of suppressing or preventing cancer metastasis. For example, it can be expected to have an effect of suppressing and destroying cancer cells present in body fluids during the metastatic process, and an effect of suppressing or destroying microscopic cancer cells immediately after implantation in any tissue. Therefore, it can be expected to have an effect of suppressing or preventing cancer metastasis, especially after surgical removal of cancer. The anti-HER2 antibody-drug conjugate of the present invention can be administered to patients as a systemic therapy, and also can be administered locally to cancer tissues to be expected to have a therapeutic effect.

[0159] Examples of cancers to which the anti-HER2 antibody-drug conjugates of the present invention can be applied include lung cancer, urothelial cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, and penile cancer. The anti-HER2 antibody-drug conjugates of the present invention are intended to treat cancer cells that express HER2 protein, which can be recognized by the antibody in the antibody-drug conjugate. As used herein, "cancer expressing HER2 protein" refers to cancer containing cells bearing HER2 protein on their cell surface. HER2 protein is overexpressed in various human tumors, and immunohistochemical staining (IHC) is used to assess HER2 protein overexpression, and fluorescence in situ hybridization (FISH) is used to assess HER2 gene amplification. The evaluation can be performed using methods commonly used in this field, such as FISH. Furthermore, the anti-HER2 antibody-drug conjugate of the present invention exerts an anti-tumor effect by the anti-HER2 antibody recognizing and further internalizing the HER2 protein expressed on the surface of cancer cells. Therefore, the therapeutic targets of the anti-HER2 antibody-drug conjugate of the present invention are not limited to "cancers expressing HER2 protein," and can also be, for example, leukemia, malignant lymphoma, plasma cell carcinoma, myeloma, or sarcoma.

[0160] The anti-HER2 antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0161] Substances used in pharmaceutical compositions containing the anti-HER2 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.

[0162] The anti-HER2 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.

[0163] Various delivery systems are known and can be used to administer the anti-HER2 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.

[0164] 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.

[0165] The pharmaceutical composition of the present invention may contain only the anti-HER2 antibody-drug conjugate of the present application, or may contain the anti-HER2 antibody-drug conjugate and at least one other cancer therapeutic agent. The anti-HER2 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 anti-cancer agent used for such a purpose may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or sequentially, or may be administered at different administration intervals. Examples of such cancer therapeutic agents include 5-FU, pertuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, sorafenib, vinblastin, vinorelbine, everolims, tanespimycin, bevacizumab, oxaliplatin, lapatinib, and ado-t. Rastuzumab emtansine (T-DM1) or International Publication No. 2003 / 0 38043, as well as LH-RH analogs (leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), etc., but are not limited thereto as long as they have antitumor activity.

[0166] 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.

[0167] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the anti-HER2 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]

[0168] 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.

[0169] Reference Example 1 Preparation of trastuzumab Fourteen 440 mg vials of Herceptin (Genentech, Inc.) were dissolved in 2 L of cation exchange chromatography buffer A (25 mM citrate buffer, 30 mM NaCl, pH 5.0) and filtered through a 0.2 μm filter (Millipore Co.: Stericup 0.22 μm, GV PVDF Membrane). The sample was applied to a cation exchange chromatography column (SP Sepharose HP 240 ml, XK50 column) and eluted with cation exchange chromatography buffer B (25 mM citrate buffer, 500 mM NaCl, pH 5.0) using a linear gradient of 30 mM to 500 mM NaCl to fractionate the IgG monomer. Samples with a monomer purity of 98% or higher based on size exclusion chromatography were combined, concentrated using UF30K (Millipore Co.: PELLICON XL Filter, BIOMAX 30K, PXB030A50), and eluted with CBS buffer (10 mM citrate / 140 mM NaCl, pH 6.0). The eluate was then filtered through a 0.2 μm filter (Sartorius: Minisart-Plus 0.2 μm, 17823K).

[0170] Reference Example 2: Production of trastuzumab emtansine T-DM1 Conversion of antibody to SMCC: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-2 (PBS 6.5 / EDTA was used as the buffer solution), common procedure A, and common procedure B (280 nm extinction coefficient of 1.37 mL mg -1 cm -1The buffer was exchanged to PBS6.5 / EDTA using a PBS6.5 / EDTA buffer solution (using PBS6.5 / EDTA), and a solution of trastuzumab (160.0 mg) dissolved in PBS6.5 / EDTA (7.60 mL) was prepared in a 15 mL polypropylene tube. Next, a DMSO solution of SMCC (1.84 mg) (0.40 mL; approximately 5.1 equivalents per antibody molecule) was added at room temperature, and the antibody concentration of the reaction solution was adjusted to 20 mg / mL. The reaction solution was then reacted at room temperature for 2 hours using a tube rotator (MTR-103, AS ONE Corporation). This reaction solution was purified according to common procedure D-2 (using PBS6.5 / EDTA as the buffer), and a solution containing 154.9 mg of SMCC-derivatized antibody was collected in 12 mL of the tube. Got it. Conjugation of antibody and drug linker: Add the above solution to a 50 mL polypropylene tube and add PBS6.5 / EDTA (2.56 mL) and N 2 -Deacetyl-N 2 -(3-mercapto-1-oxopropyl)-maytansine (4.67 mg; DM1, Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14) A solution of 0.93 mL of SMCC-derivatized antibody in DMA (dimethylacetamide) (equivalent to approximately 5.8 equivalents per SMCC-derivatized antibody molecule) was added at room temperature, and the antibody concentration of the reaction solution was adjusted to 10 mg / mL. The reaction was carried out at room temperature for 16.5 hours using a tube rotator. Purification procedure: The above solution was purified by common procedure D-1 using sodium phosphate buffer (10 mM, pH 6.5) containing sodium chloride (137 mM) to obtain 35 mL of a solution containing the target Reference Example compound. Characterization: The following characterization values ​​were obtained using the common procedure E using UV absorbance at two wavelengths, 252 nm and 280 nm. Antibody concentration: 4.14 mg / mL, antibody yield: 144.9 mg (91%), average number of drugs bound per antibody molecule (n): 3.0.

[0171] Example 1 Intermediate (1) [ka]

[0172] 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 methanesulfonate (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 1 day. The solvent was removed 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 (0.595 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),1.82-1.89(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=18.8Hz),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) +

[0173] 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 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 trifluoroacetate salt of the title compound (0.343 g, quantitative yield). 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.79-1.92(4H,m),2.10-2.17(2 H,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. 15(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) +

[0174] Example 2 Antibody-drug conjugate (2) [ka]

[0175] Step 1: 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. This reaction solution was added dropwise to a solution of the compound obtained in Step 2 of Example 1 (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 give 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) +

[0176] Step 2: 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 1 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 to the residue to form an azeotrope. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) partitioned organic layer] to obtain the trifluoroacetate salt of 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 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) +

[0177] Step 3: 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 (337 mg, 0.353 mmol) obtained in step 2 above 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) +

[0178] Step 4: Antibody-drug conjugate (2) Antibody reduction: Trastuzumab prepared in Reference Example 1 was reduced to 1.37 mL / min as an extinction coefficient at 280 nm by common procedure C-1 and common procedure B described in Production Method 1. -1 cm -1 The antibody was diluted to 10 mg / mL with PBS 6.0 / EDTA (using 100% EDTA). 3.0 mL of this solution was placed in a 15 mL polypropylene tube, and 0.0934 mL of 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Tokyo Chemical Industry Co., Ltd.) aqueous solution (4.6 equivalents per antibody molecule) and 0.150 mL of 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.) were added. After confirming that the pH of the solution was within 7.4 ± 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 at 22°C for 10 minutes, a DMSO solution (0.187 mL; 9.2 equivalents per antibody molecule) containing 10 mM of the compound obtained in step 3 above was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, an N-acetylcysteine ​​(NAC, Sigma-Aldrich Co. LLC) aqueous solution (0.0374 mL; 18.4 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (using PBS 6.0 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: Using the common procedure E described in Preparation Method 1, the following property values ​​were obtained. Antibody concentration: 3.21 mg / mL, antibody yield: 22.5 mg (75%), average number of drugs bound per antibody molecule (n): 2.6.

[0179] Example 3 Antibody-drug conjugate (3) [ka]

[0180] Step 1: Antibody-drug conjugate (3) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.487 mL / mg). -1 cm -1 The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (1.25 mL) was placed in a 1.5 mL polypropylene tube, and 10 mM TCEP aqueous solution (0.039 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.0625 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: DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound from Step 3 of Example 2 (0.078 mL; 9.2 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was stirred at room temperature for 40 minutes using a tube rotator to conjugate the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.0155 mL; 18.4 equivalents per antibody molecule) was added, and the mixture was stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) to obtain 6 mL of a solution containing the target compound. The solution was further concentrated using common procedure A, and then the following property values ​​were obtained using common procedure E. Antibody concentration: 9.85 mg / mL, antibody yield: 6.9 mg (55%), average number of drugs bound per antibody molecule (n): 7.3.

[0181] Example 4 Antibody-drug conjugate (4) [ka]

[0182] Step 1: N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)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 of Example 1 (80 mg, 0.084 mmol) was reacted in the same manner as in Step 3 of Example 2, except that N-succinimidyl 3-maleimidopropionate (24.6 mg, 0.0924 mmol) was used instead of N-succinimidyl 6-maleimidohexanoate, to obtain the title compound (60.0 mg, 73%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.89(3H,t,J=7.3Hz),1.70-1.78(2H,m),1.81- 1.94(2H,m),2.12-2.23(4H,m),2.42(3H,s),2.81(1H,dd,J=13.7,9.8Hz), 3.01-3.15(3H,m),3.16-3.23(2H,m),3.30-3.35(1H,m),3.58-3.71(6H,m) ,3.71-3.79(1H,m),4.44-4.51(1H,m),5.19(1H,d,J=19.0Hz),5.27(1H,d, J=19.0Hz),5.43(1H,d,J=17.6Hz),5.47(1H,d,J=17.6Hz),5.57-5.63(1H, m),6.56(1H,s),7.02(2H,s),7.17-7.22(1H,m),7.22-7.30(5H,m),7.34(1 H,s),7.73(1H,t,J=5.6Hz),7.83(1H,d,J=10.7Hz),8.08(1H,t,J=5.6Hz), 8.15(1H,d,J=7.8Hz),8.30(2H,dt,J=18.7,5.7Hz),8.49(1H,d,J=8.8Hz). MS(APCI)m / z:990(M+H) +

[0183] Step 2: Antibody-drug conjugate (4) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1 The medium was replaced with PBS 6.0 / EDTA using a PBS containing 100 mM TCEP solution (0.015%), and the antibody concentration was adjusted to 10 mg / mL. This solution (1 mL) was placed in a 1.5 mL polypropylene tube, and 10 mM TCEP aqueous solution (0.015%) was added. 5 mL of HCl (2.3 equivalents per antibody molecule) and 1 M aqueous solution of dipotassium hydrogen phosphate (0.050 mL) were added. After confirming that the pH of this solution was within 7.4 ± 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: DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound from Step 3 of Example 2 (0.031 mL; 4.6 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was stirred at room temperature for 40 minutes using a tube rotator to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.0078 mL; 9.2 equivalents per antibody molecule) was added, and the mixture was stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) to obtain 6 mL of a solution containing the target compound. Using common procedure E, the following property values ​​were obtained. Antibody concentration: 1.32 mg / mL, antibody yield: 7.9 mg (79%), average number of drugs bound per antibody molecule (n): 3.1.

[0184] Example 5 Antibody-drug conjugate (5) [ka]

[0185] Step 1: Antibody-drug conjugate (5) The amount of 10 mM TCEP aqueous solution added was adjusted so that the molar ratio of TCEP to antibody during antibody reduction would be 4.6, the amount of 10 mM drug linker solution added was adjusted so that the molar ratio of the compound of Example 4, Step 1 to antibody during drug linker binding would be 9.2, and the amount of 100 mM NAC aqueous solution added was adjusted so that the molar ratio of NAC to antibody when the reaction was stopped would be 18.4. By performing the same procedures as in Example 4, Step 2, 6 mL of a solution containing the title antibody-drug conjugate was obtained, and the following characteristic values ​​were obtained. Antibody concentration: 1.23 mg / mL, antibody yield: 7.4 mg (74%), average number of drugs bound per antibody molecule (n): 6.1.

[0186] Example 6 Antibody-drug conjugate (6) [ka]

[0187] 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 2 of Example 2 (100 mg, 0.119 mmol) was reacted in the same manner as in Step 3 of Example 2, except that diisopropylethylamine (20.8 μL, 0.119 mmol) was used instead of triethylamine, and N-succinimidyl 3-(2-(2-(3-maleimidopropanamido)ethoxy)ethoxy)propanoate (50.7 mg, 0.119 mmol) was used 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(2H,m),2.38(3H,s),2.76(1H,dd,J=13.7,9.8Hz),2.96-3.18(9H,m),3.42-3.44(4H,m),3.53-3.76(10H,m),4.43(1H,t d,J=8.6,4.7Hz),5.14(1H,d,J=18.8Hz),5.23(1H,d,J=18.8Hz),5.38(1H,d,J=17.2Hz),5.42(1H,d,J=17.2Hz),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) +

[0188] Step 2: Antibody-drug conjugate (6) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1 The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (1.25 mL) was placed in a 1.5 mL polypropylene tube, to which was added 10 mM aqueous TCEP solution (0.019 mL; 2.3 equivalents per antibody molecule) and 1 M aqueous dipotassium hydrogen phosphate solution (0.0625 mL). 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: DMSO (Sigma-Aldrich Co., LLC; 0.109 mL) and a DMSO solution containing 10 mM of the compound from step 1 (0.039 mL; 4.6 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was stirred at room temperature for 40 minutes using a tube rotator to allow the drug linker to bind to the antibody. Next, 100 mM NAC solution (0.008 mL) was added, and the mixture was stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) to obtain 6 mL of a solution containing the target compound. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.76 mg / mL, antibody yield: 10.6 mg (85%), average number of drugs bound per antibody molecule (n): 3.6.

[0189] Example 7 Antibody-drug conjugate (7) [ka]

[0190] Step 1: Antibody-drug conjugate (7) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1 (Use) to make the medium PBS6. The antibody concentration was adjusted to 10 mg / mL by replacing the EDTA with 0 / EDTA. This solution (1.25 mL) was placed in a 1.5 mL polypropylene tube, and 10 mM TCEP aqueous solution (0.039 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.0625 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: DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound from Step 1 of Example 6 (0.078 mL; 9.2 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was stirred at room temperature for 40 minutes using a tube rotator to bind the drug linker to the antibody. Next, 100 mM NAC aqueous solution (0.0155 mL) was added, and the mixture was further stirred 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) to obtain 6 mL of a solution containing the target compound. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.93 mg / mL, antibody yield: 11.6 mg (93%), average number of drugs bound per antibody molecule (n): 6.9.

[0191] Example 8 Antibody-drug conjugate (8) [ka]

[0192] Step 1: Antibody-drug conjugate (8) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1 The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (1.25 mL) was placed in a 1.5 mL polypropylene tube, and 10 mM TCEP aqueous solution (0.039 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.0625 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: DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound from Step 1 of Example 6 (0.078 mL; 9.2 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was stirred at room temperature for 40 minutes using a tube rotator to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.0155 mL) was added. mL) was added and stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (using ABS as the buffer) to obtain 5.7 mL of a solution containing the target compound. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.50 mg / mL, antibody yield: 8.55 mg (86%), average number of drugs bound per antibody molecule (n): 6.2.

[0193] Example 9 Antibody-drug conjugate (9) [ka]

[0194] Step 1: N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxo-16-azanonadecane-1-oyl]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 2 of Example 2 (90 mg, 0.107 mmol) was reacted in the same manner as in Step 3 of Example 2, except that diisopropylethylamine (18.7 μL, 0.107 mmol) was used instead of triethylamine, and 1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoic-19-oate N-succinimidyl (55.1 mg, 0.107 mmol) was used instead of 6-maleimidohexanoic acid N-succinimidyl to obtain the title compound (50 mg, 37%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.85(3H,t,J=7.2Hz),1.64-1.74(2H,m),1.77-1.90(2H,m),2.06-2.19(4H,m),2.27-2.32(2H,m),2.33-2.37(2H, m),2.38(3H,s),2.72-2.80(3H,m),2.96-3.19(6H,m),3.39-3.48(10H,m),3.52-3.75(10H,m),4.39-4.48(1H, m),5.14(1H,d,J=18.8Hz),5.23(1H,d,J=18.8Hz),5.38(1H,d,J=17.0Hz),5.42(1H,d,J=17.0Hz),5.52-5.58( 1H,m),6.52(1H,s),6.98(1H,s),7.13-7.24(5H,m),7.29(1H,s),7.69(1H,t,J=5.5Hz),7.78(1H,d,J=10.9Hz) ,7.98-8.03(2H,m),8.10(1H,d,J=7.8Hz),8.16(1H,t,J=5.7Hz),8.23(1H,t,J=5.7Hz),8.44(1H,d,J=8.6Hz). MS(APCI)m / z:1237(M+H) +

[0195] Step 2: Antibody-drug conjugate (9) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 1.75 mg / mL, antibody yield: 10.5 mg (84%), average number of drugs bound per antibody molecule (n): 3.4.

[0196] Example 10 Antibody-drug conjugate (10) [ka]

[0197] Step 1: Antibody-drug conjugate (10) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7 using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 of Example 9. Antibody concentration: 1.79 mg / mL, antibody yield: 10.7 mg (86%), average number of drugs bound per antibody molecule (n): 6.0.

[0198] Example 11 Intermediate (11) [ka]

[0199] Step 1: tert-butyl [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)ethyl]carbamate The methanesulfonate salt of exatecan (3.10 g, 5.47 mol) was reacted in the same manner as in Step 1 of Example 1, except that {2-[(tert-butoxycarbonyl)amino]ethoxy}acetic acid (J. Med. Chem., 1992, Vol. 35, p. 2928; 1.55 g, 6.01 mmol) was used instead of 4-(tert-butoxycarbonylamino)butanoic acid, to obtain the title compound (2.56 g, 73%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.3Hz),1.26(9H,s),1.81-1.91(2H,m),2.13-2.22(2H,m),2 .40(3H,s),3.08-3.26(4H,m),3.43-3.53(2H,m),4.00(1H,d,J=15.1Hz),4.05(1H,d,J=15.1Hz),5.14 (1H,d,J=18.7Hz),5.22(1H,d,J=18.7Hz),5.40(1H,d,J=16.6Hz),5.44(1H,d,J=16.6Hz),5.59-5.66( 1H,m),6.53(1H,s),6.86(1H,t,J=5.4Hz),7.31(1H,s),7.79(1H,d,J=10.9Hz),8.49(1H,d,J=9.1Hz). MS(APCI)m / z:637(M+H) +

[0200] Step 2: 2-(2-aminoethoxy)-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]acetamide The compound obtained in Step 1 above (1.50 g, 2.36 mol) was reacted in the same manner as in Step 2 of Example 1 to give the trifluoroacetate salt of the title compound (1.50 g, quantitative) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.5Hz),1.81-1.92(2H,m),2.15-2.23( 2H,m),2.41(3H,s),3.05(2H,t,J=5.1Hz),3.15-3.23(2H,m),3.71(2H,t,J=5.1Hz ),4.10(2H,s),5.19(1H,d,J=18.7Hz),5.24(1H,d,J=18.7Hz),5.43(2H,s),5.58 -5.66(1H,m),6.55(1H,s),7.33(1H,s),7.73-7.84(4H,m),8.55(1H,d,J=9.1Hz). MS(APCI)m / z:537(M+H) +

[0201] Example 12 Antibody-drug conjugate (12) [ka]

[0202] Step 1: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-[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)ethyl]glycinamide The compound of Step 2 of Example 11 (554 mg, 0.85 mmol) was reacted in the same manner as in Step 1 of Example 2 to obtain the title compound (775 mg, 95%). 1 H-NMR(400MHz,DMSO-d6)δ:0.85(3H,t,J=7.3Hz),1.36(9H,s),1.78-1.89(2H,m),2.13-2.22(2H,m),2.39(3H,s),2.71(1H,dd,J=13.4,9.8Hz),2 .95(1H,dd,J=13.4,4.3Hz),3.09-3.23(1H,m),3.23-3.32(2H,m),3.40- 3.62(8H,m),3.73(1H,dd,J=16.5,5.5Hz),4.03(2H,s),4.39-4.47(1H,m) ,5.17(1H,d,J=18.9Hz),5.25(1H,d,J=18.9Hz),5.41(1H,d,J=16.8Hz), 5.45(1H,d,J=16.8Hz),5.57-5.64(1H,m),6.54(1H,s),6.99(1H,t,J=5.8 Hz),7.13-7.26(5H,m),7.31(1H,s),7.76-7.82(2H,m),7.90(1H,t,J=5. 2Hz), 8.13(1H,d,J=7.9Hz),8.27(1H,t,J=5.8Hz),8.49(1H,d,J=8.5Hz). MS(APCI)m / z:955(M+H) +

[0203] Step 2: Glycylglycyl-L-phenylalanyl-N-[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)ethyl]glycinamide The compound obtained in Step 1 above (630 mg, 0.659 mmol) was reacted in the same manner as in Step 2 of Example 2 to give the trifluoroacetate salt of the title compound (588 mg, 92%). 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.3Hz),1.79-1.90(2H,m),2.13-2.22(2H,m),2.39(3H,s),2.71(1H,dd,J=13.4,10.1Hz), 2.99(1H,dd,J=13.4,4.3Hz),3.09-3.23(1H,m),3.24-3.32(3H,m),3.41-3.71(7H,m),3.86(1H,dd,J=16.8,5.8Hz),4.04(2H,s),4.5 2(1H,td,J=9.0,4.1Hz),5.17(1H,d,J=18.9Hz),5.25(1H,d,J=18.9Hz),5.41(1H,d,J=16.5Hz),5.45(1H,d,J=16.5Hz),5.56-5.65( 1H,m),6.55(1H,s),7.13-7.26(5H,m),7.32(1H,s),7.80(1H,d,J=11.0Hz),7.87-8.01(4H,m),8.29-8.36(2H,m),8.46-8.55(2H,m). MS(APCI)m / z:855(M+H) +

[0204] Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[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)ethyl]glycinamide The compound obtained in Step 2 above (240 mg, 0.247 mmol) was reacted in the same manner as in Step 3 of Example 2 to obtain the title compound (162 mg, 62%). 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.6Hz),1.13-1.22(2H,m),1.40-1.51( 4H,m),1.78-1.90(2H,m),2.09(2H,t,J=7.6Hz),2.14-2.21(2H,m),2.39(3H,s), 2.74(1H,dd,J=13.6,9.7Hz),2.96(1H,dd,J=13.6,4.5Hz),3.08-3.24(1H,m),3. 24-3.30(1H,m),3.33-3.40(4H,m),3.47-3.68(7H,m),3.72(1H,dd,J=16.6,5.7H z),4.03(2H,s),4.42(1H,td,J=8.6,4.2Hz),5.17(1H,d,J=18.7Hz),5.25(1H,d, J=18.7Hz),5.40(1H,d,J=17.2Hz),5.44(1H,d,J=17.2Hz),5.57-5.64(1H,m),6. 52(1H,s),6.99(2H,s),7.13-7.25(5H,m),7.31(1H,s),7.74-7.81(2H,m),7.99( 1H,t,J=5.7Hz),8.03-8.11(2H,m),8.22(1H,t,J=5.7Hz),8.47(1H,d,J=9.1Hz). MS(APCI)m / z:1048(M+H) +

[0205] Step 4: Antibody-drug conjugate (12) Using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 3 above, the title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6. After concentrating the solution using standard procedure E, the following properties were obtained: Antibody concentration: 10.77 mg / mL, antibody yield: 7.5 mg (60%), average number of drugs bound per antibody molecule (n): 3.7.

[0206] Example 13 Antibody-drug conjugate (13) [ka]

[0207] Step 1: Antibody-drug conjugate (13) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 3 of Example 12. The solution was then concentrated using Common Procedure A, and the following characteristic values ​​were obtained using Common Procedure E. Antibody concentration: 10.69 mg / mL, antibody yield: 7.5 mg (60%), average number of drugs bound per antibody molecule (n): 6.9.

[0208] Example 14 Intermediate (14) [ka]

[0209] Step 1: tert-butyl (3-{[(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-oxopropyl)carbamate The methanesulfonate salt of exatecan (500 mg, 0.941 mmol) was reacted in the same manner as in Step 1 of Example 1, except that N-(tert-butoxycarbonyl)-β-alanine was used instead of 4-(tert-butoxycarbonylamino)butanoic acid, to obtain the title compound (616 mg, quantitative) as a yellow-brown solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.29(9H,s),1.86(2H,dt,J=15.1,7.3Hz),2 .04-2.22(2H,m),2.31(2H,t,J=6.8Hz),2.40(3H,s),3.10-3.26(4H,m),5.15(1H,d,J=18.8Hz),5.26(1H,d,J=19.2Hz),5.42(2H,dd,J=1 8.8,16.4Hz),5.57(1H,dt,J=8.5,4.2Hz),6.53(1H,s),6.78(1H,t,J=5.5Hz),7.30(1H,s),7.80(1H,d,J=11.0Hz),8.46(1H,d,J=8.6Hz). MS(ESI)m / z:607(M+H) +

[0210] Step 2: 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]-β-alaninamide The compound obtained in Step 1 above was reacted in the same manner as in Step 2 of Example 1 to give the trifluoroacetate salt of the title compound (499 mg, 86%) as a yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.86(2H,dquin,J=14.6,7.2,7.2,7.2,7. 2Hz),2.06-2.27(1H,m),2.41(3H,s),2.46-2.57(2H,m),3.08(2H,t,J=6.8Hz),3.14-3.24( 2H,m),5.22(1H,d,J=18.8Hz),5.29(1H,d,J=18.8Hz),5.43(2H,s),5.58(1H,dt,J=8.5,4.5 Hz),6.55(1H,s),7.32(1H,s),7.74(3H,brs),7.82(1H,d,J=11.0Hz),8.67(1H,d,J=8.6Hz). MS(ESI)m / z:507(M+H) +

[0211] Example 15 Antibody-drug conjugate (15) [ka]

[0212] Step 1: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide The compound obtained in Step 2 of Example 14 (484 mg, 0.780 mmol) was reacted in the same manner as in Step 1 of Example 2 to obtain the title compound (626 mg, 87%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.27-1.42(9H,m),1.77-1.93(2H,m),2.06-2.22(2H,m),2.36(2H,t,J=7.2Hz),2.40(3H,d,J= 1.6Hz),2.44-2.54(2H,m),2.76(1H,dd,J=14.5,10.2Hz),3.02(1H,dd,J=13.9,4.5Hz),3.12-3.22(2H,m),3.52(6H,d,J=6.3Hz),4.42-4.54(1H ,m),5.19(1H,d,J=19.2Hz),5.26(1H,d,J=18.4Hz),5.42(1H,dd,J=18. 4,16.4Hz),5.57(1H,dt,J=8.7,4.4Hz),6.53(1H,s),6.98(1H,t,J=5.9H z),7.14-7.28(5H,m),7.31(1H,s),7.77-7.84(1H,m),7.91(1H,t,J=5.5 Hz),8.16(1H,d,J=7.8Hz),8.27(1H,t,J=5.1Hz),8.52(1H,d,J=9.0Hz).

[0213] Step 2: Glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide trifluoroacetate The compound obtained in Step 1 above (624 mg, 0.675 mmol) was reacted in the same manner as in Step 2 of Example 2 to obtain the title compound (626 mg, 92%) as a yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.86(2H,tt,J=14.5,7.2Hz),2.07-2.22(2H,m),2.36(2H,t,J=7.2Hz),2.40(3H,s),2.44-2.54(2 H,m),2.75(1H,dd,J=13.7,9.8Hz),3.04(1H,dd,J=13.7,4.3Hz),3.12-3. 22(2H,m),3.58(2H,d,J=4.7Hz),3.69(3H,td,J=11.2,5.7Hz),3.87(1H,dd ,J=17.0,5.7Hz),4.54(1H,m,J=17.8,4.5Hz),5.19(1H,d,J=19.2Hz),5.2 6(1H,d,J=18.8Hz),5.43(2H,s),5.51-5.60(1H,m),6.55(1H,s),7.14-7.2 9(5H,m),7.32(1H,s),7.81(1H,d,J=10.9Hz),7.88(1H,t,J=5.7Hz),7.97 (3H,brs),8.29-8.38(2H,m),8.50(1H,t,J=5.7Hz),8.55(1H,d,J=8.6Hz). MS(ESI)m / z:825(M+H) +

[0214] Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide The compound obtained in Step 2 above (60.0 mg, 0.0646 mmol) was reacted in the same manner as in Step 3 of Example 2 to give the title compound (14.0 mg, 21%) as a solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.2Hz),1.12-1.22(2H,m),1.39-1.51(4H,m),1.79- 1.91(2H,m),2.02-2.20(2H,m),2.07(2H,t,J=7.4Hz),2.30-2.42(4H,m),2.40(3H,s),2.78(1H,dd,J=14.1,9.4Hz),3.02(1 H,dd,J=14.7,4.9Hz),3.12-3.21(2H,m),3.26-3.42(2H,m),3.50-3.80(6H,m),4.40-4.51(1H,m),5.19(1H,d,J=19.6Hz),5 .26(1H,d,J=19.2Hz),5.42(2H,brs),5.51-5.62(1H,m),6.53(1H,s),6.99(2H,s),7.13-7.28(5H,m),7.31(1H,s),7.74-7. 84(2H,m),8.01(1H,t,J=5.3Hz),8.06(1H,t,J=5.7Hz),8.14(1H,d,J=8.2Hz),8.25(1H,t,J=5.7Hz),8.53(1H,d,J=8.6Hz). MS(ESI)m / z:1018(M+H) +

[0215] Step 4: Antibody-drug conjugate (15) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-1 and common procedure B (1.37 mL / min as 280 nm extinction coefficient). -1 cm -1 The antibody was diluted to 10 mg / mL with PBS 6.0 / EDTA (using 1.0 mL of 1 mL of 1 mL of 1 mL of 10 mM TCEP solution (0.0155 mL; 2.3 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate solution (0.050 mL) were added. After confirming that the pH of the solution was within 7.4 ± 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 at 22°C for 10 minutes, a DMSO solution (0.0311 mL; 4.6 equivalents per antibody molecule) containing 10 mM of the compound obtained in step 3 above was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.00622 mL; 9.2 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (PBS 6.0 was used as the buffer solution) to obtain 6 mL of a solution containing the title antibody-drug conjugate. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.18 mg / mL, antibody yield: 7.08 mg (71%), average number of drugs bound per antibody molecule (n): 2.0.

[0216] Example 16 Antibody-drug conjugate (16) [ka]

[0217] Step 1: Antibody-drug conjugate (16) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-1 and common procedure B (1.37 mL / min as 280 nm extinction coefficient). -1 cm -1 The antibody was diluted to 10 mg / mL with PBS 6.0 / EDTA (using 1.0 mL of 1 mL of 1 mL of 10 mM TCEP solution (0.0311 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate solution (0.050 mL) were added. After confirming that the pH of the solution was within 7.4 ± 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 at 22°C for 10 minutes, a DMSO solution (0.0622 mL; 9.2 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 3 of Example 15 was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.0124 mL; 18.4 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (PBS 6.0 was used as the buffer solution) to obtain 6 mL of a solution containing the title antibody-drug conjugate. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.03 mg / mL, antibody yield: 6.18 mg (62%), average number of drugs bound per antibody molecule (n): 3.8.

[0218] Example 17 Antibody-drug conjugate (17) [ka]

[0219] Step 1: N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide The compound obtained in Step 2 of Example 15 (60.0 mg, 0.0646 mmol) was reacted in the same manner as in Step 3 of Example 2, except that N-succinimidyl 3-maleimidopropionate was used instead of N-succinimidyl 6-maleimidohexanoate, to obtain the title compound (36.0 mg, 57%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.4Hz),1.85(2H,dt,J=14.4,7.5Hz),2.05-2.22(2H,m),2.40(3H,s),2.30-2.44(5H, m),2.73-2.84(1H,m),3.02(1H,dd,J=13.9,4.5Hz),3.17(3H,d,J=5.1Hz),3.26-3.40(2H,m),3.41-3.81(6H,m),4.40-4.51(1H ,m),5.19(1H,d,J=19.2Hz),5.26(1H,d,J=18.8Hz),5.42(2H,brs),5.52-5.61(1H,m),6.53(1H,s),6.99(2H,s),7.13-7.28(5H ,m),7.31(1H,s),7.80(2H,d,J=10.2Hz),8.03(1H,t,J=5.5Hz),8.12(1H,d,J=8.2Hz),8.20-8.31(2H,m),8.52(1H,d,J=8.6Hz). MS(ESI)m / z:976(M+H) +

[0220] Step 2: Antibody-drug conjugate (17) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 1.74 mg / mL, antibody yield: 10.4 mg (83%), average number of drugs bound per antibody molecule (n): 3.7.

[0221] Example 18 Antibody-drug conjugate (18) [ka]

[0222] Step 1: Antibody-drug conjugate (18) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7 using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 of Example 17. Antibody concentration: 1.98 mg / mL, antibody yield: 11.9 mg (95%), average number of drugs bound per antibody molecule (n): 6.6.

[0223] Example 19 Antibody-drug conjugate (19) [ka]

[0224] Step 1: N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino})ethoxy]propanoyl}glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide The compound obtained in Step 2 of Example 15 (60.0 mg, 0.0646 mmol) was reacted in the same manner as in Step 3 of Example 2, except that N-succinimidyl 3-(2-(2-(3-maleimidopropanamido)ethoxy)ethoxy)propanoate was used instead of N-succinimidyl 6-maleimidohexanoate, to obtain the title compound (23.0 mg, 31%) as a solid. 1H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.4Hz),1.77-1.92(2H,m),2.07-2.21(2H,m),2.27-2.42(6H,m),2.40(3H,s), 2.74-2.84(1H,m),2.97-3.06(1H,m),3.09-3.21(4H,m),3.25-3.39(6H,m),3.45(4H,s),3.50-3.80(8H,m),4.41-4.51(1 H,m),5.19(1H,d,J=18.4Hz),5.26(1H,m,J=18.4Hz),5.42(2H,brs),5.51-5.61(1H,m),6.54(1H,s),7.00(2H,s),7.13- 7.28(5H,m),7.31(1H,s),7.74-7.87(2H,m),7.93-8.07(2H,m),8.09-8.21(2H,m),8.26(1H,brs),8.54(1H,d,J=8.6Hz). MS(ESI)m / z:1135(M+H) +

[0225] Step 2: Antibody-drug conjugate (19) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 1.60 mg / mL, antibody yield: 9.6 mg (77%), average number of drugs bound per antibody molecule (n): 1.9.

[0226] Example 20 Antibody-drug conjugate (20) [ka]

[0227] Step 1: Antibody-drug conjugate (20) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7 using trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 of Example 19. Antibody concentration: 1.69 mg / mL, antibody yield: 10.1 mg (81%), average number of drugs bound per antibody molecule (n): 3.0.

[0228] Example 21 Antibody-drug conjugate (21) [ka]

[0229] Step 1: N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonandecan-1-oyl]glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide The compound obtained in Step 2 of Example 15 (60.0 mg, 0.0646 mmol) was reacted in the same manner as in Step 3 of Example 2, except that N-succinimidyl 1-maleimido-3-oxo-7,10,13,16-tetraoxa-4-azanonadecanoate was used instead of N-succinimidyl 6-maleimidohexanoate, to obtain the title compound (23.0 mg, 29%) as a solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.0Hz),1.85(2H,tt,J=14.6,7.1Hz),2.06-2.22(2H,m),2.40(3H,s),2.28-2.43(6H,m),2.78(1H,d d,J=13.7,9.4Hz),3.02(1H,dd,J=14.1,3.9Hz),3.09-3.22(4H,m),3.27-3.41(4H,m),3.47(12H,d ,J=8.6Hz),3.53-3.81(10H,m),4.41-4.51(1H,m),5.19(1H,d,J=19.2Hz),5.26(1H,d,J=18.8Hz), 5.42(2H,brs),5.53-5.61(1H,m),6.54(1H,s),7.00(2H,s),7.12-7.29(5H,m),7.31(1H,s),7.74- 7.85(2H,m),8.03(2H,d,J=6.6Hz),8.11-8.21(2H,m),8.27(1H,t,J=5.9Hz),8.54(1H,d,J=8.6Hz). MS(ESI)m / z:1224(M+H) +

[0230] Step 2: Antibody-drug conjugate (21) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 1.77 mg / mL, antibody yield: 10.6 mg (85%), average number of drugs bound per antibody molecule (n): 3.2.

[0231] Example 22 Antibody-drug conjugate (22) [ka]

[0232] Step 1: Antibody-drug conjugate (22) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7 using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 of Example 21. Antibody concentration: 1.89 mg / mL, antibody yield: 11.3 mg (90%), average number of drugs bound per antibody molecule (n): 6.2.

[0233] Example 23 Intermediate (23) [ka]

[0234] Step 1: tert-butyl (6-{[(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}-6-oxohexyl)carbamate The methanesulfonate salt of exatecan (0.500 g, 0.882 mmol) was reacted in the same manner as in Step 1 of Example 1, except that 6-(tert-butoxycarbonylamino)hexanoic acid was used instead of 4-(tert-butoxycarbonylamino)butanoic acid, to obtain the title compound (0.620 g, quantitative). 1 H-NMR(DMSO-d6)δ:0.83(3H,t,J=7.8Hz),1.14-1.28(2H,m),1.31(9H,s),1.47-1.61(2H ,m),1.75-1.89(2H,m),2.04-2.17(4H,m),2.35(3H,s),2.81-2.88(2H,m),3.09-3.16(2 H,m),5.10(1H,d,J=19.4Hz),5.16(1H,d,J=19.4Hz),5.39(2H,s),5.48-5.55(1H,m),6. 50(1H,s),6.73-6.78(1H,m),7.26(1H,s),7.74(1H,d,J=10.9Hz),8.39(1H,d,J=9.0Hz).

[0235] Step 2: 6-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]hexanamide The compound obtained in Step 1 above (0.397 g, 0.611 mmol) was reacted in the same manner as in Step 2 of Example 1 to give the trifluoroacetate salt of the title compound (0.342 g, 84%). 1 H-NMR(DMSO-d6)δ:0.88(3H,t,J=7.2Hz),1.31-1.41(2H,m),1.52-1.70(4H,m),1.80-1.94(2H,m), 2.05-2.18(2H,m),2.21(2H,t,J=7.4Hz),2.40(3H,s),2.81(2H,t,J=7.4Hz),3.10-3.25(2H,m),3.3 3(2H,brs),5.18(1H,d,J=19.8Hz),5.22(1H,d,J=19.8Hz),5.41(2H,d,J=16.6Hz),5.45(2H,d,J=1 6.6Hz),5.53-5.60(1H,m),6.55(1H,s),7.32(1H,s),7.80(1H,d,J=10.9Hz),8.49(1H,d,J=9.2Hz).

[0236] Example 24 Antibody-drug conjugate (24) [ka]

[0237] Step 1: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(6-{[(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}-6-oxohexyl)glycinamide The compound obtained in Step 2 of Example 23 (0.170 g, 0.516 mmol) was reacted in the same manner as in Step 1 of Example 2 to obtain the title compound (0.225 g, 91%). 1H-NMR(DMSO-d6)δ:0.88(3H,t,J=7.4Hz),1.43-1.70(6H,m),1.87(2H,td,J=15.0,7.4Hz),2.10-2.22(3H, m),2.28-2.37(1H,m),2.42(3H,s),2.78-2.85(1H,m),3.01-3.10(3H,m),3.15-3.22(2H,m),3.54-3.61(5H ,m),3.62-3.69(1H,m),4.44-4.53(1H,m),5.17(1H,d,J=19.2Hz),5.25(1H,d,J=19.2Hz),5.45(2H,s),5.5 4-5.61(1H,m),6.55(1H,s),7.02(1H,t,J=6.1Hz),7.11-7.28(5H,m),7.33(1H,s),7.63-7.69(1H,m),7.82 (1H,d,J=11.0Hz),7.90-7.96(1H,m),8.17(1H,d,J=7.8Hz),8.28(1H,t,J=5.5Hz),8.46(1H,d,J=9.0Hz).

[0238] Step 2: Glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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}-6-oxohexyl)glycinamide The compound obtained in Step 1 above (0.105 g, 0.108 mmol) was reacted in the same manner as in Step 2 of Example 2 to obtain the title compound (0.068 mg, 65%). 1H-NMR(DMSO-d6)δ:0.89(3H,t,J=7.4Hz),1.15-1.67(6H,m),1.79-1.97(2H,m),2.08-2.24(4H,m),2.42(3H,s),2.76 -2.82(1H,m),3.00-3.10(5H,m),3.19(1H,s),3.50-3.63(2H,m),3.64-3.76(3H,m),3.84-3.92(1H,m),4.51-4.59(1 H,m),5.17(1H,d,J=19.4Hz),5.24(1H,d,J=19.4Hz),5.44(2H,s),5.53-5.61(1H,m),6.55(1H,brs),7.15-7.29(5H, m),7.33(1H,s),7.72-7.78(1H,m),7.82(1H,d,J=11.0Hz),7.96-8.08(2H,m),8.30-8.38(2H,m),8.46-8.56(2H,m).

[0239] Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(6-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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}-6-oxohexyl)glycinamide The compound obtained in Step 2 above (58 mg, 0.060 mmol) was reacted in the same manner as in Step 3 of Example 2 to obtain the title compound (39 mg, 62%). 1H-NMR(CD3OD)δ:0.99(3H,t,J=7.4Hz),1.27(2H,td,J=11.6,6.1Hz),1.38 -1.44(2H,m),1.50-1.63(6H,m),1.65-1.80(2H,m),1.89-1.98(2H,m),2.1 7-2.25(3H,m),2.26-2.36(3H,m),2.40(3H,s),2.95(1H,dd,J=14.3,9.2Hz ),3.12(1H,dd,J=13.7,5.7Hz),3.15-3.25(4H,m),3.44(2H,t,J=7.2Hz),3 .65(1H,d,J=17.2Hz),3.76(1H,d,J=17.2Hz),3.79-3.86(4H,m),4.43(1H, dd,J=8.9,6.0Hz),5.10(1H,d,J=18.9Hz),5.25(1H,d,J=18.9Hz),5.35(1H ,d,J=16.6Hz),5.56(1H,d,J=16.0Hz),5.60-5.64(1H,m),6.76(2H,s),7.1 2-7.24(6H,m),7.58(1H,s),7.60(1H,d,J=10.9Hz),7.68(1H,t,J=5.7Hz). MS(ESI)m / z:1060(M+H) +

[0240] Step 4: Antibody-drug conjugate (24) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-1 and common procedure B (1.37 mL / min as 280 nm extinction coefficient). -1 cm -1 This solution (9.0 mL) was placed in a 50 mL tube and diluted with 10 mM TCEP (0.140 mL; 2.3 equivalents per antibody molecule). A volume of 100 mL of 1M potassium hydrogen phosphate dihydrate solution (0.450 mL) was added. After confirming that the pH of this solution was within 7.4±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 at 22°C for 10 minutes, a DMSO solution (0.280 mL; 4.6 equivalents per antibody molecule) containing 10 mM of the compound from step 3 above was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.0559 mL; 9.2 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (PBS 7.4 was used as the buffer) to obtain a solution containing the title antibody-drug conjugate. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 3.30 mg / mL, antibody yield: 53.5 mg (59%), average number of drugs bound per antibody molecule (n): 1.7.

[0241] Example 25 Antibody-drug conjugate (25) [ka]

[0242] Step 1: Antibody-drug conjugate (25) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-1 and common procedure B (1.37 mL / min as 280 nm extinction coefficient). -1 cm -1 The antibody was diluted to 10 mg / mL with PBS 6.0 / EDTA (using 100% EDTA). 9.0 mL of this solution was placed in a 50 mL tube, and 0.280 mL of 10 mM TCEP (4.6 equivalents per antibody molecule) and 0.450 mL of 1 M potassium dihydrogen phosphate were added. After confirming that the pH of the solution was within 7.4 ± 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 at 22°C for 10 minutes, a DMSO solution (0.559 mL; 9.2 equivalents per antibody molecule) containing 10 mM of the compound from Step 3 of Example 24 was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.112 mL; 18.4 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (PBS 6.0 was used as the buffer) to obtain a solution containing the title antibody-drug conjugate. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 10.65 mg / mL, antibody yield: 55.1 mg (61%), average number of drugs bound per antibody molecule (n): 2.5.

[0243] Example 26 Antibody-drug conjugate (26) [ka]

[0244] 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 (THF; 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 mixture 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. After the solvent was distilled off under reduced pressure, 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, 6 7%) 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=7.0H z),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).

[0245] 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 THF (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).

[0246] 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 was evaporated under reduced pressure to give the title compound (1.52 g, quantitative) as a colorless solid. 1 H-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).

[0247] 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 Under ice cooling, a solution of exatecan methanesulfonate (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 (10.0 mL) was added with N,N-diisopropylethylamine (92.9 μL, 0.533 mmol) and N,N'-dicyclohexylcarbodiimide (0.143 g, 0.69 mmol). The resulting 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) partitioned organic layer) to obtain the title compound (0.352 g, 82%) as a pale brown solid. 1 H-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) +

[0248] 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.

[0249] 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 (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) +

[0250] 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 Piperidine (0.251 mL, 2.53 mmol) was added to a solution of the compound (0.269 g, 0.253 mmol) obtained in step 6 above in N,N-dimethylformamide (4.00 mL), and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to obtain a mixture containing the title compound. The mixture was used in the next reaction without further purification.

[0251] 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 (0.253 mmol) obtained in Step 7 above 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) +

[0252] Step 9: Antibody-drug conjugate (26) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 8 above. Antibody concentration: 1.61 mg / mL, antibody yield: 9.7 mg (77%), average number of drugs bound per antibody molecule (n): 2.9.

[0253] Example 27 Antibody-drug conjugate (27) [ka]

[0254] Step 1: Antibody-drug conjugate (27) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7 using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 8 of Example 26. Antibody concentration: 1.58 mg / mL, antibody yield: 9.5 mg (76%), average number of drugs bound per antibody molecule (n): 5.6.

[0255] Example 28 Antibody-drug conjugate (28) [ka]

[0256] Step 1: Antibody-drug conjugate (28) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (1.25 mL) was placed into two 1.5 mL polypropylene tubes, and 10 mM TCEP aqueous solution (0.039 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.0625 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: DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound from Step 8 of Example 26 (0.078 mL; 9.2 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was stirred at room temperature for 40 minutes using a tube rotator to bind the drug linker to the antibody. Next, 100 mM NAC aqueous solution (0.0155 mL) was added, and the mixture was stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (using ABS as the buffer), and 11.7 mL of a solution containing the target compound was obtained. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.60 mg / mL, antibody yield: 18.7 mg (94%), average number of drugs bound per antibody molecule (n): 5.2.

[0257] Example 29 Antibody-drug conjugate (29) [ka]

[0258] Step 1: Antibody-drug conjugate (29) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (6 mL) was placed in a polypropylene tube, and 10 mM TCEP aqueous solution (0.108 mL; 2.5 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.091 mL) were added. After confirming that the pH of this solution was within 7.0 ± 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: DMSO (0.146 mL) and a DMSO solution containing 10 mM of the compound from Step 8 of Example 26 (0.193 mL; 4.5 equivalents per antibody molecule) were 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 aqueous solution (0.029 mL) was added, and the mixture was stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) to obtain 24 mL of a solution containing the target compound. Characterization: Common operations E and F(ε D,280 = 5178 (measured value), ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 1.77 mg / mL, antibody yield: 42 mg (85%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 3.0; average number of drugs bound per antibody molecule (n) measured by common procedure F: 3.4.

[0259] Example 30 Antibody-drug conjugate (30) [ka]

[0260] Step 1: Antibody-drug conjugate (30) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (6 mL) was placed in a polypropylene tube, to which was added 10 mM TCEP aqueous solution (0.215 mL; 5 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.094 mL). After confirming that the pH of this solution was within 7.0 ± 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: A DMSO solution (0.370 mL; 8.6 equivalents per antibody molecule) containing 10 mM of the compound from Step 8 of Example 26 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 aqueous solution (0.056 mL) was added, and the mixture was stirred at room temperature for an additional 20 minutes to quench the drug linker reaction. Purification: The above solution was purified using common procedure D (using ABS as the buffer) to obtain 24 mL of a solution containing the target compound. Characterization: Common operations E and F(ε D,280 = 5178 (measured value), ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 1.92 mg / mL, antibody yield: 46 mg (92%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 6.2; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.1.

[0261] Example 31 Antibody-drug conjugate (31) [ka]

[0262] Step 1: Antibody-drug conjugate (31) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1The medium was replaced with PBS 6.0 / EDTA using this solution (50.00 mL) to adjust the antibody concentration to 10 mg / mL. ) was placed in a polypropylene container, and while stirring, 1 M aqueous solution of dipotassium hydrogen phosphate (0.745 mL) was added at room temperature, followed by the addition of 10 mM aqueous solution of TCEP (1.868 mL; 5.4 equivalents per antibody molecule). After confirming that the pH of this solution was within 7.0 ± 0.1, stirring was stopped and 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 cooling the above solution to 15°C, a DMSO solution (2.958 mL; 8.6 equivalents per antibody molecule) containing 10 mM of the compound from Step 8 of Example 26 was slowly added dropwise under stirring. The mixture was stirred for the first 30 minutes at 15°C, 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 (0.444 mL) was added under stirring, and the mixture was further stirred at room temperature for 20 minutes to quench the drug linker reaction. Purification: While stirring, 20% aqueous acetic acid (approximately 0.25 mL) and ABS (50 mL) were slowly added to the above solution to adjust the pH of the solution 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). Ultrafiltration purification was performed using an ultrafiltration system consisting of a Biomax 50KDa cassette, a tube pump (Masterflex Pump model 77521-40, pump head model 7518-00, Cole-Parmer, USA), and tubing (Masterflex Tube L / S16, Cole-Parmer, USA). Specifically, ultrafiltration was performed while adding ABS (800 mL total) as a purification buffer to the reaction solution. This removed unbound drug linkers and other low-molecular-weight reagents, and the buffer was replaced with ABS, leading to further concentration. The resulting purified solution was microfiltered (0.22 μm (Millipore Co. Millex-GV filter, PVDF membrane) and 0.10 μm (Millipore Co. Millex-VV filter, PVDF membrane)) to obtain a solution containing the antibody-drug conjugate. Characterization: Common operations E and F(ε D,280 = 5178 (measured value), ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 11.28 mg / mL, antibody yield: 451 mg (90%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 6.6; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.7.

[0263] Example 32 (Another synthesis method for the compound of Example 26, Step 8) [ka]

[0264] Step 1: tert-butyl N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalaninate To a solution of tert-butyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalaninate (J. Pept. Res., 1999, Vol. 53, p. 393; 0.400 g, 0.717 mmol) in THF (12.0 mL) was added 1,8-diazabicyclo[5.4.0]-7-undecene (0.400 mL) under ice cooling and stirred at room temperature for 4 days. Then, N-succinimidyl 6-maleimidohexanoate (0.221 g, 0.717 mmol) was added and stirred for 3 hours. The reaction mixture was diluted with ethyl acetate and washed with 10% aqueous citric acid, saturated aqueous sodium bicarbonate, and saturated brine. The organic layer was then dried over anhydrous magnesium sulfate. 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 (0.295 g, 78%) as a pale yellow solid. 1 H-NMR(400MHz,CDCl3)δ:1.28-1.36(2H,m),1.41(9H,s),1.57-1.71(4H,m) ,2.23(2H,t,J=7.6Hz),3.09(2H,d,J=6.0Hz),3.51(2H,t,J=7.6Hz),3.85- 4.02(4H,m),4.69-4.78(1H,m),6.15(1H,t,J=4.6Hz),6.33(1H,d,J=7.3Hz ),6.60(1H,t,J=5.0Hz),6.68(2H,s),7.10-7.16(2H,m),7.22-7.31(3H,m). MS(ESI)m / z:529(M+H) +

[0265] Step 2: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanine To a solution of the compound (0.295 g, 0.558 mmol) obtained in Step 1 in dichloromethane (8 mL), trifluoroacetic acid (4 mL) was added and the mixture was stirred at room temperature for 18 hours. The solvent was evaporated under reduced pressure to give the title compound (0.240 g, 91%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:1.15-1.23(2H,m),1.40-1.53(4H,m),2.10(2H,t,J=7.6Hz),2.88( 1H,dd,J=13.7,8.9Hz),3.04(1H,dd,J=13.7,5.0Hz),3.35-3.43(2H,m),3.58-3.77(4H,m),4.41(1H,td,J=7. 8,5.0Hz),7.00(2H,s),7.16-7.31(5H,m),8.00(1H,t,J=5.7Hz),8.06(1H,t,J=5.7Hz),8.13(1H,d,J=7.8Hz).

[0266] Step 3: 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 The compound obtained in Step 2 above (0.572 g, 1.21 mmol) was dissolved in dichloromethane (12.0 mL), and N-hydroxysuccinimide (0.152 g, 1.32 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.253 g, 1.32 mmol) were added and stirred for 1 hour. The reaction solution was added to a solution of the mixture obtained in Step 5 of Example 26 (1.10 mmol) in N,N-dimethylformamide (22.0 mL), and the mixture was stirred at room temperature for 3 hours. A 10% aqueous citric acid solution was added to the reaction solution, and the mixture was extracted with chloroform. 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 [chloroform to chloroform:methanol = 8:2 (v / v)] to give the title compound (0.351 g, 31%) as a pale yellow solid. The instrumental data was similar to that of the compound in Example 26, Step 8.

[0267] Example 33 (Another synthesis method for the compound of Example 26, Step 8) [ka]

[0268] Step 1: Benzyl [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate To a solution of the compound obtained in Step 1 of Example 26 (7.37 g, 20.0 mmol) in THF (200 mL), benzyl glycolate (6.65 g, 40.0 mmol) and p-toluenesulfonic acid monohydrate (0.381 g, 2.00 mmol) were added at 0°C and stirred at room temperature for 2 hours and 30 minutes. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution, which was extracted with ethyl acetate. The resulting organic layer was dried over sodium sulfate and filtered. The solvent was removed 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 (6.75 g, 71%) as a colorless solid. The instrumental data were similar to those of the compound obtained in Step 2 of Example 26.

[0269] Step 2: N-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine-N-{[(2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide To a solution of the compound obtained in Step 1 (6.60 g, 13.9 mmol) in N,N-dimethylformamide (140 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (2.22 g, 14.6 mmol) was added at 0°C and stirred at room temperature for 15 minutes. To the reaction solution, a solution of N-[(benzyloxy)carbonyl]glycylglycyl-L-phenylalanine (6.33 g, 15.3 mmol), N-hydroxysuccinimide (1.92 g, 16.7 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.20 g, 16.7 mmol) in N,N-dimethylformamide (140 mL) was added after stirring at room temperature for 1 hour, and the mixture was stirred at room temperature for 4 hours. The reaction solution was added with 0.1 N hydrochloric acid, extracted with chloroform, and 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 [chloroform to chloroform:methanol = 8:2 (v / v)] to obtain the title compound (7.10 g, 79%) as a colorless solid. 1 H-NMR(DMSO-d6)δ:2.78(1H,dd,J=13.9,9.6Hz),3.05(1H,dd,J=13.9,4.5Hz) ,3.56-3.80(6H,m),4.15(2H,s),4.47-4.55(1H,m),4.63(2H,d,J=6.6Hz),5.0 3(2H,s),5.15(2H,s),7.16-7.38(15H,m),7.52(1H,t,J=5.9Hz),8.03(1H,t, J=5.5Hz), 8.17(1H,d,J=8.2Hz),8.36(1H,t,J=5.7Hz),8.61(1H,t,J=6.6Hz).

[0270] Step 3: Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide To a solution of the compound obtained in Step 2 (7.00 g, 10.8 mmol) in N,N-dimethylformamide (216 mL), a palladium-carbon catalyst (7.00 g) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 24 hours. Insoluble matter was removed by filtration through Celite, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in water, and the insoluble matter was removed by filtration through Celite. The solvent was evaporated under reduced pressure. This procedure was repeated twice to obtain the title compound (3.77 g, 82%) as a colorless solid. 1 H-NMR(DMSO-d6)δ:2.84(1H,dd,J=13.7,9.8Hz),3.08(1H,dd,J=13.7,4.7Hz),3.50-3.72(4H,m),3.77-3.86(2H,m),3.87(2H,s),4.52-4.43 (1H,m),4.61(2H,d,J=6.6Hz),7.12-7.30(5H,m),8.43(1H,t,J=5.9Hz),8.54(1H,d,J=7.8Hz),8.70(1H,t,J=6.3Hz),8.79(1H,t,J=5.5Hz).

[0271] Step 4: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide To a solution of the compound obtained in step 3 above (3.59 g, 8.48 mmol) in N,N-dimethylformamide (85.0 mL) was added N-succinimidyl 6-maleimidohexanoate (2. The reaction mixture was added with 0.1 N hydrochloric acid, extracted with chloroform and a mixed solvent of chloroform and methanol [chloroform:methanol = 4:1 (v / v)], and 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 [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v)] to obtain the title compound (3.70 g, 71%) as a colorless solid. 1H-NMR(DMSO-d6)δ:1.13-1.24(2H,m),1.42-1.53(4H,m),2.11(2H,t,J=7.4Hz),2. 80(1H,dd,J=13.7,9.8Hz),3.06(1H,dd,J=13.9,4.5Hz),3.37(2H,t,J=7.2Hz),3.5 6-3.78(6H,m),3.97(2H,s),4.46-4.53(1H,m),4.61(2H,d,J=6.3Hz),7.00(2H,s), 7.15-7.29(5H,m),8.03-8.20(3H,m),8.32(1H,t,J=5.9Hz),8.60(1H,t,J=6.7Hz).

[0272] Step 5: 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 exatecan methanesulfonate (1.14 g, 2.00 mmol) in N,N-dimethylformamide (40.0 mL), triethylamine (0.202 g, 2.00 mmol), the compound obtained in Step 4 above (1.48 g, 2.40 mmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (0.993 g, 3.00 mmol) containing 16.4% water were added at 0°C and stirred at room temperature for 1 hour. The solvent was removed 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 (1.69 g, 82%) as a pale yellow solid. The instrumental data were the same as those of the compound obtained in Step 8 of Example 26.

[0273] Example 34 Intermediate (34) [ka]

[0274] Step 1: 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-oxoethyl acetate Under ice cooling, a suspension of exatecan methanesulfonate (0.500 g, 0.941 mmol) in N,N-dimethylformamide (20.0 mL) was added with N,N-diisopropylethylamine (0.492 mL, 2.82 mmol) and acetoxyacetyl chloride (0. The resulting 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:water=7:3:1 (v / v / v) partition organic layer] to obtain the title compound (0.505 g, quantitative) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.81-1.92(2H,m),2.08( 3H,s),2.08-2.22(2H,m),2.41(3H,s),3.14-3.21(2H,m),4.51(2H,dd,J=1 9.4,14.7Hz),5.22(2H,dd,J=40.1,19.0Hz),5.43(2H,s),5.56-5.61(1H,m ),6.53(1H,s),7.31(1H,s),7.81(1H,d,J=11.0Hz),8.67(1H,d,J=8.6Hz). MS(ESI)m / z:536(M+H) +

[0275] Step 2: 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]-2-hydroxyacetamide To a suspension of the compound obtained in step 1 (0.504 g, 0.941 mmol) in methanol (50 mL), THF (20 mL) and 1 N aqueous sodium hydroxide (4.00 mL, 4.00 mmol) were added and stirred at room temperature for 1 hour. The reaction was terminated by the addition of 1 N hydrochloric acid (5.00 mL, 5.00 mmol), and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) organic layer] to obtain the title compound (0.412 g, 89%) as a pale yellow solid. When antibody-drug conjugates (45) and (46) were administered to mice, this compound was detected in the tumor. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.3Hz),1.78-1.95(2H,m),2.09-2.28(2H,m),2.39(3H,s),3.07-3.27(2H,m),3.96(2H,d,J=6.0Hz), 5.11-5.26(2H,m),5.42(2H,s),5.46-5.54(1H,m),5.55-5.63(1H,m),6 .52(1H,s),7.30(1H,s),7.78(1H,d,J=10.9Hz),8.41(1H,d,J=9.1Hz). MS(ESI)m / z:494(M+H) +

[0276] Example 35 (Another synthesis method for the compound of Example 34) [ka]

[0277] Step 1: 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]-2-hydroxyacetamide Glycolic acid (0.0201 g, 0.27 mmol) was dissolved in N,N-dimethylformamide (1.0 mL), and N-hydroxysuccinimide (0.0302 g, 0.27 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.0508 g, 0.27 mmol) were added and stirred for 1 hour. The reaction solution was added to a suspension of exatecan methanesulfonate (0.1 g, 0.176 mmol) in N,N-dimethylformamide (1.0 mL) and triethylamine (0.025 mL, 0.18 mmol) was added and stirred at room temperature for 24 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 10:1 (v / v)] to obtain the title compound (0.080 g, 92%) as a pale yellow solid. The instrumental data were similar to that of the compound obtained in Step 2 of Example 34.

[0278] Example 36 Antibody-drug conjugate (36) [ka]

[0279] Step 1: N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]glycylglycyl-L-phenylalanylglycyl-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]-β-alaninamide The compound obtained in Step 2 of Example 15 (60.0 mg, 0.0646 mmol) was reacted in the same manner as in Step 3 of Example 2, except that N-succinimidyl 4-maleimidobutyrate was used instead of N-succinimidyl 6-maleimidohexanoate, to obtain the title compound (24.0 mg, 38%) as a pale white solid. 1H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.2Hz),1.68(2H,quin,J=7.4Hz),1.78-1.92(2H,m),2.06-2.22(2H,m),2.1 0(2H,t,J=7.8Hz),2.31-2.43(2H,m),2.40(3H,s),2.78(1H,dd,J=13.7,9.4Hz),3.01(1H,dd,J=13.7,4.7Hz),3.17(4H ,d,J=5.1Hz),3.29-3.40(2H,m),3.52-3.80(6H,m),4.40-4.51(1H,m),5.19(1 H,d,J=18.4Hz),5.26(1H,d,J=18.8Hz),5.42(2H,s),5.52-5.61(1H,m),6.53( 1H,s),6.99(2H,s),7.12-7.28(5H,m),7.31(1H,s),7.74-7.84(2H,m),8.02(1 H,t,J=5.9Hz),8.08-8.16(2H,m),8.25(1H,t,J=5.9Hz),8.52(1H,d,J=8.2Hz). MS(ESI)m / z:990(M+H) +

[0280] Step 2: Antibody-drug conjugate (33) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6 using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 1.75 mg / mL, antibody yield: 10.5 mg (84%), average number of drugs bound per antibody molecule (n): 4.7.

[0281] Example 37 Antibody-drug conjugate (37) [ka]

[0282] Step 1: Antibody-drug conjugate (37) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7 using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 of Example 36. Antibody concentration: 1.89 mg / mL, antibody yield: 11.3 mg (90%), average number of drugs bound per antibody molecule (n): 8.5.

[0283] Example 38 Intermediate (38) [ka]

[0284] Step 1: tert-butyl (5-{[(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}-5-oxopentyl)carbamate Exatecan methanesulfonate (500 mg, 0.941 mmol) was reacted in the same manner as in Step 1 of Example 1, except that 5-(tert-butoxycarbonylamino)valeric acid was used instead of 4-(tert-butoxycarbonylamino)butanoic acid, to obtain the title compound (571 mg, 96%) as a yellow-brown solid. This was used in the next reaction without further purification. MS(ESI)m / z:635(M+H) +

[0285] Step 2: 5-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]pentanamide The compound obtained in Step 1 above (558 mg, 0.879 mmol) was reacted in the same manner as in Step 2 of Example 1 to give the trifluoroacetate salt of the title compound (363 mg, 64%) as a yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.4Hz),1.52-1.71(4H,m),1.87(2H,tt,J=14.4, 6.9Hz),2.07-2.18(2H,m),2.22(2H,t,J=7.0Hz),2.40(3H,s),2.76-2.88(2H,m),3.13-3. 22(2H,m),5.18(1H,d,J=18.8Hz),5.24(1H,d,J=18.8Hz),5.43(2H,s),5.53-5.61(1H,m), 6.55(1H,s),7.33(1H,s),7.65(3H,br.s.),7.81(1H,d,J=11.3Hz),8.49(1H,d,J=8.6Hz). MS(ESI)m / z:535(M+H) +

[0286] Example 39 Antibody-drug conjugate (39) [ka]

[0287] Step 1: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(5-{[(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}-5-oxopentyl)glycinamide The compound obtained in Step 2 of Example 38 (348 mg, 0.537 mmol) was reacted in the same manner as in Step 1 of Example 2 to obtain the title compound (429 mg, 84%) as a pale yellow solid, which was used in the next reaction without further purification.

[0288] Step 2: Glycylglycyl-L-phenylalanyl-N-(5-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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}-5-oxopentyl)glycinamide The compound obtained in Step 1 above (427 mg, 0.448 mmol) was reacted in the same manner as in Step 2 of Example 2 to give the trifluoroacetate salt of the title compound (430 mg, 99%) as a yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.38-1.49(2H,m),1.54-1.66(2H,m),1.86( 2H,tt,J=14.5,7.0Hz),2.08-2.16(2H,m),2.19(2H,t,J=7.2Hz),2.40(3H,s),2.76(1H,dd,J=1 3.9,10.0Hz),3.00-3.12(3H,m),3.14-3.21(2H,m),3.57(2H,d,J=4.7Hz),3.60-3.75(3H,m),3 .87(1H,dd,J=16.8,5.9Hz),4.55(1H,td,J=9.0,4.7Hz),5.16(1H,d,J=18.8Hz),5.23(1H,d,J= 18.4Hz),5.44(2H,s),5.53-5.60(1H,m),6.55(1H,s),7.14-7.29(5H,m),7.32(1H,s),7.74(1 H,t,J=5.5Hz),7.81(1H,d,J=10.9Hz),7.96(3H,br.s.),8.30-8.37(1H,m),8.44-8.53(2H,m). MS(ESI)m / z:853(M+H) +

[0289] Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(5-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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}-5-oxopentyl)glycinamide The compound obtained in Step 2 above (60.0 mg, 0.0621 mmol) was reacted in the same manner as in Step 3 of Example 2 to give the title compound (16.0 mg, 25%) as a solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.13-1.21(2H,m),1.36 -1.52(6H,m),1.53-1.65(2H,m),1.79-1.92(2H,m),2.05-2.15(4H,m),2.1 9(2H,s),2.40(3H,s),2.79(1H,dd,J=13.7,10.2Hz),2.98-3.10(3H,m),3 .12-3.21(2H,m),3.29-3.37(2H,m),3.53-3.79(6H,m),4.41-4.50(1H,m), 5.16(1H,d,J=18.8Hz),5.23(1H,d,J=18.8Hz),5.43(2H,s),5.52-5.60(1 H,m),6.53(1H,s),6.99(2H,s),7.12-7.28(5H,m),7.31(1H,s),7.63(1H,t ,J=5.7Hz),7.80(1H,d,J=10.6Hz),8.02(1H,t,J=5.9Hz),8.08(1H,t,J=5 .7Hz),8.12(1H,d,J=7.8Hz),8.24(1H,t,J=5.7Hz),8.45(1H,d,J=8.6Hz). MS(ESI)m / z:1046(M+H) +

[0290] Step 4: Antibody-drug conjugate (39) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-1 and common procedure B (1.37 mL / min as 280 nm extinction coefficient).-1 cm -1 The antibody was diluted to 10 mg / mL with PBS 6.0 / EDTA (using 1.0 mL of TCEP). 1.0 mL of this solution was placed in a 2 mL tube, and 10 mM TCEP aqueous solution (0.0155 mL; 2.3 equivalents per antibody molecule) and 1 M potassium hydrogen phosphate dihydrate aqueous solution (0.050 mL) were added. After confirming that the pH of this solution was within 7.4 ± 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 at 22°C for 10 minutes, a DMSO solution (0.0311 mL; 4.6 equivalents per antibody molecule) containing 10 mM of the compound obtained in step 3 above was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.00622 mL; 9.2 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (PBS 6.0 was used as the buffer solution) to obtain 6 mL of a solution containing the title antibody-drug conjugate. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 1.12 mg / mL, antibody yield: 6.72 mg (67%), average number of drugs bound per antibody molecule (n): 1.8.

[0291] Example 40 Antibody-drug conjugate (40) [ka]

[0292] Step 1: Antibody-drug conjugate (40) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedure C-1 and common procedure B (1.37 mL / min as 280 nm extinction coefficient). -1 cm -1The antibody was diluted to 10 mg / mL with PBS 6.0 / EDTA (using 1.0 mL of 1 mL of 1 mL of 10 mM TCEP solution (0.0311 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate solution (0.050 mL) were added. After confirming that the pH of the solution was within 7.4 ± 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 at 22°C for 10 minutes, a DMSO solution (0.0622 mL; 9.2 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 3 of Example 39 was added, and the mixture was incubated at 22°C for 40 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC aqueous solution (0.0124 mL; 18.4 equivalents per antibody molecule) was added, and the mixture was further incubated at 22°C for 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified using common procedure D-1 (PBS 6.0 was used as the buffer solution) to obtain 6 mL of a solution containing the title antibody-drug conjugate. Characterization: The following characterization values ​​were obtained using standard procedure E. Antibody concentration: 0.98 mg / mL, antibody yield: 5.88 mg (59%), average number of drugs bound per antibody molecule (n): 3.4.

[0293] Example 41 Antibody-drug conjugate (41) [ka]

[0294] Step 1: tert-butyl {2-[(2-hydroxyethyl)amino]-2-oxoethyl}carbamate N-(tert-Butoxycarbonyl)glycine (4.2 g, 24 mmol) was dissolved in dimethylformamide (40 mL), aminoethanol (2.9 g, 48 mmol), 1-hydroxybenzotriazole (3.7 g, 24 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.9 g, 36 mmol) were added, and the mixture was stirred at room temperature for 12 hours. The solvent was removed under reduced pressure, and the residue was azeotroped with toluene. The resulting residue was purified by silica gel column chromatography [ethyl acetate to ethyl acetate:methanol = 10:1 (v / v)] to give the title compound (3.8 g, 72%) as a colorless oil. 1 H-NMR(400MHz,CDCl3)δ:1.44(9H,s),1.69(1H,brs),3.43(2H,td,J=5.9,5.1H z),3.71(2H,t,J=5.1Hz),3.79(2H,d,J=5.9Hz),5.22(1H,brs),6.62(1H,brs).

[0295] Step 2: 2-{[N-(tert-butoxycarbonyl)glycyl]amino}ethyl 4-nitrophenyl carbonate To a solution of the compound obtained in Step 1 (1.0 g, 4.59 mmol) in THF (23 mL), diisopropylethylamine (0.80 mL, 4.59 mmol) and bis(4-nitrophenyl)carbonate (1.32 g, 6.88 mmol) were added and the mixture was stirred at room temperature for 12 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane to hexane:ethyl acetate = 1:3 (v / v)] to obtain the title compound (1.13 g, 64%) as a pale yellow solid. 1 H-NMR(400MHz,CDCl3)δ:1.44(1H,s),3.66(2H,td,J=5.1,5.9Hz),3.81(2H,d,J=5.9Hz),4.36(2H,t ,J=5.1Hz),5.07(1H,s),6.48-6.53(1H,m),7.38(2H,dt,J=9.9,2.7Hz),8.27(2H,dt,J=9.9,2.7Hz).

[0296] Step 3: 2-({[(tert-butoxycarbonyl)amino]acetyl}amino)ethyl [(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]carbamate Dimethylformamide (23 mL) was added to exatecan methanesulfonate (0.70 g, 1.2 mmol), the compound obtained in Step 2 (0.57 g, 1.5 mmol), and 1-hydroxybenztriazole (3.7 g, 24 mmol). Diisopropylethylamine (0.43 mL, 2.5 mmol) was added and the mixture was stirred at room temperature for 12 hours. The solvent was removed under reduced pressure, and toluene was added to the residue to form an azeotrope. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol = 10:1 (v / v)] to give the title compound (0.86 g, quantitative) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.35(9H,s),1.78-1.94(1H,m),2.07-2.1 7(1H,m),2.17-2.27(1H,m),2.37(3H,s),3.05-3.16(1H,m),3.19-3.26(1H,m),3.34-3.39( 2H,m),3.50-3.56(2H,m),4.00-4.07(1H,m),4.13-4.21(1H,m),5.15-5.34(3H,m),5.44(2H ,s),6.54(1H,s),6.90-6.96(1H,m),7.32(1H,s),7.78(1H,d,J=11.0Hz),7.93-8.07(2H,m).

[0297] Step 4: 2-(Glycylamino)ethyl [(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]carbamate The compound obtained in step 3 above (0.86 g, 2.1 mmol) was dissolved in dichloromethane (15 mL). Trifluoroacetic acid (15 mL) was added and the mixture was stirred for 1 hour. The solvent was removed under reduced pressure, and toluene was added to the residue to form an azeotrope. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) partitioned organic layer] to obtain the title compound (0.86 g, 99%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz),1.79-1.95(2H,m),2.06-2.18(1H,m),2.18-2.29(1H,m),2.38(3H,s),3.07-3.17(1H,m),3. 20-3.29(1H,m),3.36-3.50(2H,m),3.51-3.62(2H,m),3.99-4.08(1H,m ),4.22-4.31(1H,m),5.16-5.35(3H,m),5.42(1H,d,J=18.8Hz),5.46(1 H,d,J=18.8Hz),6.56(1H,s),7.34(1H,s),7.65(2H,brs),7.79(1H,d,J=10.6Hz),7.99-8.06(1H,m),8.51(1H,t,J=5.5Hz). MS(APCI)m / z:939(M+H) +

[0298] Step 5: 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]carbamoyl}oxy)ethyl]glycinamide N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (JP 2002-60351 A; 0.21 g, 0.41 mmol) was dissolved in N,N-dimethylformamide (3 mL), N-hydroxysuccinimide (0.052 g, 0.45 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.086 g, 0.45 mmol) were added, and the mixture was stirred for 1 hour. The reaction solution was added dropwise to an N,N-dimethylformamide solution (2 mL) containing the compound obtained in step 4 above (0.24 g, 0.35 mmol) and triethylamine (0.078 mL, 0.45 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=8:2 (v / v)] to obtain the title compound (0.24 g, 65%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.3Hz),1.79-1.90(2H,m),2.05-2.27(2H,m),2.36(3H,s),2.73-2.81(1H,m),2.98-3.12(2H,m),3.17- 3.26(1H,m),3.35-3.42(2H,m),3.55-3.79(6H,m),4.00-4.10(1H,m),4. 12-4.23(2H,m),4.23-4.29(2H,m),4.45-4.55(1H,m),5.13-5.33(3H,m), 5.40(1H,d,J=17.2Hz),5.44(1H,d,J=17.2Hz),6.53(1H,s),7.11-7.26( 5H,m),7.26-7.33(3H,m),7.38(2H,t,J=7.6Hz),7.57(1H,t,J=5.9Hz),7. 68(2H,d,J=7.4Hz),7.77(1H,d,J=11.0Hz),7.85(2H,d,J=9.0Hz),7.91- 7.97(1H,m),7.98-8.05(2H,m),8.14(1H,d,J=7.8Hz),8.31-8.26(1H,m). MS(APCI)m / z:1063(M+H) +

[0299] Step 6: 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]carbamoyl}oxy)ethyl]glycinamide The compound obtained in Step 5 above (0.24 g, 0.35 mmol) was reacted in the same manner as in Step 7 of Example 26 to give the title compound (0.12 g, 65%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.4Hz),1.78-1.94(2H,m),2.06-2.27(2 H,m),2.37(3H,s),2.72-2.81(1H,m),2.98-3.07(1H,m),3.12-3.17(2H,m),3.57-3 .81(6H,m),4.00-4.21(3H,m),4.45-4.54(1H,m),5.15-5.35(3H,m),5.41(1H,d,J= 17.2Hz),5.45(1H,d,J=17.2Hz),6.54(1H,s),7.11-7.26(6H,m),7.32(1H,s),7.78 (1H,d,J=11.0Hz),7.93-8.00(1H,m),8.03(1H,d,J=9.4Hz),8.06-8.13(1H,m),8.21-8.27(2H,m),8.30-8.36(1H,m). MS(APCI)m / z:841(M+H) +

[0300] Step 7: 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]carbamoyl}oxy)ethyl]glycinamide The compound obtained in Step 6 above (42.0 mg, 0.0499 mmol) was reacted in the same manner as in Step 3 of Example 2 to give the title compound (38.3 mg, 74%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz),1.12-1.23(2H,m),1.40-1.51(4H,m),1.80 -1.95(2H,m),2.05-2.27(4H,m),2.38(3H,s),3.43-2.40(8H,m),3.53-3.78(6H,m),4.00-4. 21(2H,m),4.44-4.55(1H,m),5.17-5.36(3H,m),5.43(2H,s),6.54(1H,s),6.99(2H,s),7.19 (5H,d,J=23.9Hz),7.33(1H,s),7.78(1H,d,J=10.6Hz),7.91-8.16(5H,m),8.24-8.31(1H,m). MS(ESI)m / z:1034(M+H) +

[0301] Step 8: Antibody-drug conjugate (41) The title antibody-drug conjugate was obtained in the same manner as in Step 2 of Example 6, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 7 above. Antibody concentration: 1.54 mg / mL, antibody yield: 9.2 mg (74%), average number of drugs bound per antibody molecule (n): 3.7.

[0302] Example 42 Antibody-drug conjugate (42) [ka]

[0303] Step 1: Antibody-drug conjugate (42) Using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 7 of Example 41, The title antibody-drug conjugate was obtained by the same method as in Example 7, Step 1. Antibody concentration: 1.47 mg / mL, antibody yield: 8.8 mg (71%), average number of drugs bound per antibody molecule (n): 7.0.

[0304] Example 43 Antibody-drug conjugate (43) [ka]

[0305] 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-[(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 The compound obtained in Step 6 of Example 26 (53.7 mg, 50.5 μmol) was dissolved in N,N-dimethylformamide (1.50 mL), and 1,8-diazabicyclo(5.4.0)-7-undecene (7.5 μL, 50.5 μmol) was added. The mixture was stirred at room temperature for 30 minutes. Pyridinium p-toluenesulfonate (14.0 mg, 5.56 μmol) was added to the reaction solution, followed by N-succinimidyl 3-(2-(2-(3-maleinimidopropanamido)ethoxy)ethoxy)propanoate (32.3 mg, 75.8 μmol). The mixture was stirred at room temperature for 2.25 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 (27.1 mg, 47%) as a pale yellow solid. 1H-NMR(DMSO-d6)δ:0.87(3H,t,J=7.0Hz),1.79-1.91(2H,m),2.18(2H,t,J=15.1Hz),2.29-2.33(4H,m),2. 39(3H,s),2.76(1H,dd,J=13.9,9.2Hz),3.02(1H,dd,J=13.7,3.9Hz),3.13-3.15(2H,m),3.44-3.46(6H,m) ,3.57-3.59(6H,m),3.69-3.75(6H,m),4.01(2H,s),4.46-4.48(1H,m),4.63(2H,d,J=6.3Hz),5.21(2H,s), 5.42(2H,s),5.60(1H,dd,J=13.5,5.7Hz),6.54(1H,s),7.00(2H,s),7.17-7.24(6H,m),7.31(1H,s),7.79( 1H,d,J=11.0Hz),8.00-8.02(2H,m),8.13(1H,d,J=7.8Hz),8.17(1H,t,J=6.3Hz),8.52(1H,d,J=9.0Hz),8.65(1H,t,J=6.5Hz). MS(ESI) m / z=1151(M+H) +

[0306] Step 2: Antibody-drug conjugate (43) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 1 above. Antibody concentration: 1.96 mg / mL, antibody yield: 17.6 mg (88%), average number of drugs bound per antibody molecule (n): 5.6.

[0307] Example 44 Antibody-drug conjugate (44) [ka]

[0308] Step 1: tert-Butyl N-[(benzyloxy)carbonyl]glycylglycyl-D-phenylalaninate N-[(benzyloxy)carbonyl]glycylglycine (3.00 g, 11.3 mmol) was dissolved in N,N-dimethylformamide (20.0 mL), and N-hydroxysuccinimide (1.43 g, 12.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.37 g, 12.4 mmol) were added, followed by stirring for 1 hour. The mixture was stirred. A solution of tert-butyl D-phenylalanine (2.74 g, 12.38 mmol) and triethylamine (1.73 mL, 12.4 mmol) in N,N-dimethylformamide (10 mL) was added dropwise to the reaction solution, and the mixture was stirred at room temperature for 2 hours. Dichloromethane was added to the reaction solution, and the mixture was washed with water, 1 N hydrochloric acid, and saturated aqueous sodium bicarbonate. The organic layer was then dried over anhydrous sodium sulfate. 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 (4.21 g, 80%) as a colorless solid. 1 H-NMR(CDCl3)δ:1.41(9H,s),3.03-3.14(2H,m),3.86-3.97(4H,m),4.70-4.77(1H,m),5.13(2H,s),5.43(1 H,brs),6.42(1H,d,J=10.0Hz),6.64-6.71(1H,m),7.11-7.15(2H,m),7.20-7.31(4H,m),7.31-7.38(4H,m). MS(APCI)m / z:470(M+H) +

[0309] Step 2: N-[(benzyloxy)carbonyl]glycylglycyl-D-phenylalanine The compound obtained in step 1 (4.21 g, 8.97 mmol) was dissolved in ethyl acetate (20 mL), and a 4 N hydrochloric acid solution in ethyl acetate (20 mL) was added. The mixture was left standing overnight at room temperature. The solvent was removed under reduced pressure, and then toluene was added and the solvent was removed under reduced pressure. 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 (1.66 g, 45%) as a colorless solid. 1 H-NMR(CDCl3)δ:2.92-3.01(1H,m),3.10-3.18(1H,m),3.65-3.81(3H,m),3.88- 3.98(1H,m),4.64-4.73(1H,m),5.06(2H,s),5.87(1H,brs),7.10-7.37(13H,m). MS(APCI)m / z:412(M+H) -

[0310] Step 3: N-[(benzyloxy)carbonyl]glycylglycyl-D-phenylalanyl-N-{[2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide To a solution of the compound obtained in Step 1 of Example 32 (1.25 g, 2.63 mmol) in dioxane (25.0 mL), piperidine (5.00 mL) and N,N-dimethylformamide (5.00 mL) were added and stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure, and the resulting residue was dissolved in N,N-dimethylformamide (20.0 mL). The compound obtained in Step 2 above (1.20 g, 2.90 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.03 g, 3.16 mmol) containing 16.4% water were added, and the mixture was stirred at room temperature for 2 hours. Chloroform was added to the reaction solution, which was washed with water, and the organic layer was dried over anhydrous sodium sulfate. 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 (270 mg, 16%) as a colorless solid. 1H-NMR(DMSO-d6)δ:2.78(1H,dd,J=13.6,10.0Hz),3.05(1H,dd,J=13.9,4.2Hz ),3.56-3.79(6H,m),4.15(2H,s),4.47-4.54(1H,m),4.63(2H,d,J=6.7Hz),5. 03(2H,s),5.15(2H,s),7.14-7.39(15H,m),7.50(1H,t,J=5.7Hz),8.02(1H,t, J=5.4Hz), 8.16(1H,d,J=7.9Hz),8.34(1H,t,J=6.0Hz),8.60(1H,t,J=7.0Hz). MS(APCI)m / z:648(M+H) +

[0311] Step 4: Glycylglycyl-D-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide The compound obtained in Step 3 above (200 mg, 0.31 mmol) was dissolved in N,N-dimethylformamide (5.0 mL), and 5% palladium-carbon catalyst (0.12 g) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 9 hours. The reaction mixture was filtered through Celite, and the residue was washed with a mixed solvent of water and N,N-dimethylformamide. The filtrate and washings were combined and evaporated under reduced pressure to give the title compound (0.15 g, quantitative) as a colorless solid. 1 H-NMR(DMSO-d6)δ:2.85(1H,dd,J=13.3,9.7Hz),3.08(1H,dd,J=13.9,5.4Hz),3.43-3.52(4H,m),3.62-3.89(7H,m),4.36-4.44( 1H,m),4.58-4.67(2H,m),7.12-7.29(5H,m),8.44(1H,t,J=5.7Hz),8.67(1H,d,J=7.3Hz),8.78(1H,t,J=5.4Hz),8.91(1H,brs). MS(APCI)m / z:424(M+H) +

[0312] Step 5: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-D-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide The compound obtained in step 4 above (0.15 g, 0.35 mmol) was dissolved in N,N-dimethylformamide (10 mL), N-succinimidyl 6-maleimidohexanoate (0.11 g, 0.35 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Chloroform was added to the reaction mixture, and the mixture was washed with water. The organic layer was dried over anhydrous sodium sulfate. The solvent was removed 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) partitioned organic layer] to obtain the title compound (41 mg, 26%) as a colorless solid. 1 H-NMR(DMSO-d6)δ:1.13-1.24(2H,m),1.42-1.53(4H,m),2.12(2H,t,J=7.3Hz),2.82 (1H,dd,J=13.9,10.0Hz),3.09(1H,dd,J=13.9,4.8Hz),3.17(2H,d,J=4.2Hz),3.47-3 .89(8H,m),4.08-4.14(1H,m),4.41-4.49(1H,m),4.58-4.69(2H,m),7.00(2H,s),7. 14-7.27(5H,m),8.31(1H,t,J=6.0Hz),8.39(1H,brs),8.55(2H,brs),8.93(1H,brs). MS(APCI)m / z:615(MH) -

[0313] Step 6: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-D-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 exatecan methanesulfonate (22 mg, 0.388 mmol) in N,N-dimethylformamide (10 mL), triethylamine (5.42 μL, 0.388 mmol), the compound obtained in Step 5 above (29 mg, 0.466 mmol), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19 mg, 0.686 mmol) containing 16.4% water were added at 0°C and stirred at room temperature for 1 hour. The reaction mixture 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 (26 mg, 65%) as a pale yellow solid. 1 H-NMR(DMSO-d6)δ:0.87(3H,t,J=7.3Hz),1.12 -1.22(2H,m),1.40-1.51(4H,m),1.79-1.92(2H,m),2.09(2H,t,J=7.6Hz),2.13-2.23(2H,m),2.39(3H,s),2.78(1H,dd,J=13. 6,9.4Hz),2.98-3.05(1H,m),3.13-3.23(2H,m),3.54-3.78(8H,m),4.02(2H,s),4.41-4.50(1H,m),4.61-4.66(2H,m),5.21(2H ,s),5.42(2H,s),5.56-5.64(1H,m),6.53(1H,s),6.99(2H,s),7.14-7.27(5H,m),7.31(1H,s),7.79(1H,d,J=10.9Hz),8.01(1H ,t,J=5.4Hz),8.07(1H,t,J=5.7Hz),8.14(1H,d,J=7.9Hz),8.31(1H,t,J=5.7Hz),8.53(1H,d,J=9.1Hz),8.63(1H,t,J=6.3Hz). MS(APCI)m / z:1034(M+H) +

[0314] Step 7: Antibody-drug conjugate (44) The title antibody-drug conjugate was obtained in the same manner as in Step 1 of Example 7, using the trastuzumab prepared in Reference Example 1 and the compound obtained in Step 6 above. Antibody concentration: 1.87 mg / mL, antibody yield: 16.8 mg (84%), average number of drugs bound per antibody molecule (n): 6.1.

[0315] Example 45 Intermediate (45) [ka]

[0316] Step 1: tert-butyl (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-oxoethyl)carbamate To a solution of N-(tert-butoxycarbonyl)-glycine (0.395 g, 2.26 mmol) in dichloromethane (3.00 mL), N-hydroxysuccinimide (0.260 g, 2.26 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.433 mg, 2.26 mmol) were added and stirred at room temperature for 1 hour. This solution was added to a solution of exatecan methanesulfonate (1.00 g, 1.88 mmol), triethylamine (0.315 mL, 2.26 mmol), and N,N-dimethylformamide (3.00 mL) and stirred at room temperature for 16.5 hours. The reaction solution was diluted with chloroform, washed with 10% citric acid solution, and the organic layer was dried over anhydrous sodium sulfate. 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 (1.16 g, 99%) as a yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.2Hz),1.30(9H,s),1.81-1.89(2H,m),2.09-2.21(2H,m),2.38(3H,s),3.15-3.17(2H,m),3.55-3. 56(2H,m),5.15(1H,d,J=18.8Hz),5.23(1H,d,J=19.2Hz),5.41(2H,s), 5.55-5.56(1H,m),6.53(1H,s),6.95(1H,t,J=5.5Hz),7.28(1H,s),7.7 7(1H,d,J=11.0Hz),8.39(1H,d,J=8.6Hz). MS(APCI)m / z:593(M+H) +

[0317] Step 2: 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 compound obtained in Step 1 above (0.513 g, 1.01 mmol) was reacted in the same manner as in Step 2 of Example 1 to give the title compound (0.463 g, 93%) as a yellow solid. 1 H-NMR(400MHz,CD3OD)δ:0.96(3H,t,J=7.0Hz),1.89-1.91(2H,m),2.14-2.16(1H,m),2.30(3H,s),2.40-2.42(1H,m),3.15-3.21(2H,m),3. 79-3.86(2H,m),4.63-4.67(1H,m),5.00-5.05(1H,m),5.23(1H,d,J=1 6.0Hz),5.48(1H,d,J=16.0Hz),5.62-5.64(1H,m),7.40-7.45(2H,m). MS(APCI)m / z:493(M+H) +

[0318] Example 46 Antibody-drug conjugate (46) [ka]

[0319] Step 1: Antibody-drug conjugate (46) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1 The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (50 mL) was placed in a 125 mL polycarbonate Erlenmeyer flask, and while stirring magnetically at room temperature, 1 M aqueous potassium phosphate (0.750 mL) was added, followed by 10 mM aqueous TCEP (1.857 mL; 5.4 equivalents per antibody molecule). After confirming that the pH of this solution was within 7.0 ± 0.1, stirring was stopped and 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 cooling the above solution to 15°C, a DMSO solution (2.958 mL; 8.6 equivalents per antibody molecule) containing 10 mM of the compound from Step 8 of Example 26 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 (0.444 mL; 12.9 equivalents per antibody molecule) was added under stirring, and the mixture was further stirred at room temperature for 20 minutes to quench the reactivity of the unreacted drug linker. Purification: While stirring, 20% aqueous acetic acid (approximately 0.25 mL) and ABS (50 mL) were slowly added to the above solution to adjust the pH of the solution to 5.5±0.1. The solution was microfiltered (Millipore Co. Millex-HV filter, 0.45 μm, PVDF membrane) to remove cloudy matter. The solution was then filtered using an ultrafiltration membrane (Merck Co., Pellicon XL). Ultrafiltration purification was performed using an ultrafiltration apparatus consisting of a Biomax 50KDa cassette, a tube pump (Masterflex Pump model 77521-40, pump head model 7518-00, Cole-Parmer, USA), and tubing (Masterflex Tube L / S16, Cole-Parmer, USA). Specifically, ultrafiltration was performed while adding ABS as a purification buffer (total 800 mL) to the reaction solution to remove unbound drug linkers and other low-molecular-weight reagents, while also replacing the buffer with ABS, and further concentrating the solution. The resulting purified solution was microfiltered (0.22 μm (Millipore Co. Millex-GV filter, PVDF membrane) and 0.10 μm (Millipore Co. Millex-VV filter, PVDF membrane)) to obtain 42.5 mL of a solution containing the title antibody-drug conjugate. Characterization: Common operations E and F(ε D,280 = 5178 (measured value), ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 10.4 mg / mL, antibody yield: 442 mg (88.5%), average number of drugs bound per antibody molecule (n) measured by common procedure E: 6.0; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.5.

[0320] Example 47 Antibody-drug conjugate (47) [ka]

[0321] Step 1: Antibody-drug conjugate (47) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1The medium was replaced with PBS 6.0 / EDTA using a PBS buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (15 mL) was placed in a polypropylene tube, and 10 mM TCEP aqueous solution (0.567 mL; 5.5 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (0.225 mL) were added. After confirming that the pH of this solution was within 7.0 ± 0.1, it 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: DMSO (0.146 mL) and a DMSO solution containing 10 mM of the compound from Step 8 of Example 26 (0.928 mL; 9.0 equivalents per antibody molecule) were added to the above solution at room temperature, and the mixture was incubated at 15°C for 30 minutes to allow the drug linker to bind to the antibody. Next, a 100 mM NAC aqueous solution (0.133 mL; 9.0 equivalents per antibody molecule) was added. The reaction mixture was stirred at room temperature for an additional 20 minutes to terminate the reactivity of the unreacted drug linker. Purification: The above solution was purified using common procedure D (using ABS as the buffer) to obtain 49 mL of a solution containing the target compound. Characterization: Common operation E(ε D,280 =5178, ε D,370 =20217) to obtain the following characteristic values. Antibody concentration: 2.91 mg / mL, antibody yield: 143 mg (95%), average number of drugs bound per antibody molecule (n): 6.2

[0322] Example 48 Antibody-drug conjugate (48) [ka]

[0323] Step 1: Antibody-drug conjugate (48) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1The medium was replaced with PBS 6.0 / EDTA using PBS (prepared with PBS-based PBS containing 10 mg / mL of antibody). This solution (280 mL) was placed in a 1000 mL polycarbonate Erlenmeyer flask. Under magnetic stirring at room temperature, 1 M dipotassium hydrogen phosphate (4.200 mL) was added, followed by 10 mM TCEP (10.594 mL; 5.5 equivalents per antibody molecule). After confirming that the pH of the solution was within 7.0 ± 0.1, stirring was stopped and the solution 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 (17.335 mL; 9.0 equivalents per antibody molecule) containing 10 mM of the compound from Step 8 of Example 26 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 (2.485 mL; 12.9 equivalents per antibody molecule) was added under stirring, and the mixture was further stirred at room temperature for 20 minutes to quench the reactivity of any unreacted drug linker. Purification: While stirring, 20% aqueous acetic acid (approximately 1.4 mL) and ABS (280 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 (0.45 μm, PVDF membrane) to remove the cloudy matter, and approximately 600 mL of filtrate was obtained. The ultrafiltration was carried out using an ultrafiltration apparatus consisting of a membrane (Merck, Pellicon XL Cassette, Biomax 50KDa), a tube pump (Cole-Parmer, USA, Masterflex Pump model 77521-40, pump head model 7518-00), and tubing (Cole-Parmer, USA, Masterflex Tube L / S16). The solution was purified by ultrafiltration while adding 800 mL of HCl (aqueous suspension) dropwise to remove unbound drug linkers and other low-molecular-weight reagents, and the buffer solution was replaced with ABS. The solution was then concentrated. The purified solution was microfiltered (twice, 0.22 μm and 0.10 μm, PVDF membrane) to obtain 70 mL of a solution containing the title antibody-drug conjugate. Characterization: Common operation E(ε D,280 =5178 、 ε D,370 =20217) to obtain the following characteristic values. Antibody concentration: 35.96 mg / mL, antibody yield: 2517 mg (90%), average number of drugs bound per antibody molecule (n): 6.2

[0324] Example 49 Antibody-drug conjugate (49) [ka]

[0325] Step 1: Antibody-drug conjugate (49) Antibody reduction: Trastuzumab prepared in Reference Example 1 was subjected to common procedures C-1 and B (280 nm extinction coefficient of 1.48 mL / mg). -1 cm -1 The medium was replaced with PBS 6.0 / EDTA using a PBS-based antibody buffer (used in Example 1), and the antibody concentration was adjusted to 10 mg / mL. This solution (280 mL) was placed in a 1000 mL polycarbonate Erlenmeyer flask, and while stirring magnetically at room temperature, 1 M aqueous potassium phosphate (4.200 mL) was added, followed by 10 mM aqueous TCEP (10.594 mL; 5.5 equivalents per antibody molecule). After confirming that the pH of the solution was within 7.0 ± 0.1, stirring was stopped and the solution 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 (17.335 mL; 9.0 equivalents per antibody molecule) containing 10 mM of the compound from Step 8 of Example 26 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 (2.485 mL; 12.9 equivalents per antibody molecule) was added under stirring, and the mixture was further stirred at room temperature for 20 minutes to quench the reactivity of any unreacted drug linker. Purification: While stirring, 20% aqueous acetic acid (approximately 1.4 mL) and ABS (280 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 (0.45 μm, PVDF membrane) to remove the cloudy matter, and approximately 600 mL of filtrate was obtained. The ultrafiltration was carried out using an ultrafiltration apparatus consisting of a membrane (Merck, Pellicon XL Cassette, Ultracell 30KDa), a tube pump (Cole-Parmer, USA, Masterflex Pump model 77521-40, pump head model 7518-00), and a tube (Cole-Parmer, USA, Masterflex Tube L / S16). The resulting purified solution was subjected to microfiltration (twice, 0.22 μm and 0.10 μm, PVDF membrane) to obtain 130 mL of a solution containing the antibody-drug conjugate. Characterization: Common operation E(ε D,280 =5178 、 ε D,370 =20217) to obtain the following characteristic values. Antibody concentration: 21.00 mg / mL, antibody yield: 2730 mg (97.5%), average number of drugs bound per antibody molecule (n): 6.3

[0326] Example 50 Antibody-drug conjugate (50)

[0327] Step 1: Antibody-drug conjugate (50) The antibody-drug conjugates (47), (48), and (49) prepared in Examples 47, 48, and 49 were mixed (243 mL), and ABS (39.75 mL) was added to obtain 283 mL of a solution containing the title antibody-drug conjugate. Characterization: Common operations E and F(ε D,280 =5178 、 ε D,370=20217) to obtain the following characteristic values. Antibody concentration: 20.0 mg / mL, antibody yield: 5655 mg, measured using common procedure E Average number of drugs bound per antibody molecule (n): 6.3; average number of drugs bound per antibody molecule (n) measured by common procedure F: 7.8.

[0328] Evaluation Example 1 Anti-cellular effect of antibody-drug conjugates (1) HER2 antigen-positive human breast cancer cell line KPL-4 (Kawasaki Medical School, Professor Junichi Kurebayashi, British Journal of Cancer, (1999) 79(5 / 6). 707-717) and antigen-negative cell line MCF7 (European Collection of Cell Cultures; ECACC) were cultured in RPMI 1640 medium (GIBCO; hereafter referred to as medium) containing 10% fetal bovine serum (MOREGATE). KPL-4 and MCF7 were cultured at 2.5 × 10 4 Each solution was prepared to give a concentration of 100 μL / mL, and 100 μL of each solution was added to a 96-well cell culture microplate and cultured overnight. The next day, 10 μL of trastuzumab or antibody-drug conjugate diluted with medium to 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, or 0.064 nM was added to each microplate. 10 μL of medium was added to wells to which no antibody was added. Culture was performed at 37°C and 5% CO2 for 5 to 7 days. After incubation, the microplate was removed from the incubator and allowed to stand at room temperature for 30 minutes. An equal volume of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added and mixed. After allowing to stand at room temperature for 10 minutes, luminescence was measured using a plate reader (PerkinElmer). IC 50 The value was calculated using the following formula: I C 50 (nM)=antilog((50-d)×(LOG 10 (b)-LOG 10 (a))÷(dc)+LOG 10 (b)) a: concentration of sample a b: concentration of sample b c: Viable cell rate of sample a d: Viable cell rate of sample b The cell viability at each concentration was calculated using the following formula. Cell viability (%)=a÷b×100 a: Average luminescence intensity of sample wells (n=2) b: Average luminescence intensity in wells without antibody (n=10)

[0329] Antibody-drug conjugates (2), (3), (5), (7), (10), (12), (13), (16), (18), (40), and (42) showed IC 50 It showed an anti-cellular effect of <0.1 (nM). Antibody-drug conjugates (4), (6), (9), (15), (17), (21), (22), (25), (36), (37), (39), (41), and (43) were 0.1 <IC 50 It showed an anti-cellular effect of <1 nM. Antibody-drug conjugates (20), (24), and (27) were synthesized by <IC 50 Antibody-drug conjugates (19) and (26) showed no anti-cellular effect (>100 nM). On the other hand, (5), (13), and (43) were 1 in MCF7 cells. <IC 50 showed anti-cellular effects of <100 nM, whereas antibody-drug conjugates (2), (3), (4), (6), (7), (9), (10), (12), (15), (16), (17), (18), (25), (26), (27), (39), (40), (41), (42), (44) did not show anti-cellular effects (>100 nM). In addition, trastuzumab did not show any anti-cellular effect on either KPL-4 or MCF7 cells (>100 nM).

[0330] Evaluation Example 2 Antitumor Test (1) Mice: Five- to six-week-old female nude mice (Charles River Japan) were acclimated under SPF conditions for 4 to 7 days before use. Mice were fed sterilized solid food (FR-2, Funabashi Farms Co., Ltd.) and provided with sterilized tap water (prepared with 5-15 ppm sodium hypochlorite solution). Measurement and calculation formula: In all studies, the major and minor diameters of tumors were measured twice a week using an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and tumor volume (mm 3 ) was calculated using the following formula: Tumor volume (mm 3 ) = 1 / 2 x major axis (mm) x [minor axis (mm)] 2

[0331] All antibody-drug conjugates and antibodies were diluted with physiological saline (Otsuka Pharmaceutical Factory, Inc.) and administered into the tail vein at a volume of 10 mL / kg. KPL-4 cells were suspended in saline and diluted to 1.5 x 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 15. The antibody-drug conjugate (27) or the anti-HER2 antibody trastuzumab (Reference Example 1) as a control group was administered intravenously at a dose of 10 mg / kg on Days 15 and 22. An untreated group was set up as a control group.

[0332] The results are shown in Figure 3. Administration of trastuzumab inhibited tumor growth, but administration of antibody-drug conjugate (27) had a more pronounced tumor growth inhibitory effect. In the figure, the horizontal axis represents the number of days after cell transplantation, and the vertical axis represents tumor volume. Furthermore, mice administered trastuzumab or antibody-drug conjugate (27) did not show any particularly noticeable findings such as weight loss, suggesting that antibody-drug conjugate (27) is highly safe. In the following evaluation examples of antitumor tests, unless otherwise specified, the tests were conducted using the same methods as in these evaluation examples.

[0333] Evaluation Example 3 Antitumor Test (2) Human gastric cancer cell line NCI-N87 cells purchased from ATCC (American Type Culture Collection) were suspended in saline and cultured at 1 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 7. Antibody-drug conjugates (8), (28), or trastuzumab emtansine (Reference Example 2) were administered intravenously to the tail vein at a dose of 10 mg / kg on Day 7. An untreated group served as a control.

[0334] The results are shown in Figure 4. Antibody-drug conjugates (8) and (28) inhibited the activity of trastuzumab. A strong antitumor effect was observed, with tumor regression comparable to that observed with mutansine. Furthermore, administration of antibody-drug conjugates (8), (28), or trastuzumab emtansine did not result in weight loss in mice.

[0335] Evaluation Example 4 Antitumor Test (3) DSMZ(Deutsche Sammlung von Mikroorganism) JIMT-1 human breast cancer cell line purchased from the company En und Zellkulturen GmbH was suspended in saline and incubated at 3 × 10 6 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 12. Antibody-drug conjugates (8), (29), and (30), or trastuzumab or trastuzumab emtansine were administered intravenously at a dose of 10 mg / kg on Days 12 and 19. A saline-treated group served as a control.

[0336] The results are shown in Figure 5. Administration of trastuzumab or trastuzumab emtansine did not inhibit tumor growth in JIMT-1 tumors. In contrast, administration of antibody-drug conjugates (8), (29), and (30) significantly inhibited tumor growth. Furthermore, administration of antibody-drug conjugates (8), (29), and (30), trastuzumab, or trastuzumab emtansine did not result in weight loss in mice.

[0337] Evaluation Example 5 Anticellular Effect of Antibody-Drug Conjugates (2) The human non-small cell lung cancer cell line Calu-3 (ATCC) was cultured in Eagle's Minimum Essential Medium (GIBCO; hereinafter referred to as MEM medium) containing 10% fetal bovine serum (MOREGATE). The human gastric cancer lines NCI-N87 (ATCC) and MKN-45 (Human Science Research Resources Bank) were cultured in RPMI1640 Medium (GIBCO; hereinafter referred to as RPMI medium) containing 10% fetal bovine serum. Human breast cancer lines MDA-MB-453 (ATCC) and MDA-MB-468 (ATCC) were cultured in Leibovitz's L-15 Medium (GIBCO; hereafter referred to as Leibovitz's medium) containing 10% fetal bovine serum. Of these five cell lines, Calu-3, NCI-N87, and MDA-MB-453 are HER2-positive cells, while MKN-45 and MDA-MB-468 are HER2-negative cells. Calu-3, NCI-N87, and MKN-45 were cultured in MEM or RPMI medium at 4 × 10 4 The cells were diluted to 4 × 10 cells / mL, and 25 μL of each was added to a 96-well cell culture microplate containing 65 μL of medium. The cells were cultured overnight at 37°C under 5% CO2. MDA-MB-453 and MDA-MB-468 were also cultured in Leibovitz's medium at a concentration of 4 × 10 cells / mL. 4 The solution was adjusted to a concentration of 1000 cells / mL, and 25 μL of each was added to a 96-well cell culture microplate containing 65 μL of medium, followed by overnight culture at 37°C without adjusting the CO2 concentration. The next day, 10 μL of each sample diluted with RPMI medium or Leibovitz's medium to 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, or 0.064 nM, and 10 μL of each of RPMI medium or Leibovitz's medium were added to the microplates, and the plates were cultured at 37°C in 5% CO2 or at 37°C with no CO2 concentration setting for 6 days. Antibody-drug conjugate (46) was added to Calu-3, NCI-N87, and MDA-MB-468 cells, while antibody-drug conjugate (50) was added to the other cells. After incubation, the microplate was removed from the incubator and left at room temperature for 30 minutes. An equal volume of CellTiter-Glo Luminescent Cell Viability Assay (Promega) was added, and the cells were completely lysed by mixing with a plate mixer. After leaving the plate at room temperature for 10 minutes, the luminescence was measured using a plate reader. The viable cell rate was calculated using the following formula. Viable cell rate (%)=a÷b×100 a: Average luminescence intensity of sample-added wells b: Average luminescence intensity of wells containing medium I C 50 The value was calculated using the following formula: I C 50 (nM)=antilog((50-d)×(LOG 10 (b)-LOG 10 (a))÷(dc)+LOG 10 (b)) a: sample concentration a b: sample concentration b c: Viable cell rate at sample concentration a d: Viable cell rate at sample concentration b a and b are two points that sandwich a cell viability of 50%, and a>b.

[0338] The antibody-drug conjugate (46) showed IC 50 It showed an anti-cellular effect of <1 nM, whereas it showed no anti-cellular effect (>100 nM) against HER2-negative MDA-MB-468 cells.

[0339] The antibody-drug conjugate (50) showed IC 50 It showed an anti-cellular effect of <1 nM, whereas it showed no anti-cellular effect (>100 nM) against HER2-negative MKN-45 cells.

[0340] Evaluation Example 6 Antitumor Test (4) HER2 low-expressing human pancreatic cancer cell line Capan-1 cells (ATCC) were suspended in saline and cultured at 4 × 10 7 Capan-1 cells were subcutaneously transplanted into the right flank of female nude mice to create Capan-1 solid tumors. These solid tumors were then maintained in female nude mice for multiple passages and used in this study. Solid tumor fragments were subcutaneously transplanted into the right flank of female nude mice (Day 0), and randomized to groups on Day 20. The antibody-drug conjugate (31), trastuzumab, or trastuzumab emtansine was administered intravenously to the tail vein at a dose of 10 mg / kg on Day 20. A saline-administered group served as a control group. The results are shown in Figure 6. Administration of trastuzumab and trastuzumab emtansine did not inhibit tumor growth in Capan-1 tumors. In contrast, administration of antibody-drug conjugate (31) significantly inhibited tumor growth, confirming the efficacy of antibody-drug conjugate (31) even in tumors with low HER2 expression. Antibody-drug conjugate (31) did not inhibit tumor growth in the HER2-nonexpressing gastric cancer line GCIY tumor. Regarding HER2 expression in tumors, based on the results of immunohistochemical staining described in the HER2 Testing Guide, Third Edition (created by the Trastuzumab Pathology Committee of the Japanese Society of Pathology), a score of 3+ was classified as high expression, 2+ as moderate expression, and 1+ as low expression. Furthermore, even if the score was 0 by this measurement method, if it was positive by other measurement methods, such as measurement by flow cytometer, it was classified as low expression.

[0341] Evaluation Example 7 Antitumor Test (5) Human gastric cancer cell line NCI-N87 cells purchased from ATCC were suspended in saline at 1 × 10 7 The cells were subcutaneously transplanted into the right flank of female nude mice (Day 0), and the mice were randomly assigned to groups on Day 6. The antibody-drug conjugate (50) was administered intravenously to each group at doses of 0.3, 1, 3, or 10 mg / kg on Day 6. A control group was administered acetate buffer. The results are shown in Figure 7. Antibody-drug conjugate (50) exhibited a dose-dependent antitumor effect. Furthermore, administration of antibody-drug conjugate (50) did not result in weight loss in mice.

[0342] Evaluation Example 8 Antitumor Test (6) This test was carried out in the following manner. Mice: Female nude mice (Charles River) aged 6-12 weeks were used in the experiment. Measurement and calculation: The major and minor diameters of the tumor were measured twice a week using an electronic digital caliper, and the tumor volume (mm 3 ) was calculated using the following formula: Tumor volume (mm 3 ) = 0.52 x major axis (mm) x [minor axis (mm)] 2 The antibody-drug conjugates, trastuzumab, and trastuzumab emtansine were diluted with acetate buffer and administered into the tail vein at a volume of 10 mL / kg.

[0343] The tumor used in this study was a breast cancer patient tumor (ST225; South Texas Accelerated Research Therapeutics (START)) that had been maintained for multiple passages by transplantation into female nude mice. This tumor was HER2-neutral (2+ as determined by immunohistochemical staining). Solid tumor fragments were subcutaneously implanted into the flanks of female nude mice until the tumor volume reached 100-300 mm. 3 Mice were randomly assigned to groups at the time of reaching 10 mg / kg. The day of group assignment was designated Day 0, and antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was administered intravenously to the tail vein at a dose of 10 mg / kg. An acetate buffer-administered group served as a control group. The results are shown in Figure 8. In HER2-expressing breast cancer ST225 tumors, administration of trastuzumab did not inhibit tumor growth. In contrast, administration of trastuzumab emtansine or antibody-drug conjugate (50) significantly inhibited tumor growth.

[0344] Evaluation Example 9 Antitumor Test (7) The tumors used in this study were excised from breast cancer patients and maintained for multiple passages by transplantation into female nude mice (ST910; START). These tumors exhibit low HER2 expression (1+ as determined by immunohistochemical staining). Solid tumor fragments were subcutaneously implanted into the flanks of female nude mice until the tumor volume reached 100-300 mm. 3 Mice were randomly assigned to groups at the time of reaching 10 mg / kg. The day of group assignment was designated Day 0, and antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was administered intravenously to the tail vein at a dose of 10 mg / kg. An acetate buffer-administered group served as a control group. The results are shown in Figure 9. Administration of trastuzumab and trastuzumab emtansine did not inhibit tumor growth in breast cancer ST910 tumors, which have low HER2 expression. In contrast, administration of antibody-drug conjugate (50) significantly inhibited tumor growth, confirming the effectiveness of antibody-drug conjugate (50) against breast cancer tumors with low HER2 expression. Evaluation Example 9 was performed using the same method as Evaluation Example 8.

[0345] Evaluation Example 10 Antitumor Test (8) This test was carried out by the following method. Furthermore, Evaluation Examples 11 to 13 were also carried out by this method. Mice: 5-8 week old female nude mice (Harlan Laboratories) were used in the experiment. Measurement and calculation: The major and minor diameters of the tumor were measured twice a week using an electronic digital caliper, and the tumor volume (mm 3 ) was calculated using the following formula: Tumor volume (mm 3 ) = 0.52 x major axis (mm) x [minor axis (mm)] 2 The antibody-drug conjugates, trastuzumab, and trastuzumab emtansine were diluted with acetate buffer and administered into the tail vein at a volume of 10 mL / kg.

[0346] The tumor used in this study was a tumor (CTG-0401; Champion Cancer Center) excised from a colon cancer patient and maintained for multiple passages by transplanting it into female nude mice. This tumor has low-to-moderate HER2 expression (immunohistochemical staining determined as 1+ or 2+). Solid tumor fragments were subcutaneously implanted into the left flank of female nude mice until the tumor volume reached 100-300 mm. 3 Mice were randomly assigned ...

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[Claim 1] The invention described herein.

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