BR122020020973B1 - ANTI-HER2-DRUG ANTIBODY CONJUGATE, USE THEREOF, ANTI-TUMOR AND/OR ANTI-CANCER DRUGS, AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

As an antitumor drug having an excellent antitumor effect and safety and also having an excellent therapeutic effect, an antibody-drug conjugate is provided, which is characterized by the fact that an antitumor compound represented by formula (I) is linked to an anti-tumor antibody. -HER2 via a linker having a structure represented by the formula: -L1-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O)- (wherein the anti-HER2 antibody is linked at the L1 terminus, and the antitumor compound is attached to a carbonyl group on a -(CH2)n2-C(=O)- moiety in which a nitrogen atom on an amino group located at the -1 position in the antitumor compound serves as the a connecting part).

Description

TECHNICAL FIELD

[001] The present invention relates to an antibody-drug conjugate having an antitumor drug conjugated to an anti-HER2 antibody via a linker backbone moiety, the conjugate being useful as an antitumor drug.

BACKGROUND TECHNIQUE

[002] An antibody-drug conjugate (ADC) having a drug with cytotoxicity conjugated to an antibody whose antigen is similarly expressed on the surface of cancer cells and which binds to an antigen capable of cellular internalization, and therefore can release the drug selectively to cancer cells, is thus expected to cause the drug to accumulate within the cancer cells and kill the cancer cells (see, Non-Patent Literatures 1 to 3). As an ADC, Mylotarg (trademark; Gentuzumab ozogamycin) in which calicamycin is conjugated to an anti-CD33 antibody is approved as a therapeutic agent for acute myeloid leukemia. In addition, Adcetris (trademark; Brentuximab vedotin) in which auristatin E is conjugated to an anti-CD30 antibody, was recently approved as a therapeutic agent for Hodgkin lymphoma and anaplastic large cell lymphoma (see, Non-Patent Literature 4) . Drugs contained in ADCs that have been approved so far target DNA or tubulin.

[003] With respect to an antitumor agent, camptothecin derivatives, low molecular weight compounds that inhibit topoisomerase I from exhibiting an antitumor effect, are known. Among these, an antitumor compound represented by the formula below

[004] (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]quinolin-10,13(9H, 15H)-dione) is a water-soluble derivative of camptothecin (Patent Literatures 1 and 2 ). Unlike irinotecan currently used in clinical applications, this compound does not require activation by an enzyme to exhibit its antitumor effect. Furthermore, its inhibitory activity on topoisomerase I was observed to be higher than SN-38 which is the main pharmaceutically active substance of irinotecan and topotecan used in clinical applications, and the highest in vitro cytocidal activity was confirmed against various cells. cancerous. In particular, it was confirmed to have the effect against cancer cells having resistance to SN-38 or the like due to P-glycoprotein expression. Furthermore, in a mouse model subcutaneously transplanted from a human tumor, it was confirmed to have a potent antitumor effect, and thus has undergone clinical studies, but has not yet been placed on the market (see, Non-Patent Literatures 5 to 10) . It remains unclear whether or not the Exatecan effectively acts as an ADC.

[005] DE-310 is a complex in which exatecan is conjugated to a biodegradable carboxymethyldextran polyalcohol polymer via a GGFG peptide spacer (Patent Literature 3). By converting exatecan into a polymer prodrug form, a high blood retention property can be maintained and likewise a high targeting property to tumor areas is passively increased using increased vessel permeability. newly formed blood vessels within tumors and retention property in tumor tissues. With DE-310, by enzyme cleavage of the peptide spacer, exatecan and exatecan with glycine attached to an amino group are continuously released as the main active substance, and as a result, the pharmacokinetics are improved. DE-310 was found to have higher effectiveness than exatecan administered alone despite the fact that the total dosage of exatecan contained in D310 is lower than in the case of administration of exatecan alone according to various tumor assessment models in non-clinical studies. A clinical study was conducted for DE-310, and actual cases were similarly confirmed, including a report suggesting that the main active substance accumulates in tumors larger than normal tissues. However, there is also a report indicating that the accumulation of DE-310 and the main active substance in tumors is not very different from the accumulation in normal tissues in humans, and thus no passive targeting is observed in humans (see, Literatures from Non-Patent 11 to 14). As a result, DE-310 was not similarly marketed, and it remains unclear whether or not Exatecan effectively acts as a drug targeting such targeting.

[006] As a compound relative to DE-310, a complex in which a moiety of structure represented by -NH-(CH2)4-C(=O)- is inserted between the -GGFG- spacer and exatecan to form - GGFG-NH-(CH2)4-C(=O)- used as a spacer structure is likewise known (Patent Literature 4). However, the antitumor effect of said complex is not known in all.

[007] HER2 is one of the products of a typical growth factor receptor-like oncogene identified as human epidermal cell growth factor 2 receptor-related oncogene, and is a transmembrane receptor protein having a molecular weight of 185 kDa and having a tyrosine kinase domain (Non-Patent Literature 15). The DNA sequence and amino acid sequence of HER2 are described in a public database, and may refer to, for example, under No. Accession M11730 (GenBank), NP_004439.2 (NCBI), or the like.

[008] HER2 (neu, ErbB-2) is one of the members of the EGFR (epidermal growth factor receptor) family and is activated by autophosphorylation at intracellular tyrosine residues by its homodimer formation or heterodimer formation with another receptor of EGFR, HER1 (EGFR, ErbB-1), HER3 (ErbB-3), or HER4 (ErbB-4) (Non-Patent Literatures 16 to 18), thereby playing an important role in cell growth, differentiation, and survival in normal cells and cancer cells (Non-Patent Literatures 19 and 20). HER2 is overexpressed in several types of cancer such as breast cancer, gastric cancer, and ovarian cancer (Non-patent Literatures 21 to 26) and has been reported to be a negative prognostic factor for breast cancer (Non-patent Literatures 27 and 28). ).

[009] Trastuzumab is a humanized antibody of a mouse anti-HER2 antibody 4D5 (Non-Patent Literature 29 and Patent Literature 5), named as recombinant humanized anti-HER2 monoclonal antibody (huMAb4D5-8, rhuMAb HER2, Herceptin(R) )) (Patent Literature 6). Trastuzumab specifically binds to the extracellular domain IV of HER2 and induces antibody-dependent cellular cytotoxicity (ADCC) or shows an anti-cancer effect by inhibiting signal transduction from HER2 (Non-Patent Literatures 30 and 31). Trastuzumab is highly effective for HER2 overexpressing tumors (Non-Patent Literature 32) and as such, it was launched in 1999 in the USA and in 2001 in Japan as a therapeutic agent for patients with HER2 overexpressing metastatic breast cancer.

[0010] Although the therapeutic effect of trastuzumab on breast cancer has been adequately proven (Non-Patent Literature 33), reportedly about 15% of patients with HER2 overexpressing breast cancer who received a wide range of conventional anticancer therapies are responders to trastuzumab. About 85% of patients in this population have no or merely poor response to trastuzumab treatment.

[0011] Thus, the need for a therapeutic agent targeting diseases related to HER2 expression has been recognized by patients affected by tumors overexpressing HER2 without either a poor response to trastuzumab or HER2-related disorders. T-DM1 (trastuzumab emtansine, Kadcyla(R); Non-Patent Literature 34) having an antitumor drug conjugated to trastuzumab via a linker structure, and pertuzumab (Perjeta(R); Non-Patent Literature 35 and Patent Literature 7) designed to target the extracellular domain II of HER2 and inhibit heterodimer formation was developed. However, its responsibility, activity intensity, and accepted indications are still insufficient, and there are unmet needs to target HER2.

CITATION LIST PATENT LITERATURES

[0012] [Patent Literature 1] Japanese Patent Open to Public Inspection No. 5-59061

[0013] [Patent Literature 2] Japanese Patent Open to Public Inspection No. 8-337584

[0014] [Patent Literature 3] International Publication No. WO 1997/46260

[0015] [Patent Literature 4] International Publication No. WO 2000/25825

[0016] [Patent Literature 5] U.S. Patent At the. 5677171

[0017] [Patent Literature 6] U.S. Patent At the. 5821337

[0018] [Patent Literature 7] International Publication No. WO 01/00244

NON-PATENTS LITERATURES

[0019] [Non-Patent Literature 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13.

[0020] [Non-Patent Literature 2] Alley, S.C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537.

[0021] [Non-Patent Literature 3] Damle N.K. Expert Opinion. Biol. The R. (2004) 4, 1445-1452.

[0022] [Non-Patent Literature 4] Senter P.D., et al., Nature Biotechnology (2012) 30, 631-637.

[0023] [Non-Patent Literature 5] Kumazawa, E., Tohgo, A., Exp. opinion Invest. Drugs (1998) 7, 625-632.

[0024] [Non-Patent Literature 6] Mitsui, I., et al., Jpn J. Cancer Res. (1995) 86, 776-782.

[0025] [Non-Patent Literature 7] Takiguchi, S., et al., Jpn J. Cancer Res. (1997) 88, 760-769.

[0026] [Non-Patent Literature 8] Joto, N. et al. Int J Cancer (1997) 72, 680-686.

[0027] [Non-Patent Literature 9] Kumazawa, E. et al., Cancer Chemother.Pharmacol. (1998) 42, 210-220.

[0028] [Non-Patent Literature 10] De Jager, R., et al., Ann N Y Acad Sci (2000) 922, 260-273.

[0029] [Non-Patent Literature 11] Inoue, K. et al., Polymer Drugs in the Clinical Stage, Edited by Maeda et al. (2003) 145-153.

[0030] [Non-Patent Literature 12] Kumazawa, E. et al., Cancer Sci (2004) 95, 168-175.

[0031] [Non-Patent Literature 13] Soepenberg, O. et al., Clinical Cancer Research, (2005) 11, 703-711.

[0032] [Non-Patent Literature 14] Wente M.N. et al., Investigational New Drugs (2005) 23, 339-347.

[0033] [Non-Patent Literature 15] Coussens L, et al., Science. 1985; 230(4730):1132-1139.

[0034] [Non-Patent Literature 16] Graus-Porta G, et al., EMBO J. 1997;16;1647-1655.

[0035] [Non-Patent Literature 17] Karnagaran D, et al., EMBO J. 1996;15:254-264.

[0036] [Non-Patent Literature 18] Sliwkowski MX, et al., J. Biol. Chem. 1994; 269:14661-14665.

[0037] [Non-Patent Literature 19]Di Fore PP, et al., Science. 1987; 237:178-182.

[0038] [Non-Patent Literature 20] Hudziak RM, et al., Proc Natl Acad Sci U S A. 1987;84:7159-7163.

[0039] [Non-Patent Literature 21] Hardwick R, et al., Eur. J Surg Oncol. 1997 (23): 30-35.

[0040] [Non-Patent Literature 22] Korkaya H, et al., Oncogene. 2008; 27(47): 6120-6130.

[0041] [Non-Patent Literature 23] Yano T, et al., Oncol Rep. 2006; 15(1):65-71.

[0042] [Non-Patent Literature 24] Slamon DJ, et al., Science. 1987;235:177-182.

[0043] [Non-Patent Literature 25] Gravalos C, et al., Ann Oncol 19: 1523-1529, 2008.

[0044] [Non-Patent Literature 26] Fukushige S, et al., Mol Cell Biol 6: 955-958, 1986.

[0045] [Non-Patent Literature 27] Slamon DJ, et al. Science. 1989; 244:707-712.

[0046] [Non-Patent Literature 28] Kaptain S et al., Diagn Mol Pathol 10:139-152, 2001.

[0047] [Non-Patent Literature 29] Fendly. et al., Cancer Research1990(50):1550-1558.

[0048] [Non-Patent Literature 30] Sliwkowski MX, et al., Semin Oncol. 1999; 26(4, Suppl 12):60-70.

[0049] [Non-Patent Literature 31] Hudis CA, et al., N Engl J Med. 357: 39-51, 2007.

[0050] [Non-Patent Literature 32] Vogel CL, et al., J Clin Oncol. 2002; 20(3):719-726.

[0051] [Non-Patent Literature 33] Baselga et al., J. Clin. oncol. 14:737-744 (1996).

[0052] [Non-Patent Literature 34] Howard A. et al., J Clin Oncol 2011; 29:398-405.

[0053] [Non-Patent Literature 35] Adams CW, et al., Cancer Immunol Immunother. 2006; 6:717-727.

SUMMARY OF THE INVENTION TECHNICAL PROBLEM

[0054] With respect to the treatment of tumors by antibodies, an insufficient antitumor effect may be observed even when the antibody recognizes an antigen to bind to tumor cells, and there are cases where a more effective antitumor antibody is needed. Furthermore, many low molecular weight antitumor compounds have safety issues such as side effects and toxicity even if the compounds have an excellent antitumor effect. It remained an objective to achieve a superior therapeutic effect while also enhancing safety. Therefore, an object of the present invention is to provide an antitumor drug having an excellent therapeutic effect which is excellent in terms of antitumor effect and safety.

SOLUTION TO THE PROBLEM

[0055] The inventors have considered that an anti-HER2 antibody is an antibody that is capable of targeting tumor cells, i.e., having a property of recognizing tumor cells, a property of binding to tumor cells, a property of internalizing within from the tumor cells, a cytotoxic activity against tumor cells, a cytocidal activity against tumor cells, or the like; thus, when the antitumor compound exatecan is converted to an antibody-drug conjugate, via a linker framework moiety, by conjugation to this antibody, the antitumor compound can be more reliably delivered to tumor cells to specifically display the antitumor effect of the compound on tumor cells, and thus the antitumor effect can be safely exhibited and likewise an enhanced cytocidal effect of the anti-HER2 antibody can be expected, and the dose of the antitumor compound can be reduced compared to the case of administering the compound alone, and thereby influences of the antitumor compound on normal cells can be alleviated so that higher safety can be obtained.

[0056] In this regard, the inventors created a linker with a specific structure and were successful in obtaining an antibody-drug conjugate in which the anti-HER2 antibody and exatecan are conjugated to each other via the linker, and confirmed a excellent antitumor effect exhibited by the conjugate to thereby complete the present invention.

[0057] Specifically, the present invention relates to the following.

[0058][1] an antibody-drug conjugate wherein an antitumor compound represented by the following formula:

[0059] is conjugated to an anti-HER2 antibody via a linker having a structure represented by the following formula: -L1-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O) -

[0060] via a thioether bond that is formed in a disulfide bonding moiety present in the hinge part of the anti-HER2 antibody.

[0061] Here, the anti-HER2 antibody is connected to the L1 terminal,

[0062] the antitumor compound is connected to the carbonyl group of the -(CH2)n2-C(=O)- moiety with the nitrogen atom of the amino group at position 1 as the connecting position,

[0063] in which

[0064] n1 represents an integer from 0 to 6,

[0065] n2 represents an integer from 0 to 5,

[0066] L1 represents -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-,

[0067] where n3 represents an integer from 2 to 8,

[0068] L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond,

[0069] where n4 represents an integer from 1 to 6,

[0070] LP represents a peptide residue consisting of 2 to 7 amino acids,

[0071] La represents -O- or a single bond, and

[0072] -(Succinimid-3-yl-N)- has a structure represented by the following formula:

[0073] which is attached to the anti-HER2 antibody at position 3 thereof and is attached to the methylene group in the linker structure containing this structure at the nitrogen atom at position 1.

[0074] The present invention also relates to each of the following.

[0075] [2] The antibody-drug conjugate according to [1], wherein the LP peptide residue is a peptide residue comprising an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid.

[0076] [3] The antibody-drug conjugate according to [1] or [2], wherein LP is a peptide residue selected from the following group: -GGF-, -DGGF-, -(D- )D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-; wherein "(D-)D" represents D-aspartic acid.

[0077] [4] The antibody-drug conjugate according to [1] or [2], wherein LP is a peptide residue consisting of 4 amino acids.

[0078] [5] The antibody-drug conjugate according to any one of [1] to [4], wherein LP is the tetrapeptide residue - GGFG-.

[0079] [6] The antibody-drug conjugate according to any one of [1] to [5], wherein n3 is an integer from 2 to 5, and L2 is a single bond.

[0080] [7] The antibody-drug conjugate according to any one of [1] to [5], wherein n3 is an integer from 2 to 5, L2 is -NH-(CH2CH2-O)n4- CH2CH2-C(=O)-, and n4 is 2 or 4.

[0081] [8] The antibody-drug conjugate according to any one of [1] to [7], wherein -NH-(CH2)n1-La-(CH2)n2-C(=O)- is a partial structure having a chain length of 4 to 7 atoms.

[0082] [9] The antibody-drug conjugate according to any one of [1] to [7], wherein -NH-(CH2)n1-La-(CH2)n2-C(=O)- is a partial structure having a chain length of 5 or 6 atoms.

[0083] [10] The antibody-drug conjugate according to any one of [1] to [9], wherein -NH-(CH2)n1-La-(CH2)n2-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 -NH-CH2CH2-O-C(=O)-.

[0084] [11] The antibody-drug conjugate according to any one of [1] to [9], wherein -NH-(CH2)n1-La-(CH2)n2-C(=O)- is -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)-.

[0085] [12] The antibody-drug conjugate according to any one of [1] to [9], wherein the portion of the drug-linker structure having the drug connected to -L1-L2-LP-NH- (CH2)n1-La-(CH2)n2-C(=O)- is a 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 )-CH2 CH2-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),

[0086] where -(Succinimid-3-yl-N)- has a structure represented by the following formula:

[0087] which is connected to the anti-HER2 antibody at position 3 thereof and is connected to the methylene group in the linker structure containing this structure at the nitrogen atom at position 1, -(NH-DX) represents a group represented by the following formula :

[0088] wherein the nitrogen atom of the amino group at position 1 is the connecting position, and -GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly -.

[0089] [13] The antibody-drug conjugate according to any one of [1] to [9], wherein the portion of the drug-linker structure having the drug connected to -L1-L2-LP-NH- (CH2)n1-La-(CH2)n2-C(=O)- is a 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).

[0090] Herein, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- is as defined above.

[0091] [14] An antibody-drug conjugate wherein an antitumor compound represented by the following formula:

[0092] is conjugated to an anti-HER2 antibody via a linker having a structure represented by the following formula: -L1-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O) -

[0093] via a thioether bond that is formed in a disulfide bonding moiety present in the hinge part of the anti-HER2 antibody,

[0094] in which

[0095] the anti-HER2 antibody is connected to the L1 terminal, the antitumor compound is connected to the carbonyl group of the - (CH2)n2-C(=O)- moiety,

[0096] in which

[0097] n1 represents an integer from 0 to 6,

[0098] n2 represents an integer from 0 to 5,

[0099] L1 represents -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-,

[00100] where n3 represents an integer from 2 to 8,

[00101] L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond,

[00102] where n4 represents an integer from 1 to 6,

[00103] LP represents the tetrapeptide residue -GGFG-,

[00104] La represents -O- or a single bond, and

[00105] -(Succinimid-3-yl-N)- has a structure represented by the following formula:

[00106] which is attached to the anti-HER2 antibody at position 3 thereof and is attached to the methylene group in the linker structure containing this structure at the nitrogen atom at position 1.

[00107] [15] The antibody-drug conjugate according to [14], wherein

[00108] n1 is 3, n2 is 0, n3 is 2, L2 is -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-, n4 is 2, and La is a single bond,

[00109] n1 is 1, n2 is 1, n3 is 5, L2 is a single bond, and La is -O-, or

[00110] n1 is 2, n2 is 1, n3 is 5, L2 is a single bond, and La is -O-.

[00111] [16] The antibody-drug conjugate according to [14] or [15], wherein n3 is 2 or 5, and L2 is a single bond.

[00112] [17] The antibody-drug conjugate according to [14] or [15], wherein n3 is 2 or 5, L2 is -NH-(CH2CH2-O)n4-CH2CH2-C(=O) -, and n4 is 2 or 4.

[00113] [18] The antibody-drug conjugate according to any one of [14] to [17], wherein -NH-(CH2)n1-La-(CH2)n2-C(=O)- is -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)-.

[00114] [19] The antibody-drug conjugate according to any one of [14] to [18], wherein the portion of the drug-linker structure having the drug connected to -L1-L2-LP-NH- (CH2)n1-La-(CH2)n2-C(=O)- is a drug-linker structure selected from the group consisting of the following: -(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), -(Succ inimid-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),

[00115] wherein, -(Succinimid-3-yl-N)- has a structure represented by the following formula:

[00116] which is attached to the anti-HER2 antibody at position 3 thereof and is attached to the methylene group in the linker structure containing this structure at the nitrogen atom at position 1, -(NH-DX) represents a group represented by the following formula :

[00117] wherein the nitrogen atom of the amino group at position 1 is the connecting moiety, and -GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly -.

[00118] [20] The antibody-drug conjugate according to any one of [14] to [18], wherein the portion of the drug-linker structure having the drug connected to -L1-L2-LP-NH- (CH2)n1-La-(CH2)n2-C(=O)- is a 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), and -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O )-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[00119] Herein, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- are as defined above.

[00120] [21] The antibody-drug conjugate according to any one of [1] to [20], wherein the average number of units of a selected drug-linker structure conjugated per antibody molecule is in the range of 1 to 10.

[00121] [22] The antibody-drug conjugate according to any one of [1] to [20], wherein the average number of units of a selected drug-linker structure conjugated per antibody molecule is in the range of 2 to 8.

[00122] [23] The antibody-drug conjugate according to any one of [1] to [20], wherein the average number of units of a selected drug-linker structure conjugated per antibody molecule is in the range of 3 to 8.

[00123] [24] A drug containing the antibody-drug conjugate according to any one of [1] to [23], a salt thereof or a hydrate thereof.

[00124] [25] An antitumor drug and/or anticancer drug containing the antibody-drug conjugate according to any one of [1] to [23], a salt thereof or a hydrate thereof.

[00125] [26] The antitumor drug and/or anticancer drug according to [25], which is for use against lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[00126] [27] A pharmaceutical composition containing the antibody-drug conjugate according to any one of [1] to [23], a salt thereof or a hydrate thereof as an active component, and a pharmaceutically formulation component acceptable.

[00127] [28] The pharmaceutical composition according to [27] which is for use against lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer , gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, cancer kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[00128] [29] A method for treating tumor and/or cancer comprising administering the antibody-drug conjugate according to any one of [1] to [23], a salt thereof or a hydrate thereof.

[00129] [30] A method for producing an antibody-drug conjugate comprising reacting a compound represented by the following formula: (maleimid-N-yl)-(CH2)n3-C(=O)-L2-LP-NH- (CH2)n1-La-(CH2)n2-C(=O)-(NH-DX)

[00130] with an anti-HER2 antibody or a reactive derivative thereof and conjugating a drug-linker moiety to the antibody by a method of forming a thioether bond at a disulfide binding site present on the hinge portion of the antibody.

[00131] In the formula, n3 represents an integer from 2 to 8,

[00132] L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond,

[00133] where n4 represents an integer from 1 to 6,

[00134] LP represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid,

[00135] n1 represents an integer from 0 to 6,

[00136] n2 represents an integer from 0 to 5,

[00137] La represents -O- or a single bond,

[00138] (maleimid-N-yl)- is a group represented by the following formula:

[00139] where the nitrogen atom is the connecting moiety, and

[00140] -(NH-DX) is a group represented by the following formula:

[00141] where the nitrogen atom of the amino group at position 1 is the connecting position.

[00142] [31] The production method according to [30], wherein the method for conjugating a drug-linker moiety to an anti-HER2 antibody is a method of reducing the antibody to convert the antibody to a reactive derivative .

[00143] [32] The production method according to [30] or [31], wherein the average number of units of a selected conjugated drug-linker structure per antibody molecule is in the range of 1 to 10.

[00144] [33] The production method according to [30] or [31], wherein the average number of units of a selected conjugated drug-linker structure per antibody molecule is in the range of 2 to 8.

[00145] [34] The production method according to [30] or [31], wherein the average number of units of a selected conjugated drug-linker structure per antibody molecule is in the range of 3 to 8.

[00146] [35] An antibody-drug conjugate obtained by the production method according to any of [30] to [34].

[00147] [36] An antibody-drug conjugate obtained by forming a thioether bond at a sulfide-binding site on the part of the antibody joint where the anti-HER2 antibody is treated in a reducing condition and thereafter reacted with a compound selected from the group shown below: (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)-CH 2CH2CH2CH2-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-CH2C H2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C( =O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2- C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N -yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- (NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2- C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)- GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2- O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O )-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)- CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX) ), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGF G-NH-CH2CH2-O-CH2-C(=O)-(NH-DX).

[00148] In the above, (maleimid-N-yl)- is a group represented by the following formula:

[00149] where the nitrogen atom is the connection position, and

[00150] -(NH-DX) is a group represented by the following formula:

[00151] wherein the nitrogen atom of the amino group at position 1 is the connecting position, and

[00152] -GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[00153] [37] An antibody-drug conjugate obtained by forming a thioether bond at a sulfide-binding site present in the part of the antibody joint where the anti-HER2 antibody is treated in a reducing condition and then further reacted with a compound selected from the group shown below: (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).

[00154] Herein, (maleimid-N-yl)-, -(NH-DX), and -GGFG- is as defined above.

[00155] [38] The antibody-drug conjugate according to [36] or [37], wherein the average number of units of a selected drug-linker structure conjugated per antibody molecule is in the range of 1 to 10 .

[00156] [39] The antibody-drug conjugate according to [36] or [37], wherein the average number of units of a selected drug-linker structure conjugated per antibody molecule is in the range of 2 to 8 .

[00157] [40] The antibody-drug conjugate according to [36] or [37], wherein the average number of units of a selected drug-linker structure conjugated per antibody molecule is in the range of 3 to 8 .

ADVANTAGEOUS EFFECTS OF THE INVENTION

[00158] With an anti-HER2 antibody-drug conjugate having the antitumor compound exatecan conjugated via a linker with a specific structure, an excellent antitumor effect and safety can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

[00159] Figure 1 shows an amino acid sequence of a heavy chain of a humanized anti-HER2 monoclonal antibody (SEQ ID NO: 1).

[00160] Figure 2 shows an amino acid sequence of a humanized anti-HER2 monoclonal antibody light chain (SEQ ID NO: 2).

[00161] Figure 3 is a diagram showing the antitumor effect of an antibody-drug conjugate (27) or trastuzumab in a nude mouse with subcutaneously transplanted human breast cancer lineage KPL-4 cells. In the drawing, the abscissa depicts days after tumor inoculation, and the ordinate describes the tumor volume.

[00162] Figure 4 is a diagram showing the antitumor effect of an antibody-drug conjugate (8), (28) or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human gastric cancer lineage NCI-N87 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00163] Figure 5 is a diagram showing the antitumor effect of an antibody-drug conjugate (8), (29), (30), trastuzumab, or trastuzumab emtansine in a nude mouse with breast cancer lineage JIMT-1 cells human subcutaneously transplanted. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00164] Figure 6 is a diagram showing the antitumor effect of an antibody-drug conjugate (31), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human pancreatic cancer lineage Capan-1 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00165] Figure 7 is a diagram showing the antitumor effect of an antibody-drug conjugate (50) in a nude mouse with subcutaneously transplanted NCI-N87 cells of human gastric cancer lineage. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00166] Figure 8 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human breast cancer ST225 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00167] Figure 9 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human breast cancer ST910 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00168] Figure 10 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human colorectal cancer lineage CTG-0401 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00169] Figure 11 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human non-small cell lung cancer CTG-0860 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00170] Figure 12 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human bile duct cancer lineage CTG-0927 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00171] Figure 13 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human esophageal cancer lineage CTG-0137 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

[00172] Figure 14 is a diagram showing the antitumor effect of an antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine in a nude mouse with subcutaneously transplanted human ovarian cancer lineage SK-OV-3 cells. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor.

DESCRIPTION OF MODALITIES

[00173] Next, preferred modes for carrying out the present invention are described with reference to the drawings. The embodiments described below are given only to illustrate an example of a typical embodiment of the present invention and are not intended to limit the scope of the present invention.

[00174] The anti-HER2 antibody-drug conjugate of the present invention is an antitumor drug in which an anti-HER2 antibody is conjugated to an antitumor compound via a linker framework moiety and is explained below in detail.

ANTIBODY

[00175] The anti-HER2 antibody used in the anti-HER2 antibody-drug conjugate of the present invention may be derived from any species, and preferred examples of the species may include humans, rats, mice, and rabbits. In the case where the antibody is derived from different human species, it is preferably chimerized or humanized using a well known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody and is preferably a monoclonal antibody.

[00176] Anti-HER2 antibody is the antibody that is capable of targeting tumor cells, i.e. it has a property of recognizing a tumor cell, a property of binding to a tumor cell, a property of internalizing into a cell of tumor, cytocidal activity against tumor cells, or the like, and may be conjugated to a drug having antitumor activity via a linker to form an antibody-drug conjugate.

[00177] Antibody binding activity against tumor cells can be confirmed using flow cytometry. Internalization of the antibody into tumor cells can be confirmed using (1) a visualization assay of a cell-incorporated antibody under a fluorescence microscope using a secondary (fluorescently labeled) antibody binding to the therapeutic antibody (Cell Death and Differentiation (Cell Death and Differentiation). 2008) 15, 751-761), (2) an assay measuring the intensity of fluorescence incorporated into cells using a secondary (fluorescently labeled) antibody binding to the therapeutic antibody ( Molecular Biology of the Cell, Vol. 15, 5268- 5282, December 2004), or (3) a Mab-ZAP assay using an immunotoxin binding to the therapeutic antibody where the toxin is released upon incorporation into cells to inhibit cell growth (Bio Techniques 28:162-165, January 2000). As the immunotoxin, a complex recombinant protein of a catalytic domain of diphtheria toxin and G protein can be used.

[00178] The antitumor activity of the antibody can be confirmed in vitro by determining the inhibitory activity against cell growth. For example, a cancer cell line overexpressing a target protein for the antibody is cultured, and the antibody is added at varying concentrations to the culture system to determine inhibitory activity against focus formation, colony formation, and spheroid growth. Anti-tumor activity can be confirmed in vivo, for example, by administering the antibody to a nude mouse with a transplanted tumor cell line highly expressing the target protein, and determining the change in the cancer cell.

[00179] Since the conjugated compound in the antibody-drug conjugate shows an antitumor effect, it is preferred, but not essential, that the antibody itself should have an antitumor effect. In order to specifically and selectively show the cytotoxic activity of the antitumor compound against tumor cells, it is important and likewise preferred that the antibody must have the property of internalization in order to migrate into tumor cells.

[00180] The anti-HER2 antibody can be obtained by a procedure known in the art. For example, the antibody of the present invention can be obtained using a method commonly performed in the art, which involves immunizing animals with an antigenic polypeptide and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to humans, and animals can be immunized with an antigen derived from a non-human animal such as a mouse, rat, and the like. In this case, the cross-reactivity of antibodies binding the heterologous antigen obtained with human antigens can be tested to evaluate an antibody applicable to a human disease.

[00181] Alternatively, antibody-producing cells that produce antibodies against the antigen are fused with myeloma cells according to a method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R.ed., Monoclonal Antibodies, p.365-367, Plenum Press, N.Y. (1980)) to establish hybridomas, from which monoclonal antibodies can in turn be obtained.

[00182] The antigen can be obtained by genetically engineering the host cells to produce a gene encoding the antigenic protein. Specifically, vectors that allow expression of the antigen gene are prepared and transferred to host cells so that the gene is expressed. The antigen so expressed can be purified. The antibody may likewise be obtained by a method of immunizing animals with the above-described genetically engineered antigen-expressing cells or a cell line expressing the antigen.

[00183] The anti-HER2 antibodies that can be used in the present invention are not particularly limited and are preferably, for example, those having properties as described below.

[00184] (1) An anti-HER2 antibody having the following properties: (a) it specifically binds to HER2, and (b) has an internalizing activity in HER2 expressing cells by binding to HER2.

[00185] (2) The antibody according to (1) above, wherein the antibody binds to the extracellular domain of HER2.

[00186] (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody.

[00187] (4) The antibody according to any one of (1) to (3) above, wherein the antibody has antibody-dependent cellular cytotoxicity (ADCC). and/or a complement-dependent cytotoxicity (CDC) activity.

[00188] (5) The antibody according to any one of (1) to (4) above, wherein the antibody is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody.

[00189] (6) The antibody according to any one of (1) to (5) above, wherein the antibody is a humanized monoclonal antibody comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a chain light consisting of the amino acid sequence represented by SEQ ID NO: 2.

[00190] (7) The antibody according to any one of (1) to (6) above, wherein the antibody requires a lysine residue at the carboxyl terminus of the heavy chain.

[00191] (8) The antibody according to (7) above, wherein the antibody comprises a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2.

[00192] (9) An antibody obtained by a method of producing the antibody according to any one of (1) to (8) above, the method comprising the steps of: culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody; and collecting the antibody of interest from the cultures obtained in the preceding step.

[00193] Next, the anti-HER2 antibody used in the invention is described.

[00194] The terms "cancer" and "tumor" when used herein are used interchangeably.

[00195] The term "gene" when used herein not only includes DNA, but also mRNA thereof, cDNA thereof and cRNA thereof.

[00196] The term "polynucleotide" when used herein is used with the same meaning as a nucleic acid and likewise includes DNA, RNA, probes, oligonucleotides, and primers.

[00197] The terms "polypeptide", "protein" and "protein" when used herein are used without distinction.

[00198] The term "cell" when similarly used herein includes cells in an individual animal and cultured cells.

[00199] The term "HER2" when used herein is used with the same meaning as the HER2 protein.

[00200] Examples of the anti-HER2 antibody when used herein may include, but are not particularly limited to, pertuzumab (International Patent Publication No. WO 01/00245) and trastuzumab (U.S. Patent No. 5,821,337). Trastuzumab is preferred. However, the anti-HER2 antibody of the present invention is not limited to this as long as it is an anti-HER2 antibody that specifically binds HER2, and more preferably has an internalizing activity in HER2 expressing cells by binding HER2.

[00201] The term "trastuzumab" when used herein is likewise called HERCEPTIN®), huMAb4D5-8, or rhuMAb4D5-8 and is a humanized antibody comprising a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 (Figure 1) and a light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2 (Figure 2).

[00202] The term "specifically binding" as used herein means binding that is not non-specific adsorption. Examples of the criterion for determining whether binding is specific or not may include the dissociation constant (hereinafter referred to as "KD"). The KD value of the antibody to the HER2 protein is preferably 1 x 10 -5 M or less, 5 x 10 -6 M or less, 2 x 10 -6 M or less, or 1 x 10 -6 M or less, more preferably 5x10-7M or less, 2x10-7M or less, or 1x10-7M or less, also preferably 5x10-8M or less, 2x10-8M or less, or 1 x 10 -8 M or less, and even more preferably 5 x 10 -9 M or less, 2 x 10 -9 M or less, or 1 x 10 -9 M or less. Binding between the HER2 protein and the antibody can be measured using a method known in the art, such as surface plasmon resonance, ELISA, or RIA.

[00203] The term "CDR" when used herein refers to a complementarity determining region (CDR). It is known that each heavy and light chain of an antibody molecule has three complementarity determining regions (CDRs). The CDR is likewise called the hypervariable domain, and is present in a variable region of each heavy and light chain of an antibody. It is a site that has extraordinarily high variability in its primary structure, and there are three separate CDRs in the primary structure of each heavy and light polypeptide chain. In this specification, according to the CDRs of an antibody, the heavy chain CDRs are represented by CDRH1, CDRH2, and CDRH3 on the amino-terminal side of the heavy chain amino acid sequence, and the light chain CDRs are represented by CDRL1, CDRL2, and CDRL3 from the amino-terminal side of the light chain amino acid sequence. These sites are close together in the tertiary structure and determine the specificity for an antigen to which the antibody binds.

[00204] The phrase "hybridization is carried out under stringent conditions" when used herein refers to a process in which hybridization is carried out under conditions under which identification can be obtained by carrying out hybridization at 68°C in a hybridization solution commercially available ExpressHyb Hybridization Solution (manufactured by Clontech, Inc.) or hybridizing at 68oC in the presence of 0.7 to 1.0M NaCl using a filter having DNA immobilized thereon, followed by washing at 68oC using 0.1 to 2 x SSC solution (1 x SSC solution is composed of 150 mM NaCl and 15 mM sodium citrate) or under conditions equivalent thereto.

1. HER2

[00205] HER2 is one of the oncogene products of a typical growth factor receptor oncogene such as human epidermal cell growth factor receptor-related oncogene 2, and is a transmembrane receptor protein having a molecular weight of 185 kDa and having a tyrosine kinase domain. HER2 is a member of the EGFR family consisting of HER1 (EGFR, ErbB-1), HER2 (neu, ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4) and is known to be autophosphorylated at residues of intracellular tyrosine by its homodimer formation or heterodimer formation with another EGFR receptor HER1, HER3, or HER4 and is itself activated in that manner, thereby playing an important role in cell growth, differentiation, and survival in normal and tumor cells.

[00206] As for the HER2 protein to be used in the present invention, the HER2 protein can be purified directly from HER2 expressing cells of a human or a non-human mammal (such as a rat or mouse) and used , or a cell membrane fraction of the cells described above can be prepared and used. Furthermore, HER2 can be obtained by in vitro synthesis of the same or production of the same in a host cell by genetic engineering. In genetic engineering, specifically, after HER2 cDNA is integrated into a vector capable of expressing HER2 cDNA, the HER2 protein can be obtained by synthesizing it in a solution containing an enzyme, a substrate and an energy substance required for transcription. and translating, or expressing HER2 in another transformed prokaryotic or eukaryotic host cell. Alternatively, the genetically engineered HER2 expressing cells described above, or a cell line expressing HER2 can be used as the HER2 protein.

[00207] The DNA sequence and amino acid sequence of HER2 are described in a public database, and may refer, for example, under No. Accession M11730 (GenBank), NP_004439.2 (NCBI), or the like.

[00208] In addition, a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted and/or added to any of the above-described amino acid sequences of HER2 and likewise have a biological activity equivalent to that of the HER2 protein is likewise included in HER2.

[00209] Human HER2 protein is composed of a signal sequence consisting of 22 N-terminal 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 23 amino acid residues. at 580 amino acid residues.

2. Production of anti-HER2 antibody

[00210] The HER2 antibody of the present invention can be obtained according to, for example, a method commonly performed in the art, which involves immunizing animals with HER2 or an arbitrary polypeptide selected from the HER2 amino acid sequence and collecting and purifying antibodies produced in vivo. The biological species of HER2 to be used as an antigen is not limited to human, and an animal can be immunized with HER2 derived from an animal other than humans such as a mouse or rat or with mouse p185neu. In this case, by examining the cross-reactivity between an antibody that binds the obtained heterologous HER2 and human HER2, an antibody applicable to a human disease can be selected.

[00211] In addition, a monoclonal antibody can be obtained from an established hybridoma by fusing antibody-producing cells that produce an antibody against HER2 with myeloma cells according to a known method (e.g., Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)).

[00212] HER2 to be used as an antigen can be obtained by expressing the HER2 gene in a host cell using genetic engineering.

[00213] Specifically, a vector capable of expressing the HER2 gene is produced, and the resulting vector is transfected into a host cell to express the gene, and then the expressed HER2 is purified.

[00214] Alternatively, the genetically engineered HER2 expression cells described above, or a cell line expressing HER2 can be used as the HER2 protein. The anti-HER2 antibody can be obtained by a procedure known in the art. In the following, a method of obtaining an antibody against HER2 is specifically described.

(1) Antigen preparation

[00215] Examples of the antigen to be used to produce the anti-HER2 antibody include HER2, or a polypeptide consisting of a partial amino acid sequence comprising at least 6 successive amino acids of HER2, or a derivative obtained by adding a certain amino acid sequence or vehicle to it.

[00216] HER2 can be purified directly from human tumor tissues or tumor cells and used. Furthermore, HER2 can be obtained by synthesizing it in vitro or by producing it in a host cell by genetic engineering.

[00217] With respect to genetic engineering, specifically, after the HER2 cDNA is integrated into a vector capable of expressing the HER2 cDNA, HER2 can be obtained by synthesizing it in a solution containing an enzyme, a substrate and a substance of energy required for transcription and translation, or by expressing HER2 in another transformed prokaryotic or eukaryotic host cell.

[00218] Furthermore, the antigen can likewise be obtained as a secretory protein by expressing a fusion protein obtained by binding the extracellular domain of HER2 which is a membrane protein, to the constant region of an antibody in a system appropriate vector host.

[00219] HER2 cDNA can be obtained, for example, by a so-called PCR method in which a polymerase chain reaction is carried out using a cDNA library expressing the HER2 cDNA as a template and primers that specifically amplify HER2 cDNA (PCR; Saiki, R.K., et al., Science, (1988) 239, pp. 487-489).

[00220] As the in vitro synthesis of the polypeptide, for example, Rapid Translation System (RTS) manufactured by Roche Diagnostics, Inc. can be exemplified, but is not limited thereto.

[00221] Examples of the prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform host cells with a target gene, the host cells are transformed by a plasmid vector comprising a replicon, i.e., an origin of replication derived from a species compatible with the host, and a regulatory sequence. Furthermore, the vector preferably has a sequence capable of imposing phenotypic selectivity on the transformed cell.

[00222] Examples of the eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. Like vertebrate cells, for example, simian COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCCCRL-1650; ATCC: American Type Culture Collection), murine fibroblasts NIH3T3 (ATCC No. CRL-1658), and dihydrofolate reductase deficient strains (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220) of Chinese hamster ovarian cells (CHO cells; ATCC: CCL-61); and the like are often used, however, cells are not limited to this.

[00223] The transformant thus obtained can be cultured according to a method commonly performed in the art, and by culturing the transformant, a target polypeptide is produced intracellularly or extracellularly.

[00224] A suitable medium to be used for cultivation can be selected from a variety of commonly used culture media depending on the host cells employed. If Escherichia coli is employed, for example, an LB medium supplemented with an antibiotic such as ampicillin or IPMG when needed can be used.

[00225] A recombinant protein produced intracellularly or extracellularly by the transformant by such cultivation may be separated and purified by any of several known separation methods utilizing the physical or chemical property of the protein.

[00226] Specific examples of the methods include treatment with a common protein precipitant, ultrafiltration, various types of liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography , dialysis, and a combination thereof.

[00227] Furthermore, by attaching a six-residue histidine tag to a recombinant protein to be expressed, the protein can be purified effectively with a nickel affinity column. Alternatively, by joining the Fc region of IgG to a recombinant protein to be expressed, the protein can be effectively purified with a protein A column.

[00228] By combining the methods described above, a large amount of a target polypeptide can be produced easily in a high yield and high purity.

[00229] The above described transformant itself can be used as the antigen. A cell line expressing HER2 can likewise be used as the antigen. Examples of such a cell line may include human breast cancer lines SK-BR-3, BT-474, KPL-4, and JIMT-1, a human gastric cancer line NCI-N87, and a human ovarian cancer line. SK-OV-3. The cell line of the present invention is not limited to these cell lines as long as it expresses HER2.

(2) Production of anti-HER2 monoclonal antibody

[00230] Examples of the antibody that specifically binds HER2 include a monoclonal antibody that specifically binds HER2, and a method of obtaining such an antibody is as described below.

[00231] The production of a monoclonal antibody generally requires the following operational steps of: (a) purifying a biopolymer to be used as an antigen, or preparing the cells for antigen expression; (b) preparing the antibody-producing cells by immunizing an animal by injecting the antigen, collecting the blood, analyzing its antibody titer to determine when the spleen is cut; (c) preparing the myeloma cells (hereinafter referred to as "myeloma"); (d) fusing the antibody-producing cells with the myeloma; (e) evaluating a group of hybridomas producing a desired antibody; (f) dividing the hybridomas into single cell clones (cloning); (g) optionally, culturing the hybridoma or raising an animal implanted with the hybridoma to produce a large amount of monoclonal antibody; (h) examining the monoclonal antibody so produced for biological activity and binding specificity, or analyzing the same for properties as a labeled reagent; and the like.

[00232] Next, the method of producing a monoclonal antibody will be described in detail following the previous steps, however, the method is not limited to these, and for example, antibody-producing cells other than spleen and myeloma cells can be used.

(a) Antigen purification

[00233] As the antigen, HER2 prepared by the method as described above or a partial peptide thereof can be used.

[00234] Furthermore, a membrane fraction prepared from recombinant cells expressing HER2 or the recombinant cells expressing HER2 by themselves, and likewise a partial peptide of the protein of the invention chemically synthesized by a method known to those skilled in the art can likewise be used as the antigen.

[00235] In addition, a HER2 expressing cell line can likewise be used as the antigen.

(b) Preparation of antibody-producing cells

[00236] The antigen obtained in step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or auxiliary agent such as potassium aluminum sulfate and the resulting mixture is used as an immunogen to immunize an experimental animal. Another method involves immunizing an experimental animal with the antigen-expressing cells as an immunogen. As the experimental animal, any animal used in a known hybridoma production method can be used without hindrance. Specifically, for example, a mouse, rat, goat, sheep, cattle, horse, or the like can be used. However, from the point of view of the ease of availability of myeloma cells to be fused with antibody-producing cells, a mouse or rat is preferably used as the animal to be immunized.

[00237] Furthermore, the strain of a mouse or rat to be used is not particularly limited, and in the case of a mouse, for example, various strains such as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129 and the like can be used, and in the case of a mouse, for example , Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer and the like can be used.

[00238] These mice and rats are commercially available from experimental animal breeders/distributors, e.g. CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

[00239] As the animal to be immunized, in view of the compatibility of the fusion with myeloma cells described below, in the case of a mouse, the BALB/c strain, and in the case of a mouse, strains of Wistar and Low are particularly favorites.

[00240] Furthermore, in view of the antigenic homology between humans and mice, it is likewise preferred to use a mouse having diminished biological function to remove autoantibodies, i.e. a mouse with an autoimmune disease.

[00241] The age of such a mouse or rat at the time of immunization is preferably 5 to 12 weeks of age, more preferably 6 to 8 weeks of age.

[00242] To immunize an animal with HER2 or a recombinant thereof, for example, a known method 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) or the like can be used.

[00243] Among these methods of immunization, a specific method preferred in the present invention is, for example, as follows.

[00244] This is, first, a membrane protein fraction that serves as the antigen or cells caused to express the antigen is/are intradermally or intraperitoneally administered to an animal. However, the combination of both routines of administration is preferred to increase the efficiency of immunization, and when intradermal administration is carried out at the last half or only at the last dosage, the efficiency of immunization can be particularly increased.

[00245] The timing of antigen administration varies depending on the type of animal to be immunized, individual difference or the like. However, in general, a schedule of administration where the frequency of antigen administration is 3 to 6 times and the dosing interval is 2 to 6 weeks is preferred, and a schedule of administration where the frequency of antigen administration is 3 to 4 times and the dosing interval is 2 to 4 weeks is more preferred.

[00246] In addition, the dose of antigen varies depending on the type of animal, individual differences or the like, however, the dose is usually fixed at 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

[00247] A booster immunization is performed 1 to 6 weeks, preferably 1 to 4 weeks, more preferably 1 to 3 weeks after antigen administration as described above. When the immunogen is cells, 1 x 106 to 1 x 107 cells are used.

[00248] The dose of antigen at the time of performing the booster immunization varies depending on the type or size of animal or the like, however, in the case of, for example, a mouse, the dose is usually fixed at 0.05 to 5 mg , preferably 0.1 to 0.5 mg, more preferably about 0.1 to 0.2 mg. When the immunogen is cells, 1 x 106 to 1 x 107 cells are used.

[00249] Spleen cells or lymphocytes including the antibody producing cells are aseptically removed from the immunized animal after 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days of the booster immunization. At this time, the antibody titer is measured, and if an animal having a sufficiently increased antibody titer is used as a source of supply for the antibody-producing cells, the subsequent procedure can be performed more effectively.

[00250] Examples of the method of measuring antibody titer to be used here include an RIA method and an ELISA method, however, the method is not limited to this. For example, if an ELISA method is employed, measurement of antibody titer in the invention can be performed according to the procedures as described below.

[00251] First, a purified or partially purified antigen is adsorbed to the surface of a solid phase such as a 96-well ELISA plate, and the surface of the solid phase that has no antigen adsorbed to it is coated with an unrelated protein to antigen such as bovine serum albumin (BSA). After washing the surface, the surface is brought into contact with a serially diluted sample (eg, mouse serum) as a primary antibody to allow the antibody in the sample to bind to the antigen.

[00252] In addition, as a secondary antibody, an enzyme labeled antibody against a mouse antibody is added and allowed to bind to the mouse antibody. After washing, a substrate for the enzyme is added and a change in absorbance that occurs due to color development induced by substrate degradation or the like is measured and the antibody titer is calculated based on the measurement.

[00253] The separation of antibody-producing cells from the spleen cells or lymphocytes of the immunized animal can be carried out according to a known method (eg, 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 general method in which antibody-producing cells are separated by homogenizing the spleen to obtain the cells by filtration through a stainless steel mesh and suspending the cells in Eagle's Minimum Essential Medium (MEM) can be used.

(c) Preparation of myeloma cells (hereinafter referred to as "myeloma")

[00254] The myeloma cells to be used for cell fusion are not particularly limited and suitable cells can be selected from known cell lines. However, for convenience when a hybridoma is selected from fused cells, it was preferred to use an HGPRT (hypoxanthine-guanine phosphoribosyl transferase) deficient strain whose selection procedure was established.

[00255] More specifically, examples of the HGPRT-deficient strain include 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, derived from mice; 210.RSY3.Ag.1.2.3(Y3) derived from rats; and U266AR(SKO-007), GM1500-GTG-A12(GM1500), UC729-6, LICR-LOW-HMy2(HMy2) and 8226AR/NIP4-1(NP41) derived from humans. These HGPRT deficient strains are available from, for example, ATCC or the like.

[00256] These cell strains are subcultured in an appropriate medium such as an 8-azaguanine medium [a medium obtained by adding 8-azaguanine to a RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and calf serum fetalis (hereinafter referred to as "FCS")], Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle's Medium (hereinafter referred to as "DMEM"). In this case, 3 to 4 days before performing cell fusion, the cells are subcultured in a normal medium (e.g., an ASF104 medium (manufactured by Ajinomoto Co., Ltd.) containing 10% FCS) to ensure no less than 2 x 10 7 cells on the day of cell fusion.

(d) Cell fusion

[00257] Fusion between antibody-producing cells and myeloma cells can suitably be carried out according to a known method (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), etc.), under conditions such that the survival rate of the cells is not excessively reduced.

[00258] As such a method, for example a chemical method in which antibody-producing cells and myeloma cells are mixed in a solution containing a polymer such as polyethylene glycol in a high concentration, a physical method using electrical stimulation , or the like can be used. Among these methods, a specific example of the chemical method is as described below.

[00259] That is, in the case where polyethylene glycol is used in the solution containing a polymer in a high concentration, the antibody producing cells and the myeloma cells are mixed in a polyethylene glycol solution having a molecular weight of 1500 to 6000 , more preferably 2000 to 4000°C at a temperature of 30 to 40°C, preferably 35 to 38°C for 1 to 10 minutes, preferably 5 to 8 minutes.

(e) Selection of a group of hybridomas

[00260] The method of selecting hybridomas obtained by the cell fusion described above is not particularly limited. Typically, a method of selecting HAT (hypoxanthine, aminopterin, thymidine) (Kohler et al., Nature (1975), 256, p. 495; Milstein et al., Nature (1977), 266, p. 550) is used. .

[00261] This method is effective when hybridomas are obtained using the myeloma cells of an HGPRT-deficient strain that cannot survive in the presence of aminopterin. That is, by growing unfused cells and hybridomas in a HAT medium, only aminopterin-resistant hybridomas are selectively allowed to survive and proliferate.

(f) Division into the single cell clone (cloning)

[00262] As a cloning method for hybridomas, a known method such as a methylcellulose method, a soft agarose method, or a limiting dilution method can be used (see, for example, Barbara, B.M. and Stanley, M.S.: Selected Methods in Cellular Immunology, W.H. Freeman and Company, San Francisco (1980)). Among these methods, particularly, a three-dimensional culture method such as a methylcellulose method is preferred. For example, the group of hybridomas produced by cell fusion is suspended in a methylcellulose medium such as ClonaCell-HY D Selection Medium (manufactured by StemCell Technologies, Inc., #03804) and cultured. Then the formed hybridoma colonies are collected, through which monoclonal hybridomas can be obtained. The respective collected hybridoma colonies are cultured, and a hybridoma that has been confirmed to have a stable antibody titer in a obtained hybridoma culture supernatant is selected as a HER2 monoclonal antibody-producing hybridoma strain.

(g) Monoclonal antibody preparation by culturing hybridoma

[00263] By culturing the hybridoma in this selected manner, a monoclonal antibody can be effectively obtained. However, before culturing, it is preferred to carry out screening of a hybridoma that produces a target monoclonal antibody.

[00264] In such an assessment, a known method may be employed.

[00265] The measurement of the antibody titer in the invention can be carried out, for example, by an ELISA method explained in item (b) described above.

[00266] The hybridoma obtained by the method described above can be stored in a frozen state in liquid nitrogen or in a freezer at -80oC or below.

[00267] Upon completion of cloning, the medium is changed from a HT medium to a normal medium, and the hybridoma is cultured.

[00268] Large scale culture is performed by spin culture using a large culture flask or by centrifuge culture. From the supernatant obtained by the broad culture, a monoclonal antibody which specifically binds to the protein of the invention can be obtained by purification using a method known to those skilled in the art such as gel filtration.

[00269] In addition, the hybridoma is injected into the abdominal cavity of a mouse of the same strain as the hybridoma (e.g., the BALB/c described above) or a Nu/Nu mouse to proliferate the hybridoma, where at least the ascites containing a large amount of the monoclonal antibody of the invention can be obtained.

[00270] In the case where the hybridoma is administered into the abdominal cavity, if a mineral oil such as 2, 6, 10, 14-tetramethyl pentadecane (pristane) is administered 3 to 7 days before that, a greater amount of ascites can be obtained .

[00271] For example, an immunosuppressant is previously injected into the abdominal cavity of a mouse of the same strain as the hybridoma to inactivate the T cells. 20 Days thereafter, 106 to 107 hybridoma clone cells are suspended in a medium free of serum (0.5 ml), and the suspension is administered into the abdominal cavity of the mouse. In general, when the abdomen is enlarged and filled with ascites, the ascites is collected from the mouse. By this method, the monoclonal antibody can be obtained at a concentration that is about 100 times or higher than that in the culture solution.

[00272] The monoclonal antibody obtained by the above-described method can be purified by a method described in, for example, Weir, D.M.: Handbook of Experimental Immunology Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978).

[00273] The monoclonal antibody thus obtained has high antigen specificity for HER2. Examples of the monoclonal antibody of the present invention may include, but are not particularly limited to, a mouse monoclonal antibody 4D5 (ATCC CRL 10463).

(h) Monoclonal antibody assay

[00274] The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows.

[00275] First, examples of the identification method include an Ouchterlony method, an ELISA method, and an RIA method.

[00276] An Ouchterlony method is simple, however when the concentration of the monoclonal antibody is low, a condensation operation is required.

[00277] On the other hand, when an ELISA method or an RIA method is used, directly reacting the culture supernatant with an antigen-absorbed solid phase and using antibodies that correspond to various types of immunoglobulin isotypes and subclass as secondary antibodies, the isotype and subclass of the monoclonal antibody can be identified.

[00278] Also, as a simpler method, a commercially available identification kit (eg, Mouse Typer Kit manufactured by Bio-Rad Laboratories, Inc.) or the like can likewise be used.

[00279] In addition, the quantitative determination of a protein can be performed by the Folin Lowry method and a calculation method based on absorbance at 280 nm (1.4 (OD280) = Immunoglobulin 1 mg/ml).

[00280] Furthermore, even when the monoclonal antibody is separately and independently obtained by performing steps (a) to (h) in (2) again, it is possible to obtain an antibody having a cytotoxic activity equivalent to that of the antibody of HER2 obtained in step (g). As an example of such an antibody, an antibody that binds to the same epitope as the HER2 antibody obtained in step (g) can be exemplified. If a newly produced monoclonal antibody binds a partial peptide or a partial tertiary structure to which the anti-HER2 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER2 antibody. In addition, by confirming that the monoclonal antibody competes with the anti-HER2 antibody for binding to HER2 (i.e., the monoclonal antibody inhibits the binding between the anti-HER2 antibody and HER2), it can be determined that the monoclonal antibody binds the same epitope as the anti-HER2 antibody even though the specific epitope sequence or structure has not been determined. When the monoclonal antibody is confirmed to bind to the same epitope as the anti-HER2 antibody, the monoclonal antibody is strongly expected to have an antigen binding affinity or biological activity equivalent to that of the anti-HER2 antibody.

(3) Other antibodies

[00281] The antibody of the invention not only includes the above-described monoclonal antibody against HER2, but also a recombinant antibody obtained by artificial modification for the purpose of decreasing heterologous antigenicity to humans such as a chimeric antibody, a humanized antibody and a human antibody. These antibodies can be produced using a known method.

[00282] Like the chimeric antibody, an antibody in which the antibody variable and constant regions are derived from different species, for example, a chimeric antibody in which a mouse or rat derived antibody variable region is connected to a region human-derived antibody constant can be exemplified (see Proc. Natl. Acad. Sci. USA, 81, 68516855, (1984)). Examples of the chimeric antibody of the present invention may include, but are not particularly limited to, chimeric antibody 4D5 comprising a human IgG1 or IgG2 heavy chain constant region.

[00283] Like the humanized antibody, an antibody obtained by integrating only a complementarity determining region (CDR) into a human-derived antibody (see Nature (1986) 321, pp. 522-525), and an antibody obtained by grafting A portion of the framework amino acid residues as well as the CDR sequence are added to a human antibody by a CDR-grafting method (WO90/07861), and a humanized antibody using gene conversion mutagenesis strategy (U.S. Patent No. 5821337) can be exemplified.

[00284] The term "various" when used herein refers to 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 two.

[00285] As the amino acid substitution in this specification, a conservative amino acid substitution is preferred. Conservative amino acid substitution refers to a substitution that occurs within a group of amino acids related to amino acid side chains. Preferred amino acid groups are as follows: an acid group (aspartic acid and glutamic acid); a basic group (lysine, arginine and histidine); a non-polar group (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan); and an uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine). More preferred amino acid groups are as follows: an aliphatic hydroxy group (serine and threonine); an amide-containing group (asparagine and glutamine); an aliphatic group (alanine, valine, leucine and isoleucine); and an aromatic group (phenylalanine, tryptophan and tyrosine). Such an amino acid substitution is preferably carried out within a range that does not impair the properties of a substance having the original amino acid sequence.

[00286] By combining a sequence that has a high homology to the heavy chain amino acid sequence described above with a sequence that has a high homology to the light chain amino acid sequence described above, it is possible to select an antibody that has a biological activity equivalent to that of each of the antibodies described above. Such homology is generally a homology of 80% or greater, preferably a homology of 90% or greater, more preferably a homology of 95% or greater, even more preferably a homology of 99% or greater. Furthermore, combining an amino acid sequence, wherein one to several amino acid residues is(are) substituted, deleted, or added to the heavy chain or light chain amino acid sequence, is of the It is likewise possible to select an antibody which has a biological activity equivalent to that of each of the antibodies described above. The term "homology" when used herein is used with the same meaning as "identity".

[00287] The homology between two amino acid sequences can be determined using basic parameters of Blast algorithm version 2.2.2 (Altschul, Stefen 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: 33893402 ). The Blast algorithm can be used in the same way over the Internet by going to www.ncbi.nlm.nih.gov/blast.

[00288] Furthermore, the antibody of the invention includes a human antibody that binds to HER2. An anti-HER2 human antibody refers to a human antibody that has only one antibody sequence derived from a human chromosome. Human anti-HER2 antibody can be obtained by a method using a human antibody-producing mouse that has a human chromosome fragment comprising heavy and light chain genes from a human antibody (see, 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., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

[00289] Such a human antibody producing mouse can be specifically bred as follows. A genetically modified animal whose endogenous immunoglobulin heavy and light chain gene sites have been disrupted, and conversely, human immunoglobulin heavy and light chain gene sites have been introduced via a yeast artificial chromosome (YAC) vector or similar is created by producing a knockout animal and a transgenic animal and mating these animals.

[00290] Furthermore, according to a recombinant DNA technique, using cDNAs encoding each of such a heavy chain and a light chain of a human antibody, and preferably a vector comprising such cDNAs, eukaryotic cells are transformed, and a transformant cell producing a recombinant human monoclonal antibody is cultured, whereby the antibody can be similarly obtained from the culture supernatant.

[00291] Here, as the host, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes or myeloma cells can be used.

[00292] Furthermore, a method of obtaining a human antibody derived from phage display selected from a human antibody library (see Wormstone, I.M. et al., Investigative Ophthalmology & Visual Science. (2002) 43(7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1(2), pp.189-203; Siriwardena, D. et al., Ophthalmology (2002) 109(3) ), pp. 427-431, etc.) is likewise known.

[00293] For example, a phage display method in which a variable region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv), and a phage that binds to an antigen is selected (Nature Biotechnology (2005), 23, (9), pp. 1105-1116) can be used.

[00294] By analyzing the phage gene selected on the basis of binding to an antigen, a DNA sequence encoding the variable region of a human antibody that binds to an antigen can be determined.

[00295] If the scFv DNA sequence that binds to an antigen is determined, a human antibody can be obtained by preparing an expression vector that comprises the sequence and introducing the vector into an appropriate host to express it ( 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 ).

[00296] As an example of another index for use in comparing antibody properties, antibody stability can be exemplified. Differential Scanning Calorimetry (DSC) is a device capable of quickly and accurately measuring a thermal denaturation center point temperature (Tm) to be used as a favorable index of the relative conformational stability of proteins. By measuring the Tm values using DSC and comparing the values, a difference in thermal stability can be compared. The storage stability of antibodies is known to show some correlation with the thermal stability of antibodies ( Lori Burton, et. al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273 ), and a preferred antibody may be selected using thermal stability as an index. Examples of other indices for selecting antibodies include the following aspects: yield in an appropriate host cell is high; and the aggregability in an aqueous solution is low. For example, an antibody that shows the highest yield does not always show the highest thermal stability, and therefore it is necessary to select an antibody most suitable for administration to humans by making a comprehensive assessment based on the indices described above.

[00297] In the present invention, a modified variant of the antibody is likewise included. Modified variant refers to a variant obtained by subjecting the antibody of the present invention to chemical or biological modification. Examples of the chemically modified variant include chemically modified variants linking a chemical moiety to an amino acid backbone, chemically modified variants having an N-linked or O-linked carbohydrate chain, and the like. Examples of the biologically modified variant include variants obtained by post-translational modification (such as N-linked or O-linked glycosylation, N- or C-terminal process, deamidation, aspartic acid isomerization, or methionine oxidation), and variants in which a methionine residue was added to the N-terminus being expressed in a prokaryotic cell host. Furthermore, a labeled antibody to allow detection or isolation of the antibody or antigen of the invention, for example, an enzyme-labeled antibody, a fluorescence-labeled antibody, and an affinity-labeled antibody are likewise included within the meaning of the modified variant. . Such a modified variant of the antibody of the invention is useful for improving the stability and blood retention of the antibody, reducing the antigenicity thereof, detecting or isolating an antibody or an antigen, and so on.

[00298] Furthermore, by regulating the modification of a glycan that is bound to the antibody of the invention (glycosylation, defucosylation, etc.), it is possible to enhance an antibody-dependent cellular cytotoxic activity. As the technique for regulating the modification of an antibody glycan, WO 99/54342, WO 00/61739, WO 02/31140, etc. They are known. However, the technique is not limited to this. In the antibody of the present invention, an antibody whose modification of a glycan is regulated is likewise included.

[00299] In the case where an antibody is produced by first isolating an antibody gene and then introducing the gene into an appropriate host, a combination of an appropriate host and an appropriate expression vector can be used. Specific examples of the antibody gene include a combination of a gene encoding a heavy chain sequence of an antibody described in this specification and a gene encoding a light chain sequence thereof. When a host cell is transformed, it is possible to insert the heavy chain sequence gene and the light chain sequence gene into the same expression vector, and likewise into different expression vectors separately.

[00300] In the case where eukaryotic cells are used as the host, animal cells, plant cells and eukaryotic microorganisms can be used. Like animal cells, mammalian cells, for example, simian COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCCCRL-1650), NIH3T3 murine fibroblasts (ATCC No. CRL-1658), and dihydrofolate reductase deficient strains (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220) of Chinese hamster ovarian cells ( CHO cells; ATCC: CCL-61) can be exemplified. In the case where prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be exemplified.

[00301] By introducing a desired antibody gene into these cells by transformation, and culturing the thereby transformed cells in vitro, the antibody can be obtained. In the above-described culturing method, the yield can sometimes vary depending on the antibody sequence, and therefore, it is possible to select an antibody that is readily produced as a pharmaceutical by using the yield as an index between antibodies having a binding activity. equivalent. Therefore, in the antibody of the present invention, an antibody obtained by a method of producing an antibody, characterized in that it includes a step of culturing the transformed host cell and a step of collecting a desired antibody from a cultured product obtained in the cultivation step is likewise included.

[00302] A lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in a cultured mammalian cell is known to be deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is known to in the same way that two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of an antibody produced in a cultured mammalian cell are deleted and a newly located proline residue at the carboxyl terminus is amidadp (Analytical Biochemistry, 360: 75- 83 (2007)). However, such deletion and modification of the heavy chain sequence do not affect the antigen binding affinity and effector function (activation of a complement, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, in the antibody according to the present invention, an antibody subjected to such modification and a functional fragment of the antibody is likewise included, and a deletion variant in which one or two amino acids have been deleted at the carboxyl terminus of the heavy chain, a variant obtained by amidation of the deletion variant (e.g., a heavy chain in which the carboxyl-terminal proline residue has been amidated), and the like are likewise included. The type of deletion variant having a deletion at the carboxyl terminus of the heavy chain of the antibody according to the invention is not limited to the above variants as long as antigen binding affinity and effector function are conserved. The two heavy chains constituting the antibody according to the invention may be of one type selected from the group consisting of a full-length heavy chain and the above-described deletion variant, or they may be of two types in selected combination thereof. . The ratio of the amount of each deletion variant can be affected by the type of cultured mammalian cells that produce the antibody according to the invention and the culture conditions, however, a case where a carboxyl-terminal amino acid residue was deleted in both the two heavy chains contained as main components in the antibody according to the invention can be exemplified.

[00303] As an isotype of the antibody of the invention, for example, IgG (IgG1, IgG2, IgG3, IgG4) can be exemplified, and IgG1 or IgG2 can preferably be exemplified.

[00304] As the biological activity of the antibody, generally an antigen-binding activity, an internalization activity in cells expressing an antigen binding to the antigen, an activity of neutralizing the activity of an antigen, an activity of enhancing the activity of an antigen, an antibody-dependent cellular cytotoxicity (ADCC) activity, a complement-dependent cytotoxicity (CDC) activity, and an antibody-dependent cell-mediated phagocytosis (ADCP) activity can be exemplified. The biological activity of the antibody of the present invention is a HER2 binding activity, and preferably an internalizing activity in HER2 expressing cells by binding HER2. Furthermore, the antibody of the present invention may have an ADCC activity, a CDC activity, and/or an ADCP activity in addition to a cell internalization activity.

[00305] The antibody obtained can be purified to homogeneity. Separation and purification of the antibody can be performed using a conventional protein separation and purification method. For example, antibody can be properly separated and purified by selecting and combining column chromatography, filter filtration, ultrafiltration, salt precipitation, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al.eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)) , however the method is not limited to this.

[00306] Examples of such chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed phase chromatography, and absorption chromatography.

[00307] Such chromatography can be performed using liquid chromatography such as HPLC or FPLC.

[00308] As a column to be used in affinity chromatography, a Protein A column and a Protein G column can be exemplified. For example, how a column using a Protein A column, Hyper D, POROS, Sepharose FF(Pharmacia Corporation) and the like can be exemplified.

[00309] Furthermore, using a carrier having an antigen immobilized thereon, the antibody can be purified using the antibody's antigen-binding property.

ANTITUMOR COMPOUND

[00310] The antitumor compound to be conjugated to the anti-HER2 antibody-drug conjugate of the present invention is explained. The antitumor compound used in the present invention is not particularly limited as long as it is a compound having an antitumor effect and a substituent group or a partial structure that allows attachment to a linker structure. When part or all of the ligand is cleaved in tumor cells, the antitumor compound moiety is released to exhibit the antitumor effect of the antitumor compound. As the ligand is cleaved at a linking position on the drug, the antitumor compound is released in an unchanged structure to exhibit its intrinsic antitumor effect.

[00311] As the antitumor compound used in the present invention, 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; shown in the following formula), one of the derivatives of camptothecin, can be used preferably.

[00312] Although having an excellent antitumor effect, Exatecan has not been marketed as an antitumor drug. The compound can be easily obtained by a known method and the amino group at position 1 can preferably be used as the attachment position to the linker structure. Furthermore, although exatecan can likewise be released into tumor cells while part of the ligand is still bound thereto, it is an excellent compound that exhibits an excellent antitumor effect even in such a structure.

[00313] Because exatecan has a camptothecin structure, it is known that the equilibrium switches to a structure with a closed lactone ring (closed ring) in an aqueous acidic medium (eg pH 3 plus or minus), however , switch to a structure with an open lactone ring (ring open) in an aqueous basic medium (eg pH 10 plus or minus). A drug conjugate to be introduced with an exatecan residue that corresponds to the closed ring structure and the open ring structure is similarly expected to have the same antitumor effect and needless to say that either of these structures are within the scope of the present invention. .

[00314] Other examples of the antitumor compound may include doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agent (cisplatin or derivatives thereof), taxol or derivatives thereof, and other camptothecins or derivatives thereof (antitumor agent described in Japanese Patent Open to Public Inspection No. 6-87746).

[00315] With respect to antibody-drug conjugate, the number of conjugated drug molecules per antibody molecule is a key factor having an influence on efficacy and safety. Production of the antibody-drug conjugate is accomplished by defining the reaction conditions including the amounts of starting materials and reagents used for the reaction to have a constant number of conjugated drug molecules. A mixture containing different numbers of conjugated drug molecules is usually obtained as opposed to the chemical reaction of a low molecular weight compound. The number of drugs conjugated to an antibody molecule is expressed or specified by the mean value, that is, the mean number of conjugated drug molecules. Unless specifically stated otherwise as a principle, the number of conjugated drug molecules means the average value except where it represents an antibody-drug conjugate having a specific number of conjugated drug molecules that is included in a mixture. of antibody-drug conjugate having different numbers of conjugated drug molecules.

[00316] The number of exatecan molecules conjugated in an antibody molecule is controllable, and as the average number of drug molecules conjugated per antibody molecule, about 1 to 10 exatecans can be connected. Preferably, it is 2 to 8, and more preferably 3 to 8. Meanwhile, a person skilled in the art can design a reaction to conjugate a required number of drug molecules to an antibody molecule based on the description of the Examples of the present application and can obtain an antibody-drug conjugate conjugated to a controlled number of molecules of exatecan.

BINDING STRUCTURE

[00317] With respect to the anti-HER2 antibody-drug conjugate of the present invention, the linker structure for conjugating an antitumor compound to the anti-HER2 antibody is explained. The linker has a structure of the following formula: -L1-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O)-.

[00318] The antibody is connected to the L1 terminal (end opposite the L2 connection), and the antitumor compound is connected to the carbonyl group of the -La-(CH2)n2-C(=O)- moiety.

[00319] n1 represents an integer from 0 to 6 and is preferably an integer from 1 to 5, and more preferably 1 to 3.

1.L1

[00320] L1 is represented by the following structure: -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-.

[00321] In the above, n3 is an integer from 2 to 8, "-(Succinimid-3-yl-N)-" has a structure represented by the following formula:

[00322] Position 3 of the above partial structure is the position of attachment to the anti-HER2 antibody. Antibody binding at position 3 is characterized by binding with thioether formation. The nitrogen atom at position 1 of the backbone moiety is connected to the methylene carbon atom that is present within the ligand including the backbone. Specifically, -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-L2 - is a structure represented by the following formula (here, "antibody-S -" originates from an antibody).

[00323] In the formula, n3 is an integer from 2 to 8, and preferably 2 to 5.

[00324] Specific examples of L1 may include -(Succinimid-3-yl-N)-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2-C(=O)-, - (Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-, and -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-.

2.L2

[00325] L2 is represented by the following structure: -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-,

[00326] L2 may not be present, and in such a case, L2 is a single bond. n4 is an integer from 1 to 6, and preferably 2 to 4. L2 is connected to L1 at its terminal amino group and is connected to LP at its carbonyl group at the other terminal.

[00327] Specific examples of L2 may 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)-.

3.LP

[00328] LP is a peptide residue consisting of 2 to 7 amino acids. Specifically, it consists of an oligopeptide residue where 2 to 7 amino acids are linked by peptide bond. LP is connected to L2 at its N-terminus and is connected to the amino group of the -NH-(CH2)n1-La-(CH2)n2-C(=O)- moiety of the ligand at its C-terminus. Here, the term " peptide residue" or "oligopeptide residue" is a group derived from a peptide consisting of two or more amino acid residues and refers to a divalent group whose N-terminal and C-terminal are connecting positions.

[00329] The amino acid constituting LP in the linker is not particularly limited, however, examples thereof include an L- or a D-amino acid, preferably an L-amino acid. And, it may be an amino acid having a structure such as β-alanine, ε-aminocaproic acid, or ε-aminobutyric acid in addition to an α-amino acid, also, it may be an unnatural type amino acid such as N-methylated amino acid.

[00330] The amino acid sequence of LP is not particularly limited, however, examples of the constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), and aspartic acid (Asp; D). Among them, preferred examples include phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. From these amino acids, LPs having an optionally selected amino acid sequence with or without overlaps can be constructed. Depending on the type of amino acids, the pattern of drug release can be controlled. The number of amino acids can be between 2 to 7.

[00331] Specific examples of LP may include -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG- , -DDGGFG-, -KDGGFG-, and -GGFGGGF-.

[00332] In the above, "(D-)D" represents a D-aspartic acid.

[00333] Particularly preferred examples of LP for the antibody-drug conjugate of the present invention may include the tetrapeptide residue -GGFG-.

4. La-(CH2)n2-C(=O)-

[00334] La in La-(CH2)n2-C(=O)- is a structure of -O- or a single bond. n2 is an integer from 0 to 5, preferably 0 to 3, and more preferably 0 or 1.

[00335] Examples of La-(CH2)n2-C(=O)- may 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)-, -O-C(=O)-.

[00336] Among these, - O-CH2-C(=O)-, - O-CH2CH2-C(=O)-, - O-C(=O)-,

[00337] or a case in which La is a single bond, and n2 is 0 is preferred.

[00338] Specific examples of the structure represented by -NH-(CH2)n1-La-(CH2)n2-C(=O)- of the linker may 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-O-C(=O)-, - NH-CH2CH2-O-C(=O)-, - NH-CH2CH2CH2-O-C(=O)-, - NH-CH2CH2CH2CH2-O-C(=O)-.

[00339] Among these, - NH-CH2CH2CH2-C(=O)-, - NH-CH2-O-CH2-C(=O)-, - NH-CH2CH2-O-CH2-C(=O)-

[00340] is more preferred.

[00341] In the linker, the chain length of -NH-(CH2)n1-La-(CH2)n2-C(=O)- is preferably a chain length of 4 to 7 atoms, and more preferably a length of chain of 5 or 6 atoms.

[00342] With respect to the anti-HER2 antibody-drug conjugate of the present invention, when it is transferred into tumor cells, it has been suggested that the linker moiety be cleaved and the drug derivative having a structure represented by NH2- (CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) is released to express an antitumor action. Examples of the antitumor derivative exhibiting an antitumor effect releasing from the antibody-drug conjugate of the present invention include an antitumor derivative having a structure portion in which one terminus of the structure represented by -NH-(CH2)n1-La-(CH2) n 2 -C(=O)- of the linker has an amino group, and particularly preferred ones include 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).

[00343] Meanwhile, in the case of NH2-CH2-O-CH2-C(=O)-(NH-DX), it was confirmed that when the aminal structure in the molecule is unstable, it again undergoes self-degradation to release the following HO-CH2 -C(=O)-(NH-DX). Such compounds may likewise be used preferably as an intermediate for producing the antibody-drug conjugate of the present invention.

[00344] For the antibody-drug conjugate of the present invention where exatecan is used as the drug, it is preferable that the [-L1-L2-LP-NH-(CH2)n1-La- (CH2)n2-C(=O)-(NH-DX)] having the following structure is connected to an antibody. The average conjugate number of said drug-linker structure moieties per antibody molecule 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-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)-CH 2CH2-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).

[00345] Among these, the most preferred are the following. -(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-CH2CH2CH2-C(=O)-(NH-DX), -( Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C (=O)-(NH-DX).

[00346] Particularly preferred are the following. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N )-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[00347] With respect to the linker structure for conjugating the anti-HER2 antibody and the drug in the antibody-drug conjugate of the present invention, the preferred linker can be constructed by connecting the preferred structures shown for each part of the linker explained above. As for the linker structure, those with the following structure can preferably be used. Meanwhile, the left end of these structures is at the antibody connection position and the right end is the drug connection position. -(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)-.

[00348] Among these, most preferred are the following. -(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-O-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-CH2CH2CH2-C(=O)-.

[00349] Particularly preferred include the following. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C (=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O -CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

PRODUCTION METHOD

[00350] Next, explanations are given for the representative method for producing the antibody-drug conjugate of the present invention or an intermediate for producing the same. Meanwhile, the compounds are described below with the compound number shown in each reaction formula. Specifically, they are referred to as a "compound of formula (1)", a "compound (1)", or the like. Compounds with numbers other than those are similarly described in the same way.

1. PRODUCTION METHOD 1

[00351] The antibody-drug conjugate represented by formula (1) wherein the antibody is attached to the drug-linker structure by means of thioether can be produced by the following method, for example.

[00352] [In the formula, AB represents an antibody having a sulfhydryl group, and L1' represents L1 linker structure wherein the terminus of the linker is a maleimidyl group (formula shown below)

[00353] (in the formula, the nitrogen atom is the connecting position), and specifically represents a group in which the -(Succinimid-3-yl-N)- moiety in -(Succinimid-3-yl-N)- (CH2)n3-C(=O)- of L1 is a maleimidyl group. Also, the -(NH-DX) represents a structure represented by the following formula:

[00354] and represents a group that is derived by removing a hydrogen atom from the amino group at position 1 of exatecan.]

[00355] Furthermore, the compound of formula (1) in the above reaction formula can be interpreted as a structure in which a portion of the structure corresponding from the drug to the terminus of the linker connects to an antibody. However, it is only a description given for the sake of convenience, and in fact there are many cases in which a plurality of framework moieties are connected to an antibody molecule. The same applies to the explanation of the production method described below.

[00356] The antibody-drug conjugate (1) can be produced by reacting the compound (2) which is obtainable by the method described below, with the antibody (3a) having a sulfhydryl group.

[00357] The antibody (3a) having a sulfhydryl group can be obtained by a method well known in the art (Hermanson, G.T, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). Examples include: Traut's reagent is reacted with the amino group of the antibody; N-succinimidyl S-acetylthioalkanoates are reacted with the amino group of the antibody followed by reaction with hydroxylamine; after reacting with N-succinimidyl 3-(pyridyldithio)propionate, the antibody is reacted with a reducing agent; the antibody is reacted with a reducing agent such as dithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bond at the hinge part on the antibody to form a sulfhydryl group, however, is not limited to this.

[00358] Specifically, by using 0.3 to 3 molar equivalents of TCEP as a disulfide-reducing agent on the joint part of the antibody and reacting with the antibody in a buffering solution containing a chelating agent, the antibody disulfide partially or completely reduced in the joint part in the antibody can be obtained. Examples of the chelating agent include ethylenediamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA). It can be used in a concentration of 1 mM to 20 mM. Examples of the buffer solution that can be used include a solution of sodium phosphate, sodium borate, or sodium acetate. Specifically, by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours, the antibody (3a) having a partially or completely reduced sulfhydryl group can be obtained.

[00359] Meanwhile, by conducting the reaction to add a sulfhydryl group to a drug-linker moiety, the drug-linker moieties can be conjugated by a thioether bond.

[00360] Using 2 to 20 molar equivalents of compound (2) per antibody (3a) having a sulfhydryl group, the antibody-drug conjugate (1) in which 2 to 8 drug molecules are conjugated per antibody molecule can be produced. Specifically, it is sufficient that the solution containing the compound (2) dissolved therein is added to a buffer solution containing the antibody (3a) having a sulfhydryl group for the reaction. Here, examples of the buffering solution that can be used include sodium acetate solution, sodium phosphate, and sodium borate. The pH for the reaction is 5 to 9, and more preferably the reaction is carried out near pH 7. Examples of the solvent for dissolving compound (2) include an organic solvent such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethyl acetamide (DMA), and N-methyl-2-pyridone (NMP).

[00361] It is sufficient that the organic solvent solution containing the compound (2) dissolved therein is added at 1 to 20% v/v to a buffering solution containing the antibody (3a) having a sulfhydryl group for the reaction. 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 the unreacted compound (2) with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine and N-acetyl-L-cysteine (NAC). More specifically, 1 to 2 molar equivalents of NAC are added to the compound (2) used and, by incubating at room temperature for 10 to 30 minutes, the reaction can be terminated.

[00362] The produced antibody-drug conjugate (1) can, after concentration, buffer exchange, purification, and measurement of antibody concentration and average number of conjugated drug molecules per antibody molecule according to the common procedures described below, be subjected to antibody-drug conjugate identification (1).

Common Procedure A: Concentration of Aqueous Antibody Solution or Antibody-Drug Conjugate

[00363] In an Amicon Ultra vessel (50,000 MWCO, Millipore Co.), an antibody or antibody-drug conjugate solution was added and the antibody or antibody-drug conjugate solution was concentrated by centrifugation (centrifuge for 5 to 20 minutes at 2000G to 3800G) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

Common Procedure B: Measurement of Antibody Concentration

[00364] Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.), the measurement of antibody concentration was performed according to the method defined by the manufacturer. At that time, a different absorption coefficient of 280 nm for each antibody was used (1.3 mLmg-1cm-1 to 1.8 mLmg-1cm-1).

Common Procedure C-1: Changing Buffer for Antibody

[00365] Column NAP-25 (Cat. No.17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 vehicle was equilibrated with phosphate buffer (10 mM, pH 6.0; is referred to as PBS6.0 /EDTA in specification) containing sodium chloride (137 mM) and ethylene diamine tetraacetic acid (EDTA, 5 mM) according to the method defined by the manufacturer. The aqueous solution of the antibody was applied in an amount of 2.5 mL to a single NAP-25 column, and then the fraction (3.5 mL) eluted with 3.5 mL of PBS6.0/EDTA was collected. The resulting fraction was concentrated by Standard Procedure A. After measuring the antibody concentration using Standard Procedure B, the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

Common Procedure C-2: Changing Buffer for Antibody

[00366] Column NAP-25 (Cat. No.17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 vehicle was equilibrated with phosphate buffer (50 mM, pH 6.5; is referred to as PBS6.5 /EDTA in specification) containing sodium chloride (50 mM) and EDTA (2 mM) according to the method defined by the manufacturer. Aqueous antibody solution was applied in an amount of 2.5 mL to the plain NAP-25 column, and then the fraction (3.5 mL) eluted with 3.5 mL of PBS6.5/EDTA was collected. The resulting fraction was concentrated by Standard Procedure A. After measuring the antibody concentration using Standard Procedure B, the antibody concentration was adjusted to 20 mg/mL using PBS6.5/EDTA.

Common Procedure D: Purification of Antibody-Drug Conjugate

[00367] Column NAP-25 was equilibrated with any buffer selected from commercially available phosphate buffer (PBS7.4, Cat.No.10010-023, Invitrogen), sodium phosphate buffer (10mM, pH 6.0; is referred to as PBS6.0) containing sodium chloride (137mM), and acetate buffer containing sorbitol (5%) (10mM, pH 5.5; is referred to as ABS in the specification). Aqueous solution of the antibody-drug conjugate reaction was applied in an amount of about 1.5 mL to the NAP-25 column, and then eluted with the buffer in an amount defined by the manufacturer to collect the antibody fraction. The collected fraction was applied again to the NAP-25 column and, repeating the gel filtration purification process 2 to 3 times in total to elute with buffer, the antibody-drug conjugate excluding the unconjugated drug binder and a A low molecular weight compound (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide) was obtained.

[00368] Common Procedure E: Measurement of antibody concentration in antibody-drug conjugate and average number of conjugated drug molecules per antibody molecule (1).

[00369] The concentration of drug conjugate in the antibody-drug conjugate can be calculated by measuring the UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths of 280 nm and 370 nm, followed by performing the calculation shown below.

[00370] Because the total absorbance at any wavelength is equal to the sum of the absorbance of all light-absorbing chemical species that are present in the system (absorbance additivity), when the molar absorption coefficients of the antibody and the drug remain the same before and after the conjugation between the antibody and the drug, the antibody concentration and the drug concentration in the antibody-drug conjugate is expressed by the following equations. A280 = AD,280 + AA,280 = SD,28OCD + SA,28OCA EQUATION (I) A370 = AD,370+AA,370 = SD,37OCD+SA,37OCA Equation (II)

[00371] In the above, A280 represents the absorbance of an aqueous solution of the Antidrug-Drug Conjugate at 280 nm, A370 represents the absorbance of AN aqueous solution of the Anti-drug-Drug Conjugate at 370 nm, AA, 280 represents the absorbance of One antibody at 280 nm, AA,370 represents the absorbance of An antibody at 370 nm, AD,280 represents the absorbance of A conjugate precursor at 280 nm, AD, 370 represents the absorbance of A conjugate precursor at 370 nm, SA, 280 represents the molar absorption coefficient of an antibody at 280 nm, SA,370 represents the molar absorption coefficient of an antibody at 370 nm, SD,28Q represents the molar absorption coefficient of a conjugated precursor at 280 nm, SD,37Q represents the molar absorption coefficient of a conjugate precursor at 370 nm, CA represents the concentration of antibody in an antibody-drug conjugate, and CD represents the concentration of drug in an antibody-drug conjugate.

[00372] As for SA,28Q, SA,37Q, SD,28Q, and SD,37Q in the above, previously prepared values (estimated values based on calculation or measurement values obtained by UV measurement of the compounds) are used. For example, SA,28Q can be calculated from the amino acid sequence of an antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). SA.37Q is usually zero. In the Examples, for the molar absorption coefficient of trastuzumab, SA,28Q = 215400 (estimated value based on calculation) and SA,37Q = 0 were used. SD,28Q and SD,37Q can be obtained based on the Lambert-Beer law (Absorbance = molar concentration x molar absorption coefficient x cell track length) by measuring the absorbance of a solution in which the precursor of conjugate a to be used is dissolved in a certain molar concentration. As for the molar absorption coefficient of a drug binder in the Examples, SD,28Q = 5000 (mean measured value) and SD,37Q = 19000 (mean measured value) were used unless otherwise specified. By measuring A28q and A37q of an aqueous solution of the antibody-drug conjugate and solving simultaneous equations (I) and (II) using the values, CA and CD can be obtained. Furthermore, by dividing CD by CA, the average number of conjugated drug molecules per antibody molecule can be obtained.

[00373] Common Procedure F: Measurement (2) of the average number of conjugated drug molecules per antibody molecule in antibody-drug conjugate.

[00374] The average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate can likewise be determined by high performance liquid chromatography (HPLC) analysis using the following method in addition to the aforementioned Common Procedure E.

F-1. Sample preparation for HPLC analysis (antibody-drug conjugate reduction)

[00375] An antibody-drug conjugate solution (about 1 mg/mL, 60 µL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 µL). A sample in which the disulfide bond between the L-chain and H-chain of the antibody-drug conjugate has been cleaved by incubating the mixture for 30 minutes at 37°C is used for HPLC analysis.

F-2. HPLC analysis

[00376] HPLC analysis is performed under the following measurement conditions: HPLC System: Agilent 1290 HPLC System (Agilent Technologies, Inc.) Detector: Ultraviolet Absorption Spectrometer (measuring wavelength: 280 nm) Column : PLRP-S (2.1 x 50 mm, 8 μm, 1000 angstroms; Agilent Technologies, Inc., P/N PL1912-1802) Column Temperature: 80oC Mobile Phase A: aqueous solution containing 0.04% trifluoroacetic acid (TFA) Mobile phase B: acetonitrile solution containing 0.04% TFA Gradient program: 29%-36% (0-12.5 min), 36%-42% (12.5-15 min), 42 %-29% (15-15.1 min), and 29%-29% (15.1-25 min) Sample injection volume: 15 μL

F-3. Data analysis

[00377] [F-3-1] Compared with unconjugated antibody, L (L0) and H (H0) chains, L (L chain connected to a drug molecule: L1) and H chains (H chain connected to a drug molecule: H1, H chain connected to two drug molecules: H2, H chain connected to three drug molecules: H3) conjugated per drug exhibit higher hydrophobicity in proportion to the number of conjugated drug molecules and thus have a longer of greater retention. These chains are therefore eluted in the order of L0 and L1 or H0, H1, H2, and H3. Detection peaks can be assigned any of L0, L1, H0, H1, H2, and H3 by comparing retention times with L0 and H0.

Expressão 2

[00378] [F-3-2] Since the drug ligand has UV absorption, peak area values are corrected with respect to the number of conjugated drug ligand molecules according to the following expression using the molar absorption coefficients of the L or H chains and the drug linker. expression 1

expression 2

[00379] Here, for the molar absorption coefficient (280 nm) of the L or H chain of each antibody, an estimated value of the amino acid sequence of the L or H chain of each antibody by a known calculation method (Protein Science, 1995). , vol 4, 2411-2423) can be used. In the case of trastuzumab, a molar absorption coefficient of 26150 and a molar absorption coefficient of 81290 were used as estimated values for the L and H chains, respectively, according to their amino acid sequence. As for the molar absorption coefficient (280 nm) of the drug ligand, the measured molar absorption coefficient (280 nm) of a compound in which the maleimide group was converted to the thioether succinimide by the reaction of each drug ligand with mercaptoethanol or N- acetylcysteine was used.

[00380] [F-3-3] The peak area ratio (%) of each strand is calculated for the total of corrected peak area values according to the following expression. expression 3

[00381] [F-3-4] The average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate is calculated according to the following expression.

[00382] Average number of conjugated drug molecules = (peak area ratio of L0 x 0 + peak area ratio of L0 x 1 + peak area ratio of H0 x 0 + peak area ratio of H1 x 1 + H2 peak area ratio x 2 + H3 peak area ratio x 3) / 100 x 2

[00383] The production intermediate compound used in Production Method 1 is described below. The compound represented by the formula (2) in Production Method 1 is a compound represented by the following formula: (maleimid-N-yl)-(CH2)n3-C(=O)-L2-LP-NH-(CH2)n1 -La-(CH2)n2-C(=O)-(NH-DX).

[00384] In the formula,

[00385] n3 represents an integer from 2 to 8,

[00386] L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond,

[00387] where n4 represents an integer from 1 to 6,

[00388] LP represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid,

[00389] n1 represents an integer from 0 to 6,

[00390] n2 represents an integer from 0 to 5,

[00391] La represents -O- or a single bond,

[00392] (maleimid-N-yl)- is a maleimidyl group (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by the following formula:

[00393] where the nitrogen atom is the connection position, and

[00394] -(NH-DX) is a group represented by the following formula:

[00395] where the nitrogen atom of the amino group at position 1 is the connecting position.

[00396] When L2 is a single bond or -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-, it is preferred as a production intermediate that n4 should be an integer from 2 to 4.

[00397] As for the LP peptide residue, a compound having a peptide residue consisting of an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid is preferred as an intermediate of production. Among these peptide residues, a compound in which LP is a peptide residue consisting of 4 amino acids is preferred as a production intermediate. More specifically, a compound in which LP is the tetrapeptide residue -GGFG- is preferred as a production intermediate.

[00398] Further, as for -NH-(CH2)n1-La-(CH2)n2 -, a compound having -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2- , -NH-CH2-O-CH2 -, or -NH-CH2CH2-O-CH2 - is preferred as a production intermediate. A compound having -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2 is more preferred.

[00399] A compound represented by formula (2) wherein n3 is an integer from 2 to 5, L2 is a single bond, and -NH-(CH2)n1- La-(CH2)n2 - is -NH-CH2CH2 -, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2 -, or -NH-CH2CH2-O-CH2 - is preferred as a production intermediate. A compound wherein -NH-(CH2)n1-La-(CH2)n2 - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2-O-CH2 -, or -NH-CH2CH2-O- CH2- is more preferred. A compound in which n3 is an integer of 2 or 5 is also preferred.

[00400] A compound represented by formula (2) wherein n3 is an integer from 2 to 5, L2 is -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-, n4 is an integer of 2 to 4, and -NH-(CH2)n1-La-(CH2)n2 - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2- O-CH2 -, or -NH-CH2 CH2 -O-CH2 - is preferred as a production intermediate. A compound in which n4 is an integer of 2 or 4 is more preferred. A compound wherein -NH-(CH 2 )n 1 -La-(CH 2 )n 2 - is -NH-CH 2 CH 2 CH 2 -, -NH-CH 2 -O-CH 2 -, or -NH-CH 2 CH 2 -O-CH 2 - is also preferred.

[00401] Preferred examples of an intermediate useful in the production of such a compound of the present invention may 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-CH2CH 2CH2CH2CH2-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).

[00402] The anti-HER2 antibody-drug conjugate of the present invention can be produced by reacting a drug-binding compound selected from the above-mentioned group of production intermediate compound with an anti-HER2 antibody or a reactive derivative of the even to thereby form a thioether bond to a disulfide binding site present on the hinge part of the anti-HER2 antibody. In this case, the reactive derivative of the anti-HER2 antibody is preferably used, and a reactive derivative obtained by reducing the anti-HER2 antibody is particularly preferred.

[00403] The following are more preferred compounds as a production intermediate. (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).

[00404] Among the above-mentioned intermediate compound group, a compound represented by 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),

[00405] is another preferred compound.

[00406] To obtain the amount of conjugate, a plurality of conjugates obtained under similar production conditions to have an equivalent number of drug (eg, about ±1) can be mixed to prepare new groups. In this case, the average number of drugs falls between the average numbers of drugs in the conjugates before mixing.

2. PRODUCTION METHOD 2

[00407] The compound represented by formula (2) as an intermediate used in the above production method and a pharmacologically acceptable salt thereof can be produced by the following method, for example.

[00408] In the formula, L1' represents a terminal maleimidyl group, and P1, P2, and P3 each represent a protecting group.

[00409] Compound (6) can be produced by deriving the carboxylic acid (5) into an active ester, mixed acid anhydride, acid halide, or the like and reacting it with NH2-DX(4) or a pharmacologically acceptable salt of the same. NH2-DX (4) indicates 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]quinolin-10,13(9H, 15H)-dione).

[00410] Reaction reagents and conditions that are generally used for peptide synthesis can be employed for the reaction. There are several types of active esters. For example, it can be produced by reacting phenols such as p-nitrophenol, N-hydroxybenzotriazole, N-hydroxysuccinimide, or the like, with the carboxylic acid (5) using a condensing agent such as N,N'-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Furthermore, the active ester can be produced in the same way by a reaction of the carboxylic acid (5) with pentafluorophenyl trifluoroacetate or the like; a reaction of the carboxylic acid (5) with benzotriazolyl oxytripyrrolidinophosphonium 1-hexafluorophosphite; a reaction of the carboxylic acid (5) with diethyl cyanophosphonate (dispersion method); a reaction of the carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyl disulfide (Mukaiyama method); a reaction of the carboxylic acid (5) with a triazine derivative such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); or similar. Furthermore, the reaction can be carried out in the same way, for example, by an acid halide method whereby the carboxylic acid (5) is treated with an acid halide such as thionyl chloride and oxalyl chloride in the presence of a base.

[00411] Reacting the active ester, mixed acid anhydride, or carboxylic acid acid halide (5) obtained as above with compound (4) in the presence of a suitable base in an inert solvent at -78°C to 150°C, the compound (6) can be produced. (Meanwhile, "inert solvent" indicates a solvent that does not inhibit a reaction for which the solvent is used.)

[00412] Specific examples of the base used for each step described above include a carbonate, alkoxide, hydroxide or hydride of an alkali metal or an alkaline earth metal such as sodium cabonate, potassium carbonate, sodium ethoxide, potassium butoxide, hydroxide sodium, potassium hydroxide, sodium hydride, and potassium hydride; an organometallic base represented by an alkyl lithium such as n-butyl lithium, or a dialkylamino lithium such as lithium diisopropylamide; a bisilylamine organometallic base such as lithium bis(trimethylsilyl)amide; and an organic base including a tertiary amine or a nitrogen-containing heterocyclic compound such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methyl morpholine, diisopropylethylamine, and diazabicyclo[5.4.0]undec- 7-ene (DBU).

[00413] Examples of the inert solvent that is used for the reaction of the present invention include a halogenated hydrocarbon solvent such as dichloromethane, chloroform, and carbon tetrachloride; an ether solvent such as tetrahydrofuran, 1,2-dimethoxyethane, and dioxane; an aromatic hydrocarbon solvent such as benzene and toluene; and an amide solvent such as N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidin-2-one. In addition to these, a sulfoxide solvent such as dimethyl sulfoxide and sulfolane; and a ketone solvent such as acetone and methyl ethyl ketone and an alcohol solvent such as methanol and ethanol may be used in some cases. Furthermore, a mixed solvent thereof may likewise be used.

[00414] As for the protecting group P1 for the terminal amino group of compound (6), a protecting group for an amino group which is generally used for peptide synthesis, e.g. a tert-butyloxycarbonyl group, a 9-fluorenylmethyloxy group carbonyl, or a benzyloxycarbonyl group, may be used. Examples of other protecting groups for an amino group include an alkanoyl group such as an acetyl group; an alkoxycarbonyl group such as a methoxycarbonyl group and an ethoxycarbonyl group; an arylmethoxycarbonyl group such as a paramethoxybenzyloxycarbonyl group, and a para (or ortho)nitrobenzyloxycarbonyl group; an arylmethyl group such as a benzyl group and a triphenyl methyl group; an aroyl group such as a benzoyl group; and an aryl sulfonyl group such as a 2,4-dinitrobenzene sulfonyl group and an orthonitrobenzene sulfonyl group. The protecting group P1 may be selected depending on, for example, the properties of the compound having the amino group to be protected.

[00415] By deprotecting the protecting group P1 to the terminal amino group of the obtained compound (6), the compound (7) can be produced. In deprotection, reagents and conditions can be selected depending on the protecting group.

[00416] Compound (9) can be produced by derivatizing the carboxylic acid peptide (8) having the N-terminus protected with P2 in an active ester, mixed acid anhydride, or the like and reacting it with the obtained compound (7). The reaction conditions, reagents, base, and inert solvent used to form a peptide bond between the peptide carboxylic acid (8) and compound (7) can be suitably selected from those described for the synthesis of compound (6). The protecting group P2 can be suitably selected from those described for the protecting group of the compound (6), and the selection can be made on the basis, for example, of the properties of the compound having the amino group to be protected. As is generally used for peptide synthesis, by sequentially repeating the reaction and deprotecting the amino acid or peptide constituting the carboxylic acid peptide (8) for elongation, compound (9) can likewise be produced.

[00417] By deprotecting the protecting group P2 to the amino group of the obtained compound (9), the compound (10) can be produced. In deprotection, reagents and conditions can be selected depending on the protecting group.

[00418] It is possible to produce the compound (2) by derivatizing the carboxylic acid (11) into an active ester, mixed acid anhydride, acid halide, or the like and reacting it with the obtained compound (10). The reaction conditions, reagents, base, and inert solvent used to form a peptide bond between the carboxylic acid (11) and compound (10) may suitably be selected from those described for the synthesis of compound (6).

[00419] Compound (9) can be produced in the same way by the following method, for example.

[00420] Compound (13) can be produced by derivatizing the peptide carboxylic acid (8) having the N-terminus protected with P2 in an active ester, mixed acid anhydride, or the like and reacting it in the presence of a base with the compound of amine (12) having the carboxy group protected with P3. The reaction conditions, reagents, base, and inert solvent used to form a peptide bond between the peptide carboxylic acid (8) and compound (12) can be suitably selected from those described for the synthesis of compound (6). The protecting group P2 for the amino group of compound (13) is not particularly limited as long as it is a generally used protecting group.

[00421] Specifically, examples of the protecting group for a hydroxyl group may include an alkoxymethyl group such as a methoxymethyl group; an arylmethyl group such as a benzyl group, a 4-methoxybenzyl group, and a triphenylmethyl group; an alkanoyl group such as an acetyl group; an aroyl group such as a benzoyl group; and a silyl group such as a tert-butyl diphenylsilyl group. Carboxy group can be protected, for example, 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 protecting group for an amino group may include: an alkyloxycarbonyl group such as a tert-butyloxycarbonyl group, a methoxycarbonyl group, and an ethoxycarbonyl group; an allyloxycarbonyl group, or an arylmethoxycarbonyl group such as a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, a paramethoxybenzyloxycarbonyl group, and a para (or ortho)nitrobenzyloxycarbonyl group; an alkanoyl group such as an acetyl group; an arylmethyl group such as a benzyl group and a triphenyl methyl group; an aroyl group such as a benzoyl group; and an aryl sulfonyl group such as a 2,4-dinitrobenzene sulfonyl group or an orthonitrobenzene sulfonyl group.

[00422] As for the protecting group P3 for a carboxy group, a protecting group generally used as a protecting group for a carboxy group in organic synthetic chemistry, in particular, peptide synthesis can be used. Specifically, it may be suitably selected from the protecting groups described above, for example, esters with an alkyl group such as a methyl group, an ethyl group, or a tert-butyl, allyl esters, and benzyl esters.

[00423] In such cases, the protecting group for an amino group and the protecting group for a carboxy group may preferably be those removed by a different method or different conditions. For example, a representative example includes a combination where P2 is a tert-butyloxycarbonyl group and P3 is a benzyl group. Protecting groups can be selected from those mentioned above depending, for example, on the properties of compounds having the amino group and the carboxy group to be protected. For removal of protecting groups, reagents and conditions can be selected depending on the protecting group.

[00424] By deprotecting the protecting group P3 to the carboxy group of the compound (13) obtained, the compound (14) can be produced. In deprotection, reagents and conditions are selected depending on the protecting group.

[00425] Compound (9) can be produced by deriving the obtained compound (14) into an active ester, mixed acid anhydride, acid halide, or the like and reacting with compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis may likewise be used, and the reaction conditions, reagents, base, and inert solvent used for the reaction may be selected appropriately from those described for the reaction. the synthesis of compound (6).

[00426] Compound (2) can be produced in the same way by the following method, for example.

[00427] By deprotecting the protecting group P2 to the amino group of compound (13), compound (15) can be produced. In deprotection, reagents and conditions can be selected depending on the protecting group.

[00428] Compound (16) can be produced by deriving the carboxylic acid derivative (11) into an active ester, mixed acid anhydride, acid halide, or the like and reacting it in the presence of a base with compound (15) obtained. The reaction conditions, reagents, base, and inert solvent used to form an amide bond between the peptide carboxylic acid (11) and compound (15) can be suitably selected from those described for the synthesis of compound (6).

[00429] By deprotecting the protecting group to the carboxy group of the compound (16) obtained, the compound (17) can be produced. Deprotection can be performed similarly to deprotection at the carboxy group to produce compound (14).

[00430] Compound (2) can be produced by deriving compound (17) into an active ester, mixed acid anhydride, acid halide, or the like and reacting it with compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis may likewise be used, and the reaction conditions, reagents, base, and inert solvent used for the reaction may be selected appropriately from those described for the reaction. the synthesis of compound (6).

3. PRODUCTION METHOD 3

[00431] The compound represented by the formula (2) of an intermediate can be produced in the same way by the following method.

[00432] In the formula, L1' corresponds to L1 having a structure in which the terminus is converted to a maleimidyl group, and P4 represents a protecting group.

[00433] Compound (19) can be produced by deriving compound (11) into an active ester, mixed acid anhydride, or the like and reacting it in the presence of a base with the carboxylic acid peptide (18) having the C-terminus protected with P4. The reaction conditions, reagents, base, and inert solvent used to form a peptide bond between the peptide carboxylic acid (18) and compound (11) can be suitably selected from those described for the synthesis of compound (6). The protecting group P4 for the carboxy group of compound (18) may suitably be selected from the protecting groups described above.

[00434] By deprotecting the protecting group to the carboxy group of the compound (19) obtained, the compound (20) can be produced. Deprotection can be performed similarly to deprotection of the carboxy group to produce compound (14).

[00435] Compound (2) can be produced by deriving the obtained compound (20) into an active ester, mixed acid anhydride, or the like and reacting it with compound (7). For the reaction, reaction reagents and conditions that are generally used for peptide synthesis may likewise be used, and the reaction conditions, reagents, base, and inert solvent used for the reaction may be selected appropriately from those described for the reaction. the synthesis of compound (6).

4. PRODUCTION METHOD 4

[00436] In the following, the method for producing the compound (10b) having n1 = 1, La = O in the production intermediate (10) described in Production Method 2 is described in detail. The compound represented by formula (10b), a salt or a solvate thereof can be produced according to the following method, for example.

[00437] In the formula, LP is as defined 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 a hydrogen atom, X and Y each represent an oligopeptide consisting of 1 to 3 amino acids, P5 and P7 each represents a protecting group for an amino group, and P6 represents a protecting group for a carboxy group.

[00438] A compound represented by formula (21) can be produced using or applying the method described in Japanese Patent Open to Public Inspection No. 2002-60351 or the literature (J. Org. Chem., Vol. 51, page 3196, 1986), and leading to removal of protecting groups or modification of functional groups, if necessary. Furthermore, it can be similarly obtained by treating an amino-terminal protected amino acid or an acidic amide of an amino-protected oligopeptide with an aldehyde or a ketone.

[00439] By reacting compound (21) with compound (22) having a hydroxyl group at a temperature ranging from under cooling temperature conditions to room temperature in an inert solvent in the presence of an acid or a base, the compound (23) can be produced.

[00440] Here, examples of the acid that can be used include an inorganic acid such as hydrofluoric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid; an organic acid such as acetic acid, citric acid, paratoluene sulfonic acid, and methanesulfonic acid; and a Lewis acid such as tetrafluoroborate, zinc chloride, tin chloride, aluminum chloride, and iron chloride. Among these, a sulfonic acid, in particular paratoluene sulfonic acid, is preferable. As for the base, any of the bases already mentioned can be properly selected and used. Preferred examples thereof include an alkali metal alkoxide such as potassium tert-butoxide, an alkali metal hydroxide such as sodium hydroxide and potassium hydroxide; an alkali metal or alkaline earth metal hydride such as sodium hydride and potassium hydride; an organometallic base represented by dialkylamino lithium such as lithium diisopropylamide; and a bisilylamine organometallic base such as lithium bis(trimethylsilyl)amide.

[00441] Examples of the solvent to be used for the reaction include an ether solvent such as tetrahydrofuran and 1,4-dioxane; and an aromatic hydrocarbon solvent such as benzene and toluene. Those solvents can be prepared as a mixture with water.

[00442] Furthermore, the protecting group for an amino group as exemplified by P5 is not particularly limited as long as it is a group generally used for protecting an amino group. Representative examples include the protecting groups for an amino group which are described in Production Method 2. However, in the present reaction, there may be cases where the protecting group for an amino group as exemplified by P5 is cleaved. In such cases, a protecting group may be reintroduced by suitably carrying out a reaction with a suitable reagent to protect an amino group as may be required.

[00443] Compound (24) can be produced by removing the protecting group P6 from compound (23). Here, representative examples of the protecting group for a carboxy group as exemplified by P6 are described in Production Method 2, and may suitably be selected therefrom. In compound (23), it is desirable in this case that the protecting group P5 for an amino group and protecting group P6 for a carboxy group are the protecting groups that can be removed by a different method or different conditions. For example, a representative example includes a combination in which P5 is a 9-fluorenylmethyloxy group and P6 is a benzyl group. Protecting groups can be selected depending on, for example, the properties of a compound having the amino group and the carboxy group to be protected. For the removal of protecting groups, reagents and conditions are selected depending on the protecting group.

[00444] Compound (26) can be produced by deriving the carboxylic acid (24) into an active ester, mixed acid anhydride, acid halide, or the like and reacting it with compound (4) or a pharmacologically acceptable salt thereof producing the compound (25) followed by removal of the protecting group P5 from the obtained compound (25). For the reaction between the compound (4) and the carboxylic acid (24) and the reaction to remove the protecting group P6, the same reagents and reaction conditions as those described for Production Method 2 can be used.

[00445] Compound (10b) can be produced by reacting compound (26) with an amino acid with a protected amino terminal group or oligopeptide (27) with a protected amino group to produce compound (9b) and removing the protecting group P7 of the obtained compound (9b). The protecting group for an amino group as represented by P7 is not particularly limited as long as it is generally used for protecting an amino group. Representative examples thereof include the protecting groups for an amino group which are described in Production Method 2. To remove the protecting group, reagents and conditions are selected depending on the protecting group. For the reaction between compound (26) and compound (27), reaction reagents and conditions that are generally used for peptide synthesis can be employed. The compound (10b) produced by the above-mentioned method can be derivatized into the compound (1) of the present invention according to the method described above.

5. PRODUCTION METHOD 5

[00446] In the following, the method for producing compound (2) having n1 = 1, n2 = 1, La = 0 in the production intermediate (2) described in Production Method 2 is described in detail. The compound represented by formula (2), a salt or a solvate thereof can be produced according to the following method, for example.

[00447] In the formula, L1', L2, LP is as defined above, Z represents an oligopeptide consisting of 1 to 3 amino acids, P8 represents a protecting group for an amino group, and P9 represents a group for a carboxy protecting group.

[00448] Compound (30) can be produced by removing the P8 protecting group from the amino acid or oligopeptide (28) with the terminal amino group protected and carboxy group to produce compound (29) and reacting the amine form obtained (29) with compound (11). The protecting group for an amino group as represented by P8 is not particularly limited as long as it is a group generally used for protecting an amino group. Representative examples include the protecting groups for an amino group which is described in Production Method 2. Furthermore, to remove the P8 protecting group, reagents and conditions can be selected depending on the protecting group. For the reaction between compound (29) and carboxylic acid (11), the same reagents and reaction conditions as those described for Production Method 2 can be used.

[00449] Production intermediate (2b) can be produced by removing the P9 protecting group from compound (30) to produce compound (31) and reacting the obtained carboxylic acid (31) with compound (26). Representative examples of the protecting group for a carboxy group as represented by P8 are described in Production Method 2. For the deprotection reaction thereof, the same reagents and reaction conditions as those described for Production Method 2 can be used. For the reaction between compound (26) and carboxylic acid (31), reaction reagents and conditions that are generally used for peptide synthesis can likewise be used. The compound (2b) produced by the above-mentioned method can be derivatized into the compound (1) of the present invention according to the method described above.

6. PRODUCTION METHOD 6

[00450] In the following, a method for producing the compound (17b) having n1 = 1, n2 = 1, La = 0 in the production intermediate (17) described in Production Method 2 is described in detail. The compound represented by formula (17b), a salt or a solvate thereof can likewise be produced according to the following method, for example.

[00451] In the formula, L1', L2, LP, X, Y, P5, P6, and P7 are as defined above.

[00452] Compound (33) can be produced by deprotecting the protecting group P5 to the amino group of compound (23) having the terminal amino group protected and carboxy group to produce compound (32) and reacting the amine derivative obtained (32) with the oligopeptide (27) having a terminal protected amino group or a protected amino group. The protecting group for an amino group as represented by P5 is not particularly limited as long as it is a group generally used for protecting an amino group. Representative examples include the protecting groups for an amino group which is described in Production Method 2. In addition, to remove the P5 protecting group, reagents and conditions can be selected depending on the protecting group. Here, however, representative examples of the protecting group for a carboxy group as represented by P6 and the protecting group for an amino group as represented by P7 include the protecting groups for a carboxy group and an amino group which are described in Production Method 2. It is desirable that in compound (33), the protecting group P6 for a carboxy group and protecting group P7 for an amino group are protecting groups that can be removed by the same method or under the same conditions. For example, a representative example includes a combination in which P6 is a benzyl ester group and P7 is a benzyloxycarbonyl group.

[00453] Compound (34) can be produced by removing the protecting group P6 for the carboxy group of the compound (33) and the protecting group P7 for the amino group of the compound (33). Compound (37) can be produced in the same way by sequentially removing the protecting group P6 for the carboxy group and the protecting group P7 for the amino group, and further, compound (34) can be produced simply by removing immediately both protecting groups P6 and P7 which can be removed by the same method or under the same conditions.

[00454] Compound (17b) can be produced by reacting the obtained compound (34) with the compound (11). For the reaction between compound (34) and compound (11), the same reagents and reaction conditions as those described for Production Method 2 can be used.

[00455] The anti-HER2 antibody-drug conjugate of the present invention, when left in the air or recrystallized or purified, may absorb moisture or may have water of absorption or transform into a hydrate, and such water-containing compounds or salts are of the likewise included in the present invention.

[00456] Compounds labeled with various radioactive or non-radioactive isotopes are likewise included in the present invention. One or more atoms constituting the antibody-drug conjugate of the present invention may contain an atomic isotope in an unnatural relationship. Examples of atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), and carbon-14 (14C). In addition, the compound of the present invention can be radioactive-labeled with a radioactive isotope such as tritium (3H), iodine-125 (125I), carbon-14 (14C), copper-64 (64Cu), zirconium-89 (89Zr ), iodine-124 (124I), fluorine-18 (18F), indium-111 (111I), carbon-11 (11C) and iodine-131 (131I). The radioactive isotope-labeled compound is useful as a therapeutic or prophylactic agent, a screening reagent such as an assay reagent, and a diagnostic agent such as an in vivo diagnostic imaging agent. Without being related to radioactivity, any isotope variant type of the antibody-drug conjugate of the present invention is within the scope of the present invention.

DRUGS

[00457] The anti-HER2 antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells, and thus, can be used as a drug, particularly as a therapeutic and/or prophylactic agent for cancer.

[00458] That is, the anti-HER2 antibody-drug conjugate of the present invention can be selectively used as a drug for chemotherapy which is a primary method of treating cancer, and as a result, can delay the development of cancer cells, inhibit their growth, and also kill cancer cells. This may allow cancer patients to be free of symptoms caused by cancer or achieve improvement in the QOL of cancer patients and achieve a therapeutic effect sustaining the lives of cancer patients. Even if the anti-HER2 antibody-drug conjugate of the present invention does not kill cancer patients, it can obtain higher QOL from cancer patients while achieving long-term survival by inhibiting or controlling cancer growth. cancer cells.

[00459] In such drug therapy, it can be used as a drug alone and moreover, it can be used as a drug in combination with an additional therapy in adjuvant therapy and can be combined with surgical operation, radiotherapy, hormone therapy, or the like . Furthermore, it can be used in the same way as a drug therapy drug in neoadjuvant therapy.

[00460] In addition to the therapeutic use as described above, an effect of suppressing the growth of small metastatic cancer cells and also killing them can likewise be expected. Particularly, when HER2 expression is confirmed in primary cancer cells, inhibition of cancer metastasis or a prophylactic effect can be expected to administer the anti-HER2 antibody-drug conjugate of the present invention. For example, an effect of inhibiting and killing cells in a body fluid in the course of metastasis or an effect, for example, of inhibiting and killing small cancer cells immediately after implantation in any tissue can be expected. Consequently, inhibition of cancer metastasis or a prophylactic effect can be expected, particularly after surgical removal of the cancer.

[00461] The anti-HER2 antibody-drug conjugate of the present invention can be expected to exert the therapeutic effect by administration as systemic therapy to patients, and additionally, by local administration to cancerous tissues.

[00462] Examples of the type of cancer to which the anti-HER2 antibody-drug conjugate of the present invention is applied may include lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, or penile cancer. The treatment object of the anti-HER2 antibody-drug conjugate of the present invention is a cancer cell expressing, in a cancer cell as a treatment subject, HER2 protein that the antibody within the antibody-drug conjugate can recognize. The term "cancer expressing the HER2 protein" when used in the present specification is a cancer containing cells having the HER2 protein on their cell surface. The HER2 protein is overexpressed in various human tumors and can be evaluated generally using a method accomplished in the art, such as immunohistochemical staining (IHC) method to assess HER2 protein overexpression, or fluorescence in situ hybridization (FISH) method. ) to assess HER2 gene amplification.

[00463] Furthermore, the anti-HER2 antibody-drug conjugate of the present invention exhibits an antitumor effect by recognizing, by its anti-HER2 antibody, the HER2 protein expressed on the surface of cancer cells and internalizing the cancer cells . Thus, the object of treatment of the anti-HER2 antibody-drug conjugate of the present invention is not limited to "cancer expressing the HER2 protein" and can likewise be, for example, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[00464] The anti-HER2 antibody-drug conjugate of the present invention may preferably be administered to a mammal, however, it is more preferably administered to a human.

[00465] Substances used in a pharmaceutical composition containing the anti-HER2 antibody-drug conjugate of the present invention can be selected appropriately and can be applied from formulation additives or the like that are generally used in the art, due to dosage or concentration administration.

[00466] The anti-HER2 antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition containing at least one pharmaceutically suitable ingredient. For example, the above pharmaceutical composition typically contains at least one pharmaceutical carrier (e.g. sterile liquid). Here, the liquid includes, for example, water and oil (petroleum oil and oil of animal origin, plant origin, or of synthetic origin. The oil may be, for example, peanut oil, soybean oil, mineral oil, or sesame oil. Water is a more typical vehicle when the above pharmaceutical composition is administered intravenously. Saline solution, an aqueous dextrose solution, and an aqueous glycerol solution can likewise be used as a liquid vehicle, in particular, for a injection solution. A suitable pharmaceutical carrier may be selected from those known in the art. If desired, the above composition may likewise contain a trace amount of a moisturizing agent, emulsifying agent, or a pH buffering agent. Examples of suitable pharmaceutical vehicle are described in "Remington's Pharmaceutical Sciences" by E. W. Martin The formulations correspond to a mode of administration.

[00467] Various delivery systems are known and they can be used to deliver the anti-HER2 antibody-drug conjugate of the present invention. Examples of routine administration may include, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routines. Administration can be by injection or bolus injection, for example. According to a specific preferred embodiment, administration of the antibody-drug conjugate is by injection. Parenteral administration is a preferred routine administration.

[00468] According to a representative embodiment, the pharmaceutical composition is prescribed, as a pharmaceutical composition suitable for intravenous administration to a human, in accordance with conventional procedures. The composition for intravenous administration is typically a solution in a sterile, isotonic aqueous buffer solution. If necessary, the drug may contain a solubilizing agent and local anesthetics to relieve pain at the injection site (eg, lignocaine). Generally, the above ingredient is supplied individually as either a lyophilized powder or an anhydrous concentrate contained in a container which is obtained by sealing in an ampoule or sachet having an amount of the active agent or as a mixture in a unit dosage form. . When the drug is to be administered by injection, it may be administered from an injection vial containing sterile pharmaceutical grade saline or water. When the drug is administered by injection, an ampoule of sterile water or saline for injection can be provided such that the aforementioned ingredients are mixed together prior to administration.

[00469] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the anti-HER2 antibody-drug conjugate of the present invention or a pharmaceutical composition containing the anti-HER2 antibody-drug conjugate and at least one anti-HER2 drug-treating agent. cancer other than conjugate. The anti-HER2 antibody-drug conjugate of the present invention can be administered with other cancer treatment agents. The anti-cancer effect can be enhanced accordingly. Other anti-cancer agents used for such purposes can be administered to a subject simultaneously with, separately from, or subsequent to the antibody-drug conjugate, and can be administered while varying the interval of administration for each. Examples of cancer treatment agents include 5-FU, pertuzumab, paclitaxel, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), paclitaxel, docetaxel, pemetrexed, sorafenib, vinblastine, vinorelbine, everolimus, tanespimycin, bevacizumab, oxaliplatin , lapatinib, ado-trastuzumab emtansine (T-DM1), or drugs described in International Publication No. WO 2003/038043, LH-RH analogues (leuprorelin, goserelin, or the like), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, or the like), and aromatase inhibitors (anastrozole, letrozole, exemestane, or the like), however, they are not limited as long as they are drug having an antitumor activity.

[00470] The pharmaceutical composition may be formulated in a lyophilization formulation or a liquid formulation as a formulation having the desired composition and required purity. When formulated as a lyophilization formulation, it may be a formulation containing suitable formulation additives that are used in the art. Likewise for a liquid formulation, it can be formulated as a liquid formulation containing various formulation additives that are used in the art.

[00471] The composition and concentration of the pharmaceutical composition may vary depending on the method of administration. However, the anti-HER2 antibody-drug conjugate contained in the pharmaceutical composition of the present invention may exhibit a pharmaceutical effect even at a small dosage when the antibody-drug conjugate has a higher affinity for an antigen, i.e., a higher affinity. high (= lowest Kd value) in terms of the dissociation constant (ie Kd value) for the antigen. Thus, to determine the dosage of the antibody-drug conjugate, the dosage can be determined due to the situation regarding the affinity between the antibody-drug conjugate and antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg/kg may be administered once or administered several times with an interval of 1 to 180 days.

[00472] once with a range of 1 to 180 days

EXAMPLES

[00473] The present invention is specifically described due to the examples shown below. However, the present invention is not limited thereto. Furthermore, it is by no means interpreted in a limited way. Furthermore, unless specifically described otherwise, the reagent, solvent, and starting material described in the specification are readily obtainable from a commercial supplier.

REFERENCE EXAMPLE 1 PREPARATION OF TRASTUZUMAB

[00474] Fourteen 440 mg/vial 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 Corp.: Stericup 0.22 μm, GVPVDF Membrane). Samples were applied to a cation exchange chromatography column (SP Sepharose HP 240 ml, XK50 column), followed by elution under a linear NaCl concentration gradient from 30 mM to 500 mM using cation exchange chromatography buffer B (25 mM Citrate Buffer, 500 mM NaCl, pH 5.0) to separate IgG monomer fractions. Monomer samples having a higher purity above 98% by size exclusion chromatography analysis were combined and concentrated with UF30K (Millipore Corp.: PELLICON XL Filter, BIOMAX 30K, PXB030A50), and the buffer was replaced with CBS buffer. (10 mM citrate/140 mM NaCl, pH 6.0). Samples replaced by CBS buffer were filtered through a 0.2 μm filter (Sartorius AG: Minisart-Plus 0.2 μm, 17823K).

REFERENCE EXAMPLE 2 PRODUCTION OF TRASTUZUMAB ENTANSINE T-DM1

[00475] The SMCC derivation of the antibody: Using Common Procedure C-2 (PBS6.5/EDTA was used as a buffering solution), Common Procedure A, and Common Procedure B (as absorption coefficient at 280 nm , 1.37 mLmg-1cm-1 was used) described in Production Method 1, buffer replacement with PBS6.5/EDTA was conducted on trastuzumab produced in Reference Example 1 to prepare a solution containing trastuzumab (160.0 mg ) dissolved in PBS6.5/EDTA (7.60 mL) in a 15 mL polypropylene tube. Subsequently, SMCC (1.84 mg) DMSO solution (0.40 ml; which corresponds to about 5.1 equivalents per antibody molecule) was added at room temperature. The reaction mixture was adjusted to have an antibody concentration of 20mg/mL, and the reaction was carried out at room temperature using a tube rotator (MTR-103, manufactured by AS ONE Corporation) for 2 hours. This reaction solution was subjected to purification according to standard Procedure D-2 (PBS6.5/EDTA was used as a buffering solution) to produce 12 ml of a solution containing 154.9 mg of the SMCC derivative antibody. Conjugation between antibody and drug ligand: Add PBS6.5/EDTA (2.56 mL) and N2-deacetyl-N2-(3-mercapto-1-oxopropyl)-maytansine (4.67 mg; DM1, Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14, page 4392) DMA (dimethylacetamide) solution (0.93 mL; which corresponds to about 5.8 equivalents per SMCC-derived antibody molecule) to the solution obtained above in a 50 mL polypropylene tube at room temperature, the reaction solution was adjusted to an antibody concentration of 10 mg/mL, and the reaction was carried out at room temperature using a tube rotator for 16.5 hours.

[00476] Purification Procedure: The above solution was subjected to purification using Common Procedure D-1 using a sodium phosphate buffer solution (10 mM, pH 6.5) containing sodium chloride (137 mM) to yield 35 mL of a solution containing the target Reference Example compound.

[00477] Physico-chemical characterization: Using the common Procedure E using UV absorbance at two wavelengths of 252 nm and 280 nm, the following characteristic values were obtained.

[00478] Antibody concentration: 4.14 mg/mL, antibody production: 144.9 mg (91%), and average number of conjugated drug molecules (n) per antibody molecule: 3.0. Example 1 Intermediate (1)

Process 1: (4-{[(1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa- tert-butyl hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)carbamate,

[00479] 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 hydrochloride -(3-dimethylaminopropyl)carbodiimide (0.216 g, 1.13 mmol) was added and stirred for 1 hour. The reaction solution was added dropwise to a solution of N,N-dimethylformamide (10 mL) charged with methanesulfonic acid salt of exatecan (0.500 g, 0.94 mmol) and triethylamine (0.157 mL, 1.13 mmol) , and stirred at room temperature for 1 day. The solvent was removed under reduced pressure and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform: methanol = 8:2 (v/v)] to yield the title compound (0.595 g, quantitative).

[00480] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.2 Hz), 1.31 (9H, s), 1.58 (1H, t, J = 7.2 Hz), 1.66 (2H, t, J = 7.2 Hz), 1.82-1.89 (2H, m), 2.12-2.21 (3H, m), 2 .39 (3H, s), 2.92 (2H, t, J = 6.5 Hz), 3.17 (2H, s), 5.16 (1H, d, J = 18.8 Hz), 5 .24 (1H, d, J = 18.8 Hz), 5.42 (2H, s), 5.59-5.55 (1H, m), 6.53 (1H, s), 6.78 ( 1H, t, J = 6.3 Hz), 7.30 (1H, s), 7.79 (1H, d, J = 11.0 Hz), 8.40 (1H, d, J = 8.6 Hz).

[00481] MS (APCI) m/z: 621 (M+H)+

Process 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

[00482] The compound (0.388 g, 0.61 mmol) obtained in Process 1 above was dissolved in dichloromethane (9 mL). Trifluoroacetic acid (9 ml) was added and stirred for 4 hours. The solvent was removed under reduced pressure, and the obtained residues were purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield trifluoroacetate of the title compound (0.343 g, quantitative).

[00483] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.2 Hz), 1.79-1.92 (4H, m), 2.10-2 .17 (2H, m), 2.27 (2H, t, J = 7.0 Hz), 2.40 (3H, s), 2.80-2.86 (2H, m), 3.15- 3.20 (2H, m), 5.15 (1H, d, J = 18.8 Hz), 5.26 (1H, d, J = 18.8 Hz), 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 = 11.0 Hz), 8.54 (1H, d, J = 8.6 Hz).

[00484] MS (APCI) m/z: 521 (M+H)+ Example 2 Antibody-Drug Conjugate (2)

Process 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

[00485] N-(tert-Butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (0.081 g, 0.19 mmol) was dissolved in dichloromethane (3 mL), N-hydroxysuccinimide (0.021 g, 0.19 mmol) and 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.036 g, 0.19 mmol) was added, then stirred for 3.5 hours. The reaction solution was added dropwise to a solution of N,N-dimethylformamide (1.5 mL) charged with the compound (0.080 g, 0.15 mmol) obtained in Process 2 of Example 1, and stirred at room temperature. for 4 hours. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 8 : 2 (v/v)] to yield the title compound (0.106 g, 73%) .

[00486] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.4 Hz), 1.36 (9H, s), 1.71 (2H, m), 1.86 (2H, t, J = 7.8 Hz), 2.15-2.19 (4H, m), 2.40 (3H, s), 2.77 (1H, dd, J = 12, 7.8.8 Hz), 3.02 (1H, dd, J = 14.1, 4.7 Hz), 3.08-3.11 (2H, m), 3.16-3.19 (2H , m), 3.54 (2H, d, J = 5.9 Hz), 3.57-3.77 (4H, m), 4.46-4.48 (1H, m), 5.16 ( 1H, d, J = 19.2 Hz), 5.25 (1H, d, J = 18.8 Hz), 5.42 (2H, s), 5.55-5.60 (1H, m), 6.53 (1H, s), 7.00 (1H, t, J = 6.3 Hz), 7.17-7.26 (5H, m), 7.31 (1H, s), 7.71 (1H, t, J = 5.7 Hz), 7.80 (1H, d, J = 11.0 Hz), 7.92 (1H, t, J = 5.7 Hz), 8.15 (1H , d, J = 8.2 Hz), 8.27 (1H, t, J = 5.5 Hz), 8.46 (1H, d, J = 8.2 Hz).

[00487] MS (APCI) m/z: 939 (M+H)+

Process 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,

[00488] The compound (1.97 g, 2.10 mmol) obtained in Process 1 above was dissolved in dichloromethane (7 mL). After adding trifluoroacetic acid (7 ml), it was stirred for 1 hour. The solvent was removed under reduced pressure, and the residues were charged with toluene by azeotropic distillation. The residues obtained were purified by column chromatography on silica gel [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield trifluoroacetate of the title compound (1.97 g , 99%).

[00489] 1 H-NMR (400 MHz, DMSO-d6) δ : 0.87 (3H, t, J = 7.4 Hz), 1.71-1.73 (2H, m), 1.82-1 .90 (2H, m), 2.12-2.20 (4H, m), 2.40 (3H, s), 2.75 (1H, dd, J = 13.7, 9.4 Hz), 3.03-3.09 (3H, m), 3.18-3.19 (2H, m), 3.58-3.60 (2H, m), 3.64 (1H, d, J = 5 .9 Hz), 3.69 (1H, d, J = 5.9 Hz), 3.72 (1H, d, J = 5.5 Hz), 3.87 (1H, dd, J = 16.8 , 5.9 Hz), 4.50-4.56 (1H, m), 5.16 (1H, d, J = 19.2 Hz), 5.25 (1H, d, J = 18.8 Hz ), 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).

[00490] MS (APCI) m/z: 839 (M+H)+

Process 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,

[00491] In a N,N-dimethylformamide solution (1.2 mL) of the compound (337 mg, 0.353 mmol) obtained in Process 2 above, triethylamine (44.3 mL, 0.318 mmol) and N-succinimidyl hexanoate 6 - maleimide (119.7 mg, 0.388 mmol) were added and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 5 : 1 (v/v)] to yield the title compound as a pale yellow solid (278 .0 mg, 76%).

[00492] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.3 Hz), 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, 9.8 Hz), 2.98-3.21 (5H, m), 3.55-3.77 (8H, m ), 4.41-4.48 (1H, m), 5.15 (1H, d, J = 18.9 Hz), 5.24 (1H, d, J = 18.9 Hz), 5.40 (1H, d, J = 17.1 Hz), 5.44 (1H, d, J = 17.1 Hz), 5.54-5.60 (1H, m), 6.53 (1H, s) , 6.99 (2H, s), 7.207.27 (5H, m), 7.30 (1H, s), 7.70 (1H, t, J = 5.5 Hz), 7.80 (1H, s). d, J = 11.0 Hz), 8.03 (1H, t, J = 5.8 Hz), 8.08 (1H, t, J = 5.5 Hz), 8.14 (1H, d, J = 7.9 Hz), 8.25 (1H, t, J = 6.1 Hz), 8.46 (1H, d, J = 8.5 Hz).

[00493] MS (APCI) m/z: 1032 (M+H)+

Process 4: Antibody-Drug Conjugate (2)

[00494] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used) described in Production Method 1. The solution (3.0 mL) was placed in a 15 mL polypropylene tube and charged with a 10 mM aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Tokyo Chemical Industry Co., Ltd.) (0.0934 mL; 4.6 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque , Inc.; 0.150 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00495] Conjugation between antibody and drug ligand: 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 the Process 3 was added to this and incubated to conjugate the drug binder to the antibody at 22°C for 40 minutes. Then, an aqueous solution (0.0374 mL; 18.4 equivalents per antibody molecule) of N-acetylcysteine (NAC, Sigma-Aldrich Co. LLC) was added thereto and incubated at 22°C to terminate the reaction. drug binder for another 20 minutes.

[00496] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS6.0 was used as the buffer solution) described in Production Method 1 to produce 6 mL of a solution containing the antibody- titrated drug. After that, the solution was concentrated by Common procedure A.

[00497] Physical-Chemical Characterization: Using the Common E procedure described in Production Method 1, the following characteristic values were obtained.

[00498] Antibody concentration: 3.21 mg/mL, antibody yield: 22.5 mg (75%), and average number of conjugated drug molecules (n) per antibody molecule: 2.6. Example 3 Antibody-Drug Conjugate (3)

Process 1: Antibody-Drug Conjugate (3)

[00499] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.487 mLmg-1cm-1 was used). The solution (1.25 mL) was placed in a 1.5 mL polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.039 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of hydrogen phosphate. of 1M dipotassium (0.0625 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00500] Conjugation between antibody and drug ligand: After adding DMSO (0.072 mL) and a DMSO solution containing 10 mM of the Process 3 compound of Example 2 (0.078 mL; 9.2 equivalents per antibody molecule) to the above solution at room temperature, was shaken using a tube rotator to conjugate drug ligand to antibody at room temperature for 40 minutes. Then, an aqueous solution (0.0155 ml; 18.4 equivalents per antibody molecule) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug-ligand reaction for another 20 minutes.

[00501] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) described in Production Method 1 to produce 6 mL of a solution containing the compound of interest. The solution was also concentrated by Common procedure A. After that, using Common procedure E, the following characteristic values were obtained.

[00502] Antibody concentration: 9.85 mg/mL, antibody yield: 6.9 mg (55%), and average number of conjugated drug molecules (n) per antibody molecule: 7.3. Example 4 Antibody-Drug Conjugate (4)

Process 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,

[00503] The compound (80 mg, 0.084 mmol) of Example 1 was reacted in the same manner as Process 3 of Example 2 using N-succinimidyl 3-maleimide propioate (24.6 mg, 0.0924 mmol) instead of N-succinimidyl 6-maleimide hexanoate to yield the title compound as a pale yellow solid (60.0 mg, 73%).

[00504] 1H-NMR (400 MHz, DMSO-d6) δ: 0.89 (3H, t, J = 7.3 Hz), 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, 9.8 Hz), 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 = 19.0 Hz), 5.27 (1H, d, J = 19.0 Hz), 5.43 (1H, d, J = 17.6 Hz), 5.47 (1H, d, J = 17.6 Hz), 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 (1H, s), 7.73 (1H, t, J = 5.6 Hz), 7.83 (1H, d, J = 10.7 Hz), 8.08 (1H, t, J = 5.6 Hz), 8.15 (1H, d, J = 7.8 Hz), 8.30 (2H, dt, J = 18.7, 5.7 Hz), 8.49 (1H, d, J = 8.8 Hz).

[00505] MS (APCI) m/z: 990 (M+H)+

Process 2: Antibody-Drug Conjugate (4)

[00506] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (1 mL) was placed in a 1.5 mL polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.0155 mL; 2.3 equivalents per antibody molecule) and an aqueous solution of hydrogen phosphate. of 1M dipotassium (0.050 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00507] Conjugation between antibody and drug ligand: After adding DMSO (0.072 mL) and a DMSO solution containing 10 mM of the Process 3 compound of Example 2 (0.031 mL; 4.6 equivalents per antibody molecule) to the above solution at room temperature, was shaken using a tube rotator to conjugate drug ligand to antibody at room temperature for 40 minutes. Then, an aqueous solution (0.0078 mL; 9.2 equivalents per antibody molecule) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug-ligand reaction for another 20 minutes.

[00508] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) to yield 6 mL of a solution containing the compound of interest. Using the Common E procedure, the following characteristic values were obtained.

[00509] Antibody concentration: 1.32 mg/mL, antibody yield: 7.9 mg (79%), and average number of conjugated drug molecules (n) per antibody molecule: 3.1. Example 5 Antibody-Drug Conjugate (5)

Process 1: Antibody-Drug Conjugate (5)

[00510] The amount of the 10 mM TCEP aqueous solution was adjusted such that the molar ratio of TCEP to antibody in antibody reduction was 4.6. And the amount of the 10 mM drug-binder solution added was adjusted, such that the molar ratio of Process 1 compound of Example 4 to antibody to drug-linker conjugation was 9.2. Then, the amount of 100 mM aqueous NAC solution added was adjusted such that the molar ratio of NAC to antibody at the termination of the reaction was 18.4. By the same procedures as Process 2 of Example 4, 6 ml of a solution containing the titrated antibody-drug conjugate was obtained, and the following characteristic values were obtained.

[00511] Antibody concentration: 1.23 mg/mL, antibody yield: 7.4 mg (74%), and average number of conjugated drug molecules (n) per antibody molecule: 6.1. Example 6: Antibody-Drug Conjugate (6)

Process 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 ,

[00512] The compound (100 mg, 0.119 mmol) obtained from Example 2 in Process 2 was reacted in the same manner as Process 3 of Example 2 using diisopropylethylamine (20.8 µL, 0.119 mmol) in place of triethylamine and N -succinimidyl 3-(2-(2-(3-maleimidepropanamide)ethoxy)ethoxy)propanoate (50.7 mg, 0.119 mmol) in place of N-succinimidyl 6-maleimide hexanoate to yield the title compound as a pale yellow solid (66.5 mg, 48%).

[00513] 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 = 7.2 Hz), 2.33-2.36 (2H, m), 2.38 (3H, s), 2.76 (1H, dd, J = 13.7, 9.8 Hz), 2.96-3.18 (9H, m), 3.42-3.44 ( 4H, m), 3.53-3.76 (10H, m), 4.43 (1H, td, J = 8.6, 4.7 Hz), 5.14 (1H, d, J = 18, 8 Hz), 5.23 (1H, d, J = 18.8 Hz), 5.38 (1H, d, J = 17.2 Hz), 5.42 (1H, d, J = 17.2 Hz ), 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 = 5.5 Hz), 7.78 (1H, d, J = 11.3 Hz), 7.98-8.03 (2H, m), 8.11 (1H, d, J = 7.8 Hz), 8.16 (1H, t, J = 5.7 Hz), 8.23 (1H , t, J = 5.9 Hz), 8.44 (1H, d, J = 9.0 Hz).

[00514] MS (APCI) m/z: 1149 (M+H)+

Process 2: Antibody-Drug Conjugate (6)

[00515] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (1.25 mL) was placed in a 1.5 mL polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.019 mL; 2.3 equivalents per antibody molecule) and an aqueous solution of hydrogen phosphate. of 1M dipotassium (0.0625 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00516] Conjugation between antibody and drug ligand: After adding DMSO (Sigma-Aldrich Co. LLC; 0.109 mL) and a DMSO solution containing 10 mM of the compound of Process 1 (0.039 mL; 4.6 equivalents per molecule of antibody) to the above solution at room temperature, was shaken using a tube rotator to conjugate the drug binder to the antibody at room temperature for 40 minutes. Then, an aqueous solution (0.008 mL) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00517] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) described in Production Method 1 to produce 6 mL of a solution containing the compound of interest.

[00518] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00519] Antibody concentration: 1.76 mg/mL, antibody yield: 10.6 mg (85%), and average number of conjugated drug molecules (n) per antibody molecule: 3.6. Example 7 Antibody-Drug Conjugate (7)

Process 1: Antibody-Drug Conjugate (7)

[00520] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (1.25 mL) was placed in a 1.5 mL polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.039 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of hydrogen phosphate. of 1M dipotassium (0.0625 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00521] Conjugation between antibody and drug ligand: After adding DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound of Process 1 of Example 6 (0.078 mL; 9.2 equivalents per antibody molecule) to the above solution at room temperature, was shaken using a tube rotator to conjugate drug ligand to antibody at room temperature for 40 minutes. Then, an aqueous solution (0.0155 mL) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00522] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) to yield 6 mL of a solution containing the compound of interest.

[00523] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00524] Antibody concentration: 1.93 mg/mL, antibody yield: 11.6 mg (93%), and average number of conjugated drug molecules (n) per antibody molecule: 6.9. Example 8 Antibody-Drug Conjugate (8)

Process 1: Antibody-Drug Conjugate (8)

[00525] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (1.25 mL) was placed in a 1.5 mL polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.039 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of hydrogen phosphate. of 1M dipotassium (0.0625 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00526] Conjugation between antibody and drug ligand: After adding DMSO (0.072 mL) and a DMSO solution containing 10 mM of the compound of Process 1 of Example 6 (0.078 mL; 9.2 equivalents per antibody molecule) to the above solution at room temperature, was shaken using a tube rotator to conjugate drug ligand to antibody at room temperature for 40 minutes. Then, an aqueous solution (0.0155 mL) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00527] Purification: The above solution was subjected to purification using the Common D-1 procedure (ABS was used as a buffering solution) to yield 5.7 mL of a solution containing the compound of interest.

[00528] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00529] Antibody concentration: 1.50 mg/mL, antibody yield: 8.55 mg (86%), and average number of conjugated drug molecules (n) per antibody molecule: 6.2. Example 9 Antibody-Drug Conjugate (9)

Process 1: N-[19-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxo-16-azanonedecan- 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

[00530] The compound (90 mg, 0.107 mmol) obtained from Example 2 in Process 2 was reacted in the same manner as Process 3 of Example 2 using diisopropylethylamine (18.7 µL, 0.107 mmol) in place of triethylamine and N -succinimidyl 1-maleimide-3-oxo-7, 10, 13, 16-tetraoxa-4-azanonecan-19-oate (55.1 mg, 0.107 mmol) in place of N-succinimidyl 6-maleimidehexanoate to yield the title compound as a pale yellow solid (50 mg, 37%).

[00531] 1H-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 = 18.8 Hz), 5.23 (1H, d, J = 18.8 Hz), 5.38 (1H, d, J = 17.0 Hz), 5.42 (1H, d, J = 17.0 Hz), 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, 5 Hz), 7.78 (1H, d, J = 10.9 Hz), 7.98-8.03 (2H, m), 8.10 (1H, d, J = 7.8 Hz), 8 .16 (1H, t, J = 5.7 Hz), 8.23 (1H, t, J = 5.7 Hz), 8.44 (1H, d, J = 8.6 Hz).

[00532] MS (APCI) m/z: 1237 (M+H)+

Process 2: Antibody-Drug Conjugate (9)

[00533] Using trastuzumab Reference 1 and the obtained compound 1 titrated antibody-drug was obtained Process 2 of Example 6.

[00534] Antibody concentration: 1.75 mg/mL, antibody yield: 10.5 mg (84%), and average number of conjugated drug molecules (n) per antibody molecule: 3.4. Example 10 Antibody-Drug Conjugate (10)

Process 1: Antibody-Drug Conjugate (10)

[00535] Using the trastuzumab produced in Reference Example 1 and the compound obtained 1 from Reference Example 1 and the compound obtained 1 from Example 9 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of the Example 7.

[00536] Antibody concentration: 1.79 mg/mL, antibody yield: 10.7 mg (86%), and average number of conjugated drug molecules (n) per antibody molecule: 6.0. Example 11 Intermediate (11)

Process 1: [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 tert-butyl,

[00537] Methanesulfonic acid salt of exatecan (3.10 g, 5.47 mol) was reacted in the same manner as Process 1 of Example 1 using {2-[(tert-butoxycarbonyl)amino]ethoxy}acetic acid (J Med. Chem., 1992, vol. 35, pp. 292; 1.55 g, 6.01 mmol) in place of 4-(tert-butoxycarbonylamino)butanoic acid to yield the title compound as a pale yellow solid (2 .56 g, 73%).

[00538] 1H-NMR (400 MHz, DMSO-d6) δ : 0.87 (3H, t, J = 7.3 Hz), 1.26 (9H, s), 1.81-1.91 (2H , m), 2.13-2.22 (2H, m), 2.40 (3H, s), 3.083.26 (4H, m), 3.43-3.53 (2H, m), 4, 00 (1H, d, J = 15.1 Hz), 4.05 (1H, d, J = 15.1 Hz), 5.14 (1H, d, J = 18.7 Hz), 5.22 ( 1H, d, J = 18.7 Hz), 5.40 (1H, d, J = 16.6 Hz), 5.44 (1H, d, J = 16.6 Hz), 5.59-5, 66 (1H, m), 6.53 (1H, s), 6.86 (1H, t, J = 5.4 Hz), 7.31 (1H, s), 7.79 (1H, d, J = 10.9 Hz), 8.49 (1H, d, J = 9.1 Hz).

[00539] MS (APCI) m/z: 637 (M+H)+

Process 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

[00540] The compound (1.50 g, 2.36 mol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 1 to yield the trifluoroacetic acid salt of the title compound as a pale yellow solid (1, 50 g, quantitative).

[00541] 1H-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 = 5.1 Hz), 3.15-3.23 (2H, m), 3.71 ( 2H, t, J = 5.1 Hz), 4.10 (2H, s), 5.19 (1H, d, J = 18.7 Hz), 5.24 (1H, d, J = 18.7 Hz), 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 = 9.1 Hz).

[00542] MS (APCI) m/z: 537 (M+H)+ Example 12 Antibody-Drug Conjugate (12)

Process 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

[00543] The compound (554 mg, 0.85 mmol) from Process 2 of Example 11 was reacted in the same manner as Process 1 of Example 2 to yield the title compound (775 mg, 95%).

[00544] 1H-NMR (400 MHz, DMSO-d6) δ: 0.85 (3H, t, J = 7.3 Hz), 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, 9.8 Hz), 2.95 ( 1H, dd, J = 13.4, 4.3 Hz), 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, 5.5 Hz), 4.03 (2H, s), 4.39-4.47 (1H, m), 5.17 (1H, d, J = 18.9 Hz), 5.25 (1H, d, J = 18.9 Hz), 5.41 (1H, d, J = 16.8 Hz), 5.45 (1H , d, J = 16.8 Hz), 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 = 5.2 Hz), 8.13 (1H, d, J = 7.9 Hz), 8.27 (1H, t, J = 5.8 Hz), 8.49 (1H, d, J = 8.5 Hz).

[00545] MS (APCI) m/z: 955 (M+H)+

Process 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

[00546] The compound (630 mg, 0.659 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 2 to produce the trifluoroacetic acid salt of the title compound (588 mg, 92%).

[00547] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.3 Hz), 1.79-1.90 (2H, m), 2.13-2 .22 (2H, m), 2.39 (3H, s), 2.71 (1H, dd, J = 13.4, 10.1 Hz), 2.99 (1H, dd, J = 13.4 , 4.3 Hz), 3.09-3.23 (1H, m), 3.243.32 (3H, m), 3.41-3.71 (7H, m), 3.86 (1H, dd, J = 16.8, 5.8 Hz), 4.04 (2H, s), 4.52 (1H, td, J = 9.0, 4.1 Hz), 5.17 (1H, d, J = 18.9 Hz), 5.25 (1H, d, J = 18.9 Hz), 5.41 (1H, d, J = 16.5 Hz), 5.45 (1H, d, J = 16 .5 Hz), 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 = 11.0 Hz), 7.87-8.01 (4H, m), 8.29-8.36 (2H, m), 8.468.55 (2H, m).

[00548] MS (APCI) m/z: 855 (M+H)+

Process 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

[00549] The compound (240mg, 0.247mmol) obtained in Process 2 above was reacted in the same manner as Process 3 of Example 2 to yield the title compound (162mg, 62%).

[00550] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.6 Hz), 1.13-1.22 (2H, m), 1.40-1 .51 (4H, m), 1.78-1.90 (2H, m), 2.09 (2H, t, J = 7.6 Hz), 2.14-2.21 (2H, m), 2.39 (3H, s), 2.74 (1H, dd, J = 13.6, 9.7 Hz), 2.96 (1H, dd, J = 13.6, 4.5 Hz), 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, 5.7 Hz), 4.03 (2H, s), 4.42 (1H, td, J = 8.6, 4.2 Hz), 5.17 (1H, d, J = 18.7 Hz), 5.25 (1H, d, J = 18.7 Hz), 5.40 (1H, d, J = 17.2 Hz), 5. 44 (1H, d, J = 17.2 Hz), 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 = 5.7 Hz), 8.03- 8.11 (2H, m), 8.22 (1H, t, J = 5.7 Hz), 8.47 (1H, d, J = 9.1 Hz).

[00551] MS (APCI) m/z: 1048 (M+H)+

Process 4: Antibody-Drug Conjugate (12)

[00552] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 3, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6. The solution was also concentrated by Common procedure A. After that, using the Common E procedure, the following characteristic values were obtained.

[00553] Antibody concentration: 10.77 mg/mL, antibody yield: 7.5 mg (60%), and average number of conjugated drug molecules (n) per antibody molecule: 3.7. Example 13 Antibody-Drug Conjugate (13)

Process 1: Antibody-Drug Conjugate (13)

[00554] Using the trastuzumab produced in Reference Example 1 and the compound obtained 3 from Example 12 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7. The solution was also concentrated by the procedure Common A. After that, using the Common E procedure, the following characteristic values were obtained.

[00555] Antibody concentration: 10.69 mg/mL, antibody yield: 7.5 mg (60%), and average number of conjugated drug molecules (n) per antibody molecule: 6.9. Example 14 Intermediate (14)

Process 1: (3-{[(1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa- tert-butyl hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-3-oxopropyl)carbamate,

[00556] Methanesulfonic acid salt of exatecan (500 mg, 0.941 mmol) was reacted in the same manner as Process 1 of Example 1 using N-(tert-butoxycarbonyl)-β-alanine in place of 4-(tert-butoxycarbonylamino acid) )butanoic acid to give the title compound as a yellowish brown solid (616 mg, quantitative).

[00557] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.2 Hz), 1.29 (9H, s), 1.86 (2H, dt, J = 15.1, 7.3 Hz), 2.04-2.22 (2H, m), 2.31 (2H, t, J = 6.8 Hz), 2.40 (3H, s), 3 .10-3.26 (4H, m), 5.15 (1H, d, J = 18.8 Hz), 5.26 (1H, d, J = 19.2 Hz), 5.42 (2H, dd, J = 18.8, 16.4 Hz), 5.57 (1H, dt, J = 8.5, 4.2 Hz), 6.53 (1H, s), 6.78 (1H, t , J = 5.5 Hz), 7.30 (1H, s), 7.80 (1H, d, J = 11.0 Hz), 8.46 (1H, d, J = 8.6 Hz).

[00558] MS (ESI) m/z: 607 (M+H)+

Process 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,

[00559] The compound obtained in Process 1 above was reacted in the same manner as Process 2 of Example 1 to yield the trifluoroacetic acid salt of the title compound as a yellow solid (499 mg, 86%).

[00560] 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, 7.2 Hz), 2.06-2.27 (1H, m), 2.41 (3H, s), 2.46-2.57 (2H, m), 3 .08 (2H, t, J = 6.8 Hz), 3.14-3.24 (2H, m), 5.22 (1H, d, J = 18.8 Hz), 5.29 (1H, d, J = 18.8 Hz), 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 = 11.0 Hz), 8.67 (1H, d, J = 8.6 Hz).

[00561] MS (ESI) m/z: 507 (M+H)+ Example 15 Antibody-Drug Conjugate (15)

Process 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,

[00562] The compound (484 mg, 0.780 mmol) obtained in Process 2 of Example 14 was reacted in the same manner as Process 1 of Example 2 to yield the title compound as a pale yellow solid (626 mg, 87%).

[00563] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.4 Hz), 1.27-1.42 (9H, m), 1.77-1 .93 (2H, m), 2.06-2.22 (2H, m), 2.36 (2H, t, J = 7.2 Hz), 2.40 (3H, d, J = 1.6 Hz), 2.44-2.54 (2H, m), 2.76 (1H, dd, J = 14.5, 10.2 Hz), 3.02 (1H, dd, J = 13.9, 4.5 Hz), 3.12-3.22 (2H, m), 3.52 (6H, d, J = 6.3 Hz), 4.42-4.54 (1H, m), 5. 19 (1H, d, J = 19.2 Hz), 5.26 (1H, d, J = 18.4 Hz), 5.42 (1H, dd, J = 18.4, 16.4 Hz), 5.57 (1H, dt, J = 8.7, 4.4 Hz), 6.53 (1H, s), 6.98 (1H, t, J = 5.9 Hz), 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 = 7.8 Hz), 8.27 (1H, t, J = 5.1 Hz), 8.52 (1H, d, J = 9.0 Hz).

Process 2: Glycylglycyl-L-phenylalanylglycyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 trifluoroacetate, 13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide

[00564] The compound (624 mg, 0.675 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 2 to yield the title compound as a yellow solid (626 mg, 92%).

[00565] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.4 Hz), 1.86 (2H, tt, J = 14.5, 7.2 Hz ), 2.07-2.22 (2H, m), 2.36 (2H, t, J = 7.2 Hz), 2.40 (3H, s), 2.44-2.54 (2H, m), 2.75 (1H, dd, J = 13.7, 9.8 Hz), 3.04 (1H, dd, J = 13.7, 4.3 Hz), 3.12-3.22 (2H, m), 3.58 (2H, d, J = 4.7 Hz), 3.69 (3H, td, J = 11.2, 5.7 Hz), 3.87 (1H, dd, J = 17.0, 5.7 Hz), 4.54 (1H, m, J = 17.8, 4.5 Hz), 5.19 (1H, d, J = 19.2 Hz), 5, 26 (1H, d, J = 18.8 Hz), 5.43 (2H, s), 5.51-5.60 (1H, m), 6.55 (1H, s), 7.14-7 .29 (5H, m), 7.32 (1H, s), 7.81 (1H, d, J = 10.9 Hz), 7.88 (1H, t, J = 5.7 Hz), 7 .97 (3H, brs), 8.29-8.38 (2H, m), 8.50 (1H, t, J = 5.7 Hz), 8.55 (1H, d, J = 8.6 Hz).

[00566] MS (ESI) m/z: 825 (M+H)+

Process 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,

[00567] The compound (60.0mg, 0.0646mmol) obtained in Process 2 above was reacted in the same manner as Process 3 of Example 2 to yield the title compound as a solid (14.0mg, 21%) .

[00568] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.2 Hz), 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 = 7.4 Hz), 2.30-2.42 (4H, m), 2.40 (3H, s), 2.78 (1H, dd, J = 14.1, 9.4 Hz), 3.02 (1H, dd, J = 14.7, 4.9 Hz), 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 = 19.6 Hz), 5.26 (1H, d, J = 19.2 Hz), 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 = 5.3 Hz), 8.06 (1H, t, J = 5.7 Hz), 8.14 (1H, d, J = 8.2 Hz), 8.25 (1H, t, J = 5.7 Hz), 8.53 (1H, d, J = 8.6 Hz) .

[00569] MS (ESI) m/z: 1018 (M+H)+

Process 4: Antibody-Drug Conjugate (15)

[00570] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used). The solution (1.0 mL) was collected in a 2 mL tube and loaded with a 10 mM aqueous solution of TCEP (0.0155 mL; 2.3 equivalents per antibody molecule) and an aqueous solution of dipotassium hydrogen phosphate. at 1M (0.050 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00571] Conjugation between antibody and drug ligand: After incubating the solution at 22°C for 10 minutes, a solution of DMSO (0.0311 mL; 4.6 equivalents per molecule of antibody) containing 10 mM of the compound obtained in Process 3 was added to this and incubated to conjugate drug binder to antibody at 22°C for 40 minutes. Then, an aqueous solution (0.00622 ml; 9.2 equivalents per antibody molecule) of 100 mM NAC was added thereto and incubated at 22°C to terminate the drug-ligand reaction for another 20 minutes.

[00572] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS6.0 was used as the buffer solution) to produce 6 mL of a solution containing the titrated antibody-drug conjugate.

[00573] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00574] Antibody concentration: 1.18 mg/mL, antibody yield: 7.08 mg (71%), and average number of conjugated drug molecules (n) per antibody molecule: 2.0. Example 16 Antibody-Drug Conjugate (16)

Process 1: Antibody-Drug Conjugate (16)

[00575] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used). The solution (1.0 mL) was collected in a 2 mL tube and loaded with a 10 mM aqueous solution of TCEP (0.0311 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of dipotassium hydrogen phosphate. at 1M (0.050 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00576] Conjugation between antibody and drug ligand: After incubating the solution at 22°C for 10 minutes, a DMSO solution (0.0622 mL; 9.2 equivalents per antibody molecule) containing 10 mM of the obtained compound 3 of Example 15 in the Process was added thereto and incubated to conjugate the drug binder to the antibody at 22°C for 40 minutes. Then, an aqueous solution (0.0124 ml; 18.4 equivalents per antibody molecule) of 100 mM NAC was added thereto and incubated at 22°C to terminate the drug-ligand reaction for another 20 minutes.

[00577] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS6.0 was used as the buffer solution) to produce 6 mL of a solution containing the titrated antibody-drug conjugate.

[00578] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00579] Antibody concentration: 1.03 mg/mL, antibody yield: 6.18 mg (62%), and average number of conjugated drug molecules (n) per antibody molecule: 3.8. Example 17 Antibody-Drug Conjugate (17)

Process 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]pyran[3',4 ':6,7]indolizino[1, 2-b]quinolin-1-yl]-β-alaninamide,

[00580] The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15 was reacted in the same manner as Process 3 of Example 2 using N-succinimidyl 3-maleimide propionate in place of N-hexanoate -succinimidyl 6-maleimidate to yield the title compound as a pale yellow solid (36.0 mg, 57%).

[00581] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.4 Hz), 1.85 (2H, dt, J = 14.4, 7.5 Hz ), 2.05-2.22 (2H, m), 2.40 (3H, s), 2.302.44 (5H, m), 2.73-2.84 (1H, m), 3.02 ( 1H, dd, J = 13.9, 4.5 Hz), 3.17 (3H, d, J = 5.1 Hz), 3.26-3.40 (2H, m), 3.41-3 .81 (6H, m), 4.40-4.51 (1H, m), 5.19 (1H, d, J = 19.2 Hz), 5.26 (1H, d, J = 18.8 Hz), 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 = 10.2 Hz), 8.03 (1H, t, J = 5.5 Hz), 8.12 (1H, d, J = 8.2 Hz), 8.20-8.31 (2H, m), 8.52 (1H, d, J = 8.6 Hz).

[00582] MS (ESI) m/z: 976 (M+H)+

Process 2: Antibody-Drug Conjugate (17)

[00583] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 1 above, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6.

[00584] Antibody concentration: 1.74 mg/mL, antibody yield: 10.4 mg (83%), and average number of conjugated drug molecules (n) per antibody molecule: 3.7. Example 18 Antibody-Drug Conjugate (18)

Process 1: Antibody-Drug Conjugate (18)

[00585] Using the trastuzumab produced in Reference Example 1 and the compound obtained 1 from Example 17 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00586] Antibody concentration: 1.98 mg/mL, antibody yield: 11.9 mg (95%), and average number of conjugated drug molecules (n) per antibody molecule: 6.6. Example 19 Antibody-Drug Conjugate (19)

Process 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-hexa -hydro-1H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-β-alaninamide,

[00587] The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15 was reacted in the same manner as Process 3 of Example 2 using N-succinimidyl 3-(2-(2-(3- maleinimidepropanamide)ethoxy)ethoxy)propanoate in place of N-succinimidyl 6-maleimidate hexanoate to yield the title compound as a solid (23.0 mg, 31%).

[00588] 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 (1H, m), 5.19 (1H, d, J = 18.4 Hz), 5.26 (1H, m, J = 18.4 Hz), 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 = 8.6 Hz).

[00589] MS (ESI) m/z: 1135 (M+H)+

Process 2: Antibody-Drug Conjugate (19)

[00590] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 1 above, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6.

[00591] Antibody concentration: 1.60 mg/mL, antibody yield: 9.6 mg (77%), and average number of conjugated drug molecules (n) per antibody molecule: 1.9. Example 20 Antibody-Drug Conjugate (20)

Process 1: Antibody-Drug Conjugate (20)

[00592] Using the trastuzumab produced in Reference Example 1 and the compound obtained 1 from Example 19 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00593] Antibody concentration: 1.69 mg/mL, antibody yield: 10.1 mg (81%), and average number of conjugated drug molecules (n) per antibody molecule: 3.0. Example 21 Antibody-Drug Conjugate (21)

Process 1: N-[19-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonedecan- 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,

[00594] The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15 was reacted in the same manner as Process 3 of Example 2 using N-succinimidyl 1-maleinimide-3-oxo-7.10 , 13,16-tetraoxa-4-azanonedecanoate in place of N-succinimidyl 6-maleimide hexanoate to yield the title compound as a solid (23.0 mg, 29%).

[00595] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.0 Hz), 1.85 (2H, tt, J = 14.6, 7.1 Hz ), 2.06-2.22 (2H, m), 2.40 (3H, s), 2.282.43 (6H, m), 2.78 (1H, dd, J = 13.7, 9.4 Hz), 3.02 (1H, dd, J = 14.1, 3.9 Hz), 3.09-3.22 (4H, m), 3.27-3.41 (4H, m), 3 .47 (12H, d, J = 8.6 Hz), 3.53-3.81 (10H, m), 4.41-4.51 (1H, m), 5.19 (1H, d, J = 19.2 Hz), 5.26 (1H, d, J = 18.8 Hz), 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 = 6.6 Hz), 8.11-8.21 (2H, m), 8.27 (1H, t, J = 5.9 Hz), 8.54 (1H, d , J = 8.6 Hz).

[00596] MS (ESI) m/z: 1224 (M+H)+

Process 2: Antibody-Drug Conjugate (21)

[00597] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 1, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6.

[00598] Antibody concentration: 1.77 mg/mL, antibody yield: 10.6 mg (85%), and average number of conjugated drug molecules (n) per antibody molecule: 3.2. Example 22 Antibody-Drug Conjugate (22)

Process 1: Antibody-Drug Conjugate (22)

[00599] Using the trastuzumab produced in Reference Example 1 and the compound obtained 1 from Example 21 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00600] Antibody concentration: 1.89 mg/mL, antibody yield: 11.3 mg (90%), and average number of conjugated drug molecules (n) per antibody molecule: 6.2. Example 23 Intermediate (23)

Process 1: (6-{[(1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa- tert-butyl hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-6-oxohexyl)carbamate

[00601] Methanesulfonic acid salt of exatecan (0.500 g, 0.882 mmol) was reacted in the same manner as Process 1 of Example 1 using 6-(tert-butoxycarbonylamino)hexanoic acid in place of 4-(tert-butoxycarbonylamino)butanoic acid to produce the title compound (0.620 g, quantitative).

[00602] 1H-NMR (DMSO-d6) δ: 0.83 (3H, t, J = 7.8 Hz), 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 (2H, m), 5.10 (1H, d, J = 19.4 Hz), 5.16 (1H, d, J = 19.4 Hz), 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 = 10.9 Hz), 8.39 (1H, d, J = 9.0 Hz).

Process 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

[00603] The compound (0.397 g, 0.611 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 1 to produce the trifluoroacetic acid salt of the title compound (0.342 g, 84%).

[00604] 1H-NMR (DMSO-d6) δ: 0.88 (3H, t, J = 7.2 Hz), 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 = 7.4 Hz), 2.40 (3H, s), 2.81 (2H, t, J = 7.4 Hz), 3.10-3.25 (2H, m), 3.33 (2H, brs), 5.18 (1H, d, J = 19.8 Hz), 5.22 (1H, d, J = 19.8 Hz), 5.41 (2H, d, J = 16.6 Hz), 5.45 (2H, d, J = 16.6 Hz), 5.53-5.60 (1H, m), 6.55 (1H, s), 7.32 (1H, s), 7.80 (1H, d, J = 10 .9 Hz), 8.49 (1H, d, J = 9.2 Hz). Example 24 Antibody-Drug Conjugate (24)

Process 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

[00605] The compound (0.170 g, 0.516 mmol) obtained 2 of Example 23 was reacted in the same manner as Process 1 of Example 2 to produce the title compound in Process (0.225 g, 91%).

[00606] 1H-NMR (DMSO-d6) δ: 0.88 (3H, t, J = 7.4 Hz), 1.43-1.70 (6H, m), 1.87 (2H, td, J = 15.0, 7.4 Hz), 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 = 19.2 Hz), 5.25 (1H, d, J = 19.2 Hz), 5.45 (2H, s), 5.54-5.61 (1H, m), 6.55 (1H, s), 7.02 (1H, t, J = 6, 1 Hz), 7.11-7.28 (5H, m), 7.33 (1H, s), 7.63-7.69 (1H, m), 7.82 (1H, d, J = 11 .0 Hz), 7.90-7.96 (1H, m), 8.17 (1H, d, J = 7.8 Hz), 8.28 (1H, t, J = 5.5 Hz), 8.46 (1H, d, J = 9.0 Hz).

Process 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

[00607] The compound (0.105 g, 0.108 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 2 to yield the title compound (0.068 mg, 65%).

[00608] 1H-NMR (DMSO-d6) δ: 0.89 (3H, t, J = 7.4 Hz), 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). 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 (1H, m), 5.17 (1H, d, J = 19.4 Hz), 5.24 (1H, d, J = 19.4 Hz), 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 = 11.0 Hz), 7.96-8.08 (2H, m), 8.30-8.38 (2H , m), 8.46-8.56 (2H, m).

Process 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,

[00609] The compound (58mg, 0.060mmol) obtained in Process 2 above was reacted in the same manner as Process 3 of Example 2 to yield the title compound (39mg, 62%).

[00610] 1H-NMR (CD3OD) δ: 0.99 (3H, t, J = 7.4 Hz), 1.27 (2H, td, J = 11.6, 6.1 Hz), 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 .17-2.25 (3H, m), 2.26-2.36 (3H, m), 2.40 (3H, s), 2.95 (1H, dd, J = 14.3, 9, 2 Hz), 3.12 (1H, dd, J = 13.7, 5.7 Hz), 3.15-3.25 (4H, m), 3.44 (2H, t, J = 7.2 Hz), 3.65 (1H, d, J = 17.2 Hz), 3.76 (1H, d, J = 17.2 Hz), 3.79-3.86 (4H, m), 4, 43 (1H, dd, J = 8.9, 6.0 Hz), 5.10 (1H, d, J = 18.9 Hz), 5.25 (1H, d, J = 18.9 Hz), 5.35 (1H, d, J = 16.6 Hz), 5.56 (1H, d, J = 16.0 Hz), 5.60-5.64 (1H, m), 6.76 (2H , s), 7.12-7.24 (6H, m), 7.58 (1H, s), 7.60 (1H, d, J = 10.9 Hz), 7.68 (1H, t, J = 5.7 Hz).

[00611] MS (ESI) m/z: 1060 (M+H)+

Process 4: Antibody-Drug Conjugate (24)

[00612] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used). The solution (9.0 mL) was collected in a 50 mL tube and loaded with a 10 mM aqueous solution of TCEP (0.140 mL; 2.3 equivalents per antibody molecule) and an aqueous solution of dipotassium hydrogen phosphate at 1 M (0.450 ml). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00613] Conjugation between antibody and drug ligand: After incubating the 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 in Process 3 was added to this and incubated to conjugate the drug binder to the antibody at 22°C for 40 minutes. Then, an aqueous solution (0.0559 ml; 9.2 equivalents per antibody molecule) of 100 mM NAC was added thereto and incubated at 22°C to terminate the drug-ligand reaction for another 20 minutes.

[00614] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS7,4 was used as the buffering solution) to produce a solution containing the titrated antibody-drug conjugate.

[00615] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00616] Antibody concentration: 3.30 mg/mL, antibody yield: 53.5 mg (59%), and average number of conjugated drug molecules (n) per antibody molecule: 1.7. Example 25 Antibody-Drug Conjugate (25)

Process 1: Antibody-Drug Conjugate (25)

[00617] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used). The solution (9.0 mL) was collected in a 50 mL tube and loaded with a 10 mM aqueous solution of TCEP (0.280 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of dipotassium hydrogen phosphate at 1 M (0.450 ml). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00618] Conjugation between antibody and drug ligand: After incubating the 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 in Process 3 of the Example 24 was added to this and incubated to conjugate drug binder to antibody at 22°C for 40 minutes. Then, an aqueous solution (0.112 ml; 18.4 equivalents per antibody molecule) of 100 mM NAC was added thereto and incubated at 22°C to terminate the drug-ligand reaction for another 20 minutes.

[00619] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS6.0 was used as the buffer solution) to produce a solution containing the titrated antibody-drug conjugate.

[00620] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00621] Antibody concentration: 10.65 mg/mL, antibody yield: 55.1 mg (61%), and average number of conjugated drug molecules (n) per antibody molecule: 2.5. Example 26 Antibody-Drug Conjugate (26)

Process 1: ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl acetate

[00622] In a mixture containing N-9-fluorenylmethoxycarbonylglycylglycine (4.33 g, 12.2 mmol), tetrahydrofuran (THF; 120 ml), and toluene (40.0 ml), pyridine (1.16 ml , 14.7 mmol) and lead tetraacetate (6.84 g, 14.7 mmol) were added and heated under reflux for 5 hours. After the reaction solution was cooled to room temperature, the insolubles were removed by filtration through Celite and concentrated under reduced pressure. The obtained residues were dissolved in ethyl acetate and washed with water and saturated brine, and then the organic layer was dried over anhydrous magnesium sulfate. After the solvent was removed under reduced pressure, the obtained residues were purified by silica gel column chromatography [hexane : ethyl acetate = 9 : 1 (v/v) - ethyl acetate] to yield the title compound as a colorless solid (3.00 g, 67%).

[00623] 1H-NMR (400 MHz, CDCl 3 ) δ : 2.07 (3H, s), 3.90 (2H, d, J = 5.1 Hz), 4.23 (1H, t, J = 7 .0 Hz), 4.46 (2H, d, J = 6.6 Hz), 5.26 (2H, d, J = 7.0 Hz), 5.32 (1H, brs), 6.96 ( 1H, brs), 7.32 (2H, t, J = 7.3 Hz), 7.41 (2H, t, J = 7.3 Hz), 7.59 (2H, d, J = 7.3 Hz), 7.77 (2H, d, J = 7.3 Hz).

Process 2: [({N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]benzyl acetate

[00624] In a THF solution (40.0 mL) of the compound (3.68 g, 10.0 mmol) obtained in Process 1 above and benzyl glycolate (4.99 g, 30.0 mmol), tert. Potassium butoxide (2.24 g, 20.0 mmol) was added at 0°C and stirred at room temperature for 15 minutes. The reaction solution was charged with ethyl acetate and water at 0°C and extracted with ethyl acetate and chloroform. The organic layer obtained was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The residues obtained were dissolved in dioxane (40.0 mL) and water (10.0 mL), charged with sodium hydrogen carbonate (1.01 g, 12.0 mmol) and 9-fluorenylmethyl chloroformate (2.59 g, 10 .0 mmol) and stirred at room temperature for 2 hours. The reaction solution was charged with water and extracted with ethyl acetate. The organic layer obtained was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [hexane : ethyl acetate = 100 : 0 (v/v) - 0 : 100] to yield the title compound as a substance colorless oil (1.88 g, 40%).

[00625] 1H-NMR (400 MHz, CDCl 3 ) δ : 3.84 (2H, d, J = 5.5 Hz), 4.24 (3H, t, J = 6.5 Hz), 4.49 ( 2H, d, J = 6.7 Hz), 4.88 (2H, d, J = 6.7 Hz), 5.15-5.27 (1H, m), 5.19 (2H, s), 6.74 (1H, brs), 7.31-7.39 (7H, m), 7.43 (2H, t, J = 7.4 Hz), 7.61 (2H, d, J = 7, 4 Hz), 7.79 (2H, d, J = 7.4 Hz).

Process 3: [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid

[00626] The compound (1.88 g, 3.96 mmol) obtained in Process 2 above was dissolved in ethanol (40.0 mL) and ethyl acetate (20.0 mL). After adding palladium carbon catalyst (376 mg), it was stirred under a hydrogen atmosphere at room temperature for 2 hours. The insolubles were removed by filtration through Celite, and the solvent was removed under reduced pressure to yield the title compound as a colorless solid (1.52 g, quantitative).

[00627] 1H-NMR (400MHz, DMSO-d6) δ: 3.62 (2H, d, J = 6.3Hz), 3.97 (2H, s), 4.18-4.32 (3H , m), 4.60 (2H, d, J = 6.7 Hz), 7.29-7.46 (4H, m), 7.58 (1H, t, J = 5.9 Hz), 7 .72 (2H, d, J = 7.4 Hz), 7.90 (2H, d, J = 7.4 Hz), 8.71 (1H, t, J = 6.5 Hz).

Process 4: (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] 9H-fluoren-9-ylmethyl amino}-2-oxoethyl)carbamate

[00628] Under ice-cooling, to a solution of N,N-dimethylformamide (10.0 mL) of methanesulfonic acid salt of exatecan (0.283 g, 0.533 mmol), N-hydroxysuccinimide (61.4 mg, 0.533 mmol) , and the compound (0.205 g, 0.533 mmol) obtained in Process 3 above, N,N-diisopropylethylamine (92.9 µL, 0.533 mmol) and N,N'-dicyclohexylcarbodiimide (0.143 g, 0.693 mmol) were added and stirred at room temperature for 3 days. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform - split organic layer of chloroform: methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a pale brown solid (0.352 g, 82%).

[00629] 1H-NMR (400MHz, DMSO-d6) δ: 0.81 (3H, t, J=7.4Hz), 1.73-1.87 (2H, m), 2.06-2 .20 (2H, m), 2.34 (3H, s), 3.01-3.23 (2H, m), 3.58 (2H, d, J = 6.7 Hz), 3.98 ( 2H, s), 4.13-4.25 (3H, m), 4.60 (2H, d, J = 6.7 Hz), 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 = 7.4 Hz), 7.53 (1H, t, J = 6.3 Hz), 7.66 (2H, d, J = 7.4 Hz), 7.75 (1H, d , J = 11.0 Hz), 7.84 (2H, d, J = 7.4 Hz), 8.47 (1H, d, J = 8.6 Hz), 8.77 (1H, t, J = 6.7 Hz).

[00630] MS (ESI) m/z: 802 (M+H)+

Process 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,

[00631] In a N,N-dimethylformamide solution (11.0 mL) of the compound (0.881 g, 1.10 mmol) obtained in Process 4 above, piperidine (1.1 mL) was added and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to produce a mixture containing the title compound. The mixture was used for the next reaction without further purification.

Process 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

[00632] Under ice-cooling, to a solution of N,N-dimethylformamide (50.0 mL) of the mixture (0.439 mmol) obtained in Process 5 above, N-hydroxysuccinimide (0.101 g, 0.878 mmol) and N-[( 9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (the compound described in Japanese Patent Open to Public Inspection No. 200260351; 0.440 g, 0.878 mmol), N,N'-dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) were added and stirred at room temperature for 4 days. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 9 : 1 (v/v)] to yield the title compound as a pale orange solid (0.269 g, 58%).

[00633] MS (ESI) m/z: 1063 (M+H)+

Process 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

[00634] In a N,N-dimethylformamide solution (4.00 mL) of the compound (0.269 g, 0.253 mmol) obtained in Process 6 above, piperidine (0.251 mL, 2.53 mmol) was added and stirred at room temperature. for 2 hours. The solvent was removed under reduced pressure to produce a mixture containing the title compound. The mixture was used for the next reaction without further purification.

Process 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]pyran [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

[00635] In a N,N-dimethylformamide solution (10.0 mL) of the compound (0.253 mmol) obtained in Process 7 above, N-succinimidyl 6-maleimide hexanoate (0.156 g, 0.506 mmol) was added and stirred in room temperature for 3 days. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 9 : 1 (v/v)] to yield the title compound as a pale yellow solid (0.100 g, 38%).

[00636] 1H-NMR (400 MHz, DMSO-d6) δ: 0.83 (3H, t, J = 7.2 Hz), 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 (3H, 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 = 6.7 Hz), 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 = 11.0 Hz), 7.97 (1H, t, J = 5.7 Hz), 8.03 (1H, t, J = 5.9 Hz ), 8.09 (1H, d, J = 7.8 Hz), 8.27 (1H, t, J = 6.5 Hz), 8.48 (1H, d, J = 9.0 Hz), 8.60 (1H, t, J = 6.5 Hz).

[00637] MS (ESI) m/z: 1034 (M+H)+

Process 9 : Antibody-Drug Conjugate (26)

[00638] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 8 above, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6.

[00639] Antibody concentration: 1.61 mg/mL, antibody yield: 9.7 mg (77%), and mean number of conjugated drug molecules (n) per antibody molecule: 2.9. Example 27 Antibody-Drug Conjugate (27)

Process 1: Antibody-Drug Conjugate (27)

[00640] Using the trastuzumab produced in Reference Example 1 and the compound obtained 8 from Example 26 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00641] Antibody concentration: 1.58 mg/mL, antibody yield: 9.5 mg (76%), and average number of conjugated drug molecules (n) per antibody molecule: 5.6. Example 28 Antibody-Drug Conjugate (28)

Process 1: Antibody-Drug Conjugate (28)

[00642] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (1.25 mL) was placed in two 1.5 mL polypropylene tubes and loaded with a 10 mM aqueous solution of TCEP (0.039 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of hydrogen phosphate. of 1M dipotassium (0.0625 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00643] Conjugation between antibody and drug ligand: After adding DMSO (0.072 mL) and a DMSO solution containing 10 mM of the Process 8 compound of Example 26 (0.078 mL; 9.2 equivalents per antibody molecule) to the above solution at room temperature, was shaken using a tube rotator to conjugate drug ligand to antibody at room temperature for 40 minutes. Then, a 100 mM aqueous solution of NAC (0.0155 mL) was added thereto and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00644] Purification: The above solution was subjected to purification using the Common D-1 procedure (ABS was used as a buffering solution) to yield 11.7 mL in total of a solution containing the compound of interest.

[00645] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00646] Antibody concentration: 1.60 mg/mL, antibody yield: 18.7 mg (94%), and average number of conjugated drug molecules (n) per antibody molecule: 5.2. Example 29 Antibody-Drug Conjugate (29)

Process 1: Antibody-Drug Conjugate (29)

[00647] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (6 mL) was placed in a polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.108 mL; 2.5 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (0.091 mL). After confirming that the solution had a pH of 7.0 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00648] Conjugation between antibody and drug ligand: After adding DMSO (0.146 mL) and a DMSO solution containing 10 mM of the Process 8 compound of Example 26 (0.193 mL; 4.5 equivalents per antibody molecule) to the above solution at room temperature, this was incubated to conjugate drug binder to antibody at 15°C for 1 hour. Then, an aqueous solution (0.029 mL) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00649] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) to yield 24 mL of a solution containing the compound of interest.

[00650] Physical-Chemical Characterization: Using Common procedures E and F (SD,280 = 5178 (measured value) and SD,3?O = 20217 (measured value) were used), the following characteristic values were obtained.

[00651] Antibody concentration: 1.77 mg/mL, antibody yield: 42 mg (85%), average number of conjugated drug molecules (n) per antibody molecule measured by procedure Common E: 3.0, and average number of conjugated drug molecules (n) per antibody molecule measured by the Common F procedure: 3.4. Example 30 Antibody-Drug Conjugate (30)

Process 1: Antibody-Drug Conjugate (30)

[00652] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (6 mL) was placed in a polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.215 mL; 5 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (0.094 mL) . After confirming that the solution had a pH of 7.0 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00653] Conjugation between antibody and drug ligand: After adding a DMSO solution containing 10 mM of the Process 8 compound of Example 26 (0.370 mL; 8.6 equivalents per antibody molecule) to the above solution at room temperature, this was incubated to conjugate drug binder to antibody at 15°C for 1 hour. Then, an aqueous solution (0.056 mL) of 100 mM NAC was added thereto and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00654] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) to yield 24 mL of a solution containing the compound of interest.

[00655] Physical-Chemical Characterization: Using Common Procedures E and F (SD,280 = 5178 (measured value) and SD,3?O = 20217 (measured value) were used), the following characteristic values were obtained.

[00656] Antibody concentration: 1.92 mg/mL, antibody yield: 46 mg (92%), mean number of conjugated drug molecules (n) per antibody molecule measured by the procedure Common E: 6.2, and average number of conjugated drug molecules (n) per antibody molecule measured by the Common F procedure: 7.1. Example 31 Antibody-Drug Conjugate (31)

Process 1: Antibody-Drug Conjugate (31)

[00657] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). A solution (50.00 mL) was placed in a polypropylene vessel and charged with a 1 M aqueous solution of dipotassium hydrogen phosphate (0.745 mL) at room temperature with stirring, followed by a 10 mM aqueous solution of TCEP. (1.868 ml; 5.4 equivalents per antibody molecule). After confirming that the solution had a pH of 7.0 ± 0.1, stirring was terminated, and disulfide binding to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00658] Conjugation between antibody and drug ligand: After cooling the above solution to 15°C, a DMSO solution containing 10 mM of the compound of Process 8 of Example 26 (2.958 mL; 8.6 equivalents per molecule of antibody ) was gradually added dropwise thereto with stirring. While the temperature was maintained at 15°C, the reaction solution was shaken for the first 30 minutes and incubated without shaking to conjugate the drug ligand to the antibody for the next 1 hour. Then, an aqueous solution (0.444 mL) of 100 mM NAC was added thereto with stirring and stirred at room temperature to terminate the drug ligand reaction for another 20 minutes.

[00659] Purification: By gradually adding 20% aqueous acetic acid solution (about 0.25 mL) and ABS (50 mL) to the above solution with stirring, the pH of the solution was adjusted to 5.5 ± 0. 1. This solution was subjected to microfiltration (Millipore Corp., Millex-HV filter, 0.45 μm, PVDF membrane) to remove whitish matter. This solution was subjected to purification by ultrafiltration using an ultrafiltration mechanism composed of an ultrafiltration membrane (Merck Japan, Pellicon XL Cassette, Biomax 50 KDa), a tube pump (Cole-Parmer International, MasterFlex Pump model 77521-40, Pump Head model 7518-00), and a tube (Cole-Parmer International, MasterFlex Tube L/S16). Specifically, at the same time that ABS was added dropwise (a total of 800 mL) as a buffering solution for purification to the reaction solution, purification by ultrafiltration was performed to remove unconjugated drug binders and other low molecular weight reagents. , likewise replacing the buffering solution with ABS, and also concentrating the solution. The purified solution obtained was subjected to microfiltration (0.22 μm (Millipore Corp., Millex-GV filter, PVDF membrane) and 0.10 μm (Millipore Corp., Millex-VV filter, PVDF membrane)) to produce a solution containing the titrated antibody-drug conjugate.

[00660] Physicochemical Characterization: Using Common procedures E and F (SD,28Q = 5178 (measured value), and SD,3?Q = 20217 (measured value) were used), the following characteristic values were obtained.

[00661] Antibody concentration: 11.28 mg/mL, antibody yield: 451 mg (90%), mean number of conjugated drug molecules (n) per antibody molecule measured by procedure Common E: 6.6, and average number of conjugated drug molecules (n) per antibody molecule measured by the Common F procedure: 7.7. Example 32 (Alternative method for synthesizing the compound of Process 8 of Example 26)

Process 1: tert-Butyl N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenyl alaninate

[00662] Under ice-cooling, to a THF solution (12.0 ml) of tert-butyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenyl alaninate (J. Pept. Res. , 1999, vol 53, pp 393; 0.400 g, 0.717 mmol), 1,8-diazabicyclo[5.4.0]-7-undecene (0.400 ml) was added and stirred at room temperature for 4 days, then N-succinimidyl 6-maleimide hexanoate (0.221 g, 0.717 mmol) was also added and stirred for 3 hours. The reaction solution was diluted with ethyl acetate and washed with a 10% aqueous solution of citric acid, a saturated aqueous solution of sodium hydrogen carbonate, and saturated brine, and then the organic layer was dried over anhydrous magnesium sulfate. After the solvent was removed under reduced pressure, the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 9 : 1 (v/v)] to yield the title compound as a pale yellow solid ( 0.295 g, 78%).

[00663] 1H-NMR (400 MHz, CDCl 3 ) δ : 1.28-1.36 (2H, m), 1.41 (9H, s), 1.57-1.71 (4H, m), 2 .23 (2H, t, J = 7.6 Hz), 3.09 (2H, d, J = 6.0 Hz), 3.51 (2H, t, J = 7.6 Hz), 3.85 -4.02 (4H, m), 4.69-4.78 (1H, m), 6.15 (1H, t, J = 4.6 Hz), 6.33 (1H, d, J = 7 .3 Hz), 6.60 (1H, t, J = 5.0 Hz), 6.68 (2H, s), 7.10-7.16 (2H, m), 7.22-7.31 (3H, m).

[00664] MS (ESI) m/z: 529 (M+H)+

Process 2: N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanine

[00665] In a dichloromethane solution (8.00 ml) of the compound (0.295 g, 0.558 mmol) obtained in Process 1 above, trifluoroacetic acid (4.00 ml) was added and stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to yield the title compound as a pale yellow solid (0.240 g, 91%).

[00666] 1H-NMR (400MHz, DMSO-d6) δ: 1.15-1.23 (2H, m), 1.40 1.53 (4H, m), 2.10 (2H, t, J = 7.6 Hz), 2.88 (1H, dd, J = 13.7, 8.9 Hz), 3.04 (1H, dd, J = 13.7, 5.0 Hz), 3.35 -3.43 (2H, m), 3.58-3.77 (4H, m), 4.41 (1H, td, J = 7.8, 5.0 Hz), 7.00 (2H, s ), 7.16-7.31 (5H, m), 8.00 (1H, t, J = 5.7 Hz), 8.06 (1H, t, J = 5.7 Hz), 8.13 (1H, d, J = 7.8 Hz).

Process 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]pyran [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

[00667] The compound (0.572 g, 1.21 mmol) obtained in Process 2 above was dissolved in dichloromethane (12.0 mL), charged with N-hydroxysuccinimide (0.152 g, 1.32 mmol) and 1-( 3-dimethylaminopropyl)-3-ethylcarbodiimide (0.253 g, 1.32 mmol), and stirred for 1 hour. The reaction solution was added to a N,N-dimethylformamide solution (22.0 mL) of the mixture (1.10 mmol) obtained from Example 26 in Process, and stirred at room temperature for 3 hours. The reaction solution was charged with 10% aqueous citric acid solution and extracted with chloroform. The organic layer obtained was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 8 : 2 (v/v)] to yield the title compound as a pale yellow solid (0.351 g, 31%). Instrumental data for the compound were the same as those for the Process 8 compound of Example 26. Example 33 (Alternative method for synthesizing the Process 8 compound of Example 26)

Process 1: [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]benzyl acetate

[00668] In a THF solution (200 ml) of the compound (7.37 g, 20.0 mmol) obtained in Process 1 of Example 26, benzyl glycolate (6.65 g, 40.0 mmol) and p-toluenesulfonic acid (0.381 g, 2.00 mmol) was added at 0°C and stirred at room temperature for 2 hours and 30 minutes. The reaction solution was charged with a saturated aqueous solution of sodium hydrogen carbonate and extracted with ethyl acetate. The organic layer obtained was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [hexane : ethyl acetate = 100 : 0 (v/v) - 0 : 100] to yield the title compound as a solid colorless (6.75 g, 71%). The instrumental data of the compound was the same as that of the compound of Process 2 of Example 26.

Process 2: N-[(Benzyloxy)carbonyl]glycylglycyl-L-phenylalanine-N-{[(2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide

[00669] In a N,N-dimethylformamide solution (140 mL) of the compound (6.60 g, 13.9 mmol) obtained in Process 1 above, 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. The reaction solution was charged with a solution of N,N-dimethylformamide (140 mL) 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) stirred in advance at room temperature for 1 hour, and stirred at room temperature for 4 hours. The reaction solution was charged with 0.1N hydrochloric acid and extracted with chloroform. The organic layer obtained was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 8 : 2 (v/v)] to yield the title compound as a colorless solid (7. 10 g, 79%).

[00670] 1H-NMR (DMSO-d6) δ: 2.78 (1H, dd, J = 13.9, 9.6 Hz), 3.05 (1H, dd, J = 13.9, 4.5 Hz), 3.56-3.80 (6H, m), 4.15 (2H, s), 4.47-4.55 (1H, m), 4.63 (2H, d, J = 6, 6 Hz), 5.03 (2H, s), 5.15 (2H, s), 7.16-7.38 (15H, m), 7.52 (1H, t, J = 5.9 Hz) , 8.03 (1H, t, J = 5.5 Hz), 8.17 (1H, d, J = 8.2 Hz), 8.36 (1H, t, J = 5.7 Hz), 8 .61 (1H, t, J = 6.6 Hz).

Process 3: Glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

[00671] In a N,N-dimethylformamide solution (216 mL) of the compound (7.00 g, 10.8 mmol) obtained in Process 2 above, palladium carbon catalyst (7.00 g) was added and stirred under an atmosphere of hydrogen at room temperature for 24 hours. The insolubles were removed by filtration through Celite, and the solvent was removed under reduced pressure. The residues obtained were dissolved in water, the insoluble material was removed by filtration through Celite, and the solvent was removed under reduced pressure. This procedure was repeated twice to yield the title compound as a colorless solid (3.77 g, 82%).

[00672] 1H-NMR (DMSO-d6) δ: 2.84 (1H, dd, J = 13.7, 9.8 Hz), 3.08 (1H, dd, J = 13.7, 4.7 Hz), 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 = 6.6 Hz), 7.12-7.30 (5H, m), 8.43 (1H, t, J = 5.9 Hz), 8.54 ( 1H, d, J = 7.8 Hz), 8.70 (1H, t, J = 6.3 Hz), 8.79 (1H, t, J = 5.5 Hz).

Process 4: N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

[00673] In a N,N-dimethylformamide solution (85.0 mL) of the compound (3.59 g, 8.48 mmol) obtained in Process 3 above, N-succinimidyl 6-maleimide hexanoate (2.88 g , 9.33 mmol) and triethylamine (0.858 g, 8.48 mmol) were added and stirred at room temperature for 1 hour. The reaction solution was charged with 0.1N hydrochloric acid and extracted with chloroform and a mixed solvent of chloroform and methanol [chloroform : methanol = 4:1 (v/v)]. The organic layer obtained was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure, and the obtained residues were purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a colorless solid (3.70 g, 71%).

[00674] 1H-NMR (DMSO-d6) δ: 1.13-1.24 (2H, m), 1.42-1.53 (4H, m), 2.11 (2H, t, J = 7 .4 Hz), 2.80 (1H, dd, J = 13.7, 9.8 Hz), 3.06 (1H, dd, J = 13.9, 4.5 Hz), 3.37 (2H , t, J = 7.2 Hz), 3.56-3.78 (6H, m), 3.97 (2H, s), 4.464.53 (1H, m), 4.61 (2H, d, J = 6.3 Hz), 7.00 (2H, s), 7.15-7.29 (5H, m), 8.03-8.20 (3H, m), 8.32 (1H, t , J = 5.9 Hz), 8.60 (1H, t, J = 6.7 Hz).

Process 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]pyran [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

[00675] In a solution of N,N-dimethylformamide (40.0 mL) of exatecan methanesulfonic acid salt (1.14 g, 2.00 mmol), triethylamine (0.202 g, 2.00 mmol), the compound (1.48 g, 2.40 mmol) obtained in Process 4 above, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (0.993 g, 3 .00 mmol) containing 16.4% of water were added at 0°C and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 8 : 2 (v/v)] to yield the title compound as a pale yellow solid (1 .69 g, 82%). The spectral data of the compound was the same as that of the compound of Process 8 of Example 26. Example 34 Intermediate (34)

Process 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-oxoethylacetate

[00676] Under ice-cooling, to a suspension of N,N-dimethylformamide (20.0 mL) of exatecan methanesulfonic acid salt (0.500 g, 0.941 mmol), N,N-diisopropylethylamine (0.492 mL, 2 .82 mmol) and acetoxyacetyl chloride (0.121 ml, 1.13 mmol) were added and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the obtained residues were purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a pale yellow solid (0.505 g, quantitative).

[00677] 1 H-NMR (400 MHz, DMSO-d6) δ : 0.87 (3H, t, J = 7.4 Hz), 1.81-1.92 (2H, m), 2.08 (3H , s), 2.08-2.22 (2H, m), 2.41 (3H, s), 3.143.21 (2H, m), 4.51 (2H, dd, J = 19.4, 14 .7 Hz), 5.22 (2H, dd, J = 40.1, 19.0 Hz), 5.43 (2H, s), 5.56-5.61 (1H, m), 6.53 (1H, s), 7.31 (1H, s), 7.81 (1H, d, J = 11.0 Hz), 8.67 (1H, d, J = 8.6 Hz).

[00678] MS (ESI) m/z: 536 (M+H)+

Process 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

[00679] In a methanol suspension (50.0 mL) of the compound (0.504 g, 0.941 mmol) obtained in Process 1 above, a THF (20.0 mL) and a 1N aqueous solution of sodium hydroxide (4 .00 ml, 4.00 mmol) were added and stirred at room temperature for 1 hour. The reaction was quenched by the addition of 1N hydrochloric acid (5.00 ml, 5.00 mmol), and the solvent was removed under reduced pressure. The obtained residues were purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a pale yellow solid ( 0.412 g, 89%). This compound was confirmed in the tumor of a mouse that received the antibody-drug conjugate (45) or (46).

[00680] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.3 Hz), 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 = 6.0 Hz), 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, m). s), 7.30 (1H, s), 7.78 (1H, d, J = 10.9 Hz), 8.41 (1H, d, J = 9.1 Hz). MS (ESI) m/z: 494 (M+H)+ Example 35 (Alternative method to synthesize the compound of Example 34)

Process 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

[00681] Glycolic acid (0.0201 g, 0.27 mmol) was dissolved in N,N-dimethylformamide (1.0 mL), charged with N-hydroxysuccinimide (0.0302 g, 0.27 mmol) and sodium hydrochloride. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.0508 g, 0.27 mmol), and stirred for 1 hour. The reaction solution was added to a suspension of N,N-dimethylformamide (1.0 mL) loaded with methanesulfonic acid salt of exatecan (0.1 g, 0.176 mmol) and triethylamine (0.025 mL, 0.18 mmol) and stirred at room temperature for 24 hours. The solvent was removed under reduced pressure, and the residues obtained were purified by silica gel column chromatography [chloroform-chloroform : methanol = 10 : 1 (v/v)] to yield the title compound as a pale yellow solid (0.080 g, 92%). The spectral data of the compound was the same as that of the compound obtained 2 of Example 34 in the Process. Example 36 Antibody-Drug Conjugate (36)

Process 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

[00682] The compound (60.0 mg, 0.0646 mmol) obtained in Process 2 of Example 15 was reacted in the same manner as Process 3 of Example 2 using N-succinimidyl 4-maleimide butyrate in place of N-hexanoate -succinimidyl 6-maleimide to yield the title compound as a pale white solid (24.0 mg, 38%).

[00683] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.2 Hz), 1.68 (2H, quin, J = 7.4 Hz), 1, 78-1.92 (2H, m), 2.06-2.22 (2H, m), 2.10 (2H, t, J = 7.8 Hz), 2.31-2.43 (2H, m), 2.40 (3H, s), 2.78 (1H, dd, J = 13.7, 9.4 Hz), 3.01 (1H, dd, J = 13.7, 4.7 Hz ), 3.17 (4H, d, J = 5.1 Hz), 3.29-3.40 (2H, m), 3.52-3.80 (6H, m), 4.40-4, 51 (1H, m), 5.19 (1H, d, J = 18.4 Hz), 5.26 (1H, d, J = 18.8 Hz), 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 (1H, t, J = 5.9 Hz), 8.08-8.16 (2H, m), 8.25 (1H, t , J = 5.9 Hz), 8.52 (1H, d, J = 8.2 Hz).

[00684] MS (ESI) m/z: 990 (M+H)+

Process 2: Antibody-Drug Conjugate (33)

[00685] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 1 above, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6.

[00686] Antibody concentration: 1.75 mg/ml, antibody yield: 10.5 mg (84%) and mean number of conjugated drug molecules (n) per antibody molecule: 4.7. Example 37 Antibody-Drug Conjugate (37)

Process 1: Antibody-Drug Conjugate (37)

[00687] Using the trastuzumab produced in Reference Example 1 and the compound obtained 1 from Example 36 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00688] Antibody concentration: 1.89 mg/mL, antibody yield: 11.3 mg (90%) and mean number of conjugated drug molecules (n) per antibody molecule: 8.5. Example 38 Intermediate (38)

Process 1: (5-{[(1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa- tert-butyl hydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-5-oxopentyl)carbamate

[00689] Methanesulfonic acid salt of exatecan (500 mg, 0.941 mmol) was reacted in the same manner as Process 1 of Example 1 using 5-(tert-butoxycarbonylamino)valeric acid in place of 4-(tert-butoxycarbonylamino)butanoic acid to yield the title compound as a yellowish brown solid (571 mg, 96%). The compound was used for the next reaction without performing further purification.

[00690] MS(ESI)m/z:635(M+H)+

Process 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]pyran[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]pentanamide

[00691] The compound (558 mg, 0.879 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 1 to yield trifluoroacetate of the title compound as a yellow solid (363 mg, 64%).

[00692] 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, 6.9 Hz), 2.07-2.18 (2H, m), 2.22 (2H, t, J = 7.0 Hz), 2.40 (3H, s), 2.76-2.88 (2H, m), 3.13-3.22 (2H, m), 5.18 (1H, d, J = 18.8 Hz), 5.24 (1H , d, J = 18.8 Hz), 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 = 11.3 Hz), 8.49 (1H, d, J = 8.6 Hz).

[00693] MS (ESI) m/z: 535 (M+H)+ Example 39 Antibody-Drug Conjugate (39)

Process 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

[00694] The compound (348 mg, 0.537 mmol) obtained from Example 38 in Process 2 was reacted in the same manner as Process 1 of Example 2 to yield the title compound as a pale yellow solid (429 mg, 84%). The compound was used for the next reaction without performing further purification.

Process 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

[00695] The compound (427 mg, 0.448 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 2 to yield trifluoroacetate of the title compound as a yellow solid (430 mg, 99%).

[00696] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.2 Hz), 1.38-1.49 (2H, m), 1.54-1 .66 (2H, m), 1.86 (2H, tt, J = 14.5, 7.0 Hz), 2.08-2.16 (2H, m), 2.19 (2H, t, J = 7.2 Hz), 2.40 (3H, s), 2.76 (1H, dd, J = 13.9, 10.0 Hz), 3.00-3.12 (3H, m), 3 .14-3.21 (2H, m), 3.57 (2H, d, J = 4.7 Hz), 3.60-3.75 (3H, m), 3.87 (1H, dd, J = 16.8, 5.9 Hz), 4.55 (1H, td, J = 9.0, 4.7 Hz), 5.16 (1H, d, J = 18.8 Hz), 5.23 (1H, d, J = 18.4 Hz), 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 (1H, t, J = 5.5 Hz), 7.81 (1H, d, J = 10.9 Hz), 7. 96 (3H, br.s.), 8.30-8.37 (1H, m), 8.44-8.53 (2H, m).

[00697] MS (ESI) m/z: 853 (M+H)+

Process 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

[00698] The compound (60.0 mg, 0.0621 mmol) obtained in Process 2 above was reacted in the same manner as Process 3 of Example 2 to yield the title compound as a solid (16.0 mg, 25%) .

[00699] 1H-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), 2.19 (2H, s), 2.40 (3H, s), 2.79 (1H, dd, J = 13.7, 10.2 Hz), 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 = 18.8 Hz), 5.23 (1H, d, J = 18.8 Hz), 5.43 (2H, s), 5.52-5.60 (1H , m), 6.53 (1H, s), 6.99 (2H, s), 7.12-7.28 (5H, m), 7.31 (1H, s), 7.63 (1H, s). t, J = 5.7 Hz), 7.80 (1H, d, J = 10.6 Hz), 8.02 (1H, t, J = 5.9 Hz), 8.08 (1H, t, J = 5.7 Hz), 8.12 (1H, d, J = 7.8 Hz), 8.24 (1H, t, J = 5.7 Hz), 8.45 (1H, d, J = 8.6 Hz).

[00700] MS (ESI) m/z: 1046 (M+H)+

Process 4: Antibody-Drug Conjugate (39)

[00701] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used). The solution (1.0 mL) was collected in a 2 mL tube and loaded with a 10 mM aqueous solution of TCEP (0.0155 mL; 2.3 equivalents per antibody molecule) and an aqueous solution of dipotassium hydrogen phosphate. at 1M (0.050 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00702] Conjugation between antibody and drug ligand: After incubating the solution for 10 minutes at 22°C, a DMSO solution (0.0311 mL; 4.6 equivalents per antibody molecule) containing 10 mM of the compound obtained in Process 3 was added to this and incubated to conjugate drug binder to antibody at 22°C for 40 minutes. Then, an aqueous solution (0.00622 ml; 9.2 equivalents per antibody molecule) of 100 mM NAC was added thereto and incubated at 22°C to terminate the drug-ligand reaction for another 20 minutes.

[00703] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS6.0 was used as the buffer solution) to produce 6 mL of a solution containing the titrated antibody-drug conjugate.

[00704] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00705] Antibody concentration: 1.12 mg/mL, antibody yield: 6.72 mg (67%), and average number of conjugated drug molecules (n) per antibody molecule: 1.8. Example 40 Antibody-Drug Conjugate (40)

Process 1: Antibody-Drug Conjugate (40)

[00706] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.37 mLmg-1cm-1 was used). The solution (1.0 mL) was collected in a 2 mL tube and loaded with a 10 mM aqueous solution of TCEP (0.0311 mL; 4.6 equivalents per antibody molecule) and an aqueous solution of dipotassium hydrogen phosphate. at 1M (0.050 mL). After confirming that the solution had a pH of 7.4 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00707] Conjugation between antibody and drug ligand: After incubating the solution at 22°C for 10 minutes, a DMSO solution (0.0622 ml; 9.2 equivalents per antibody molecule) containing 10 mM of the obtained compound 3 of Example 39 in the Process was added thereto and incubated to conjugate the drug binder to the antibody at 22°C for 40 minutes. Then, an aqueous solution (0.0124 ml; 18.4 equivalents per antibody molecule) of 100 mM NAC was added thereto and incubated at 22°C to terminate the drug-ligand reaction for another 20 minutes.

[00708] Purification: The above solution was subjected to purification using the Common D-1 procedure (PBS6.0 was used as the buffer solution) to produce 6 mL of a solution containing the titrated antibody-drug conjugate.

[00709] Physical-Chemical Characterization: Using the Common E procedure, the following characteristic values were obtained.

[00710] Antibody concentration: 0.98 mg/mL, antibody yield: 5.88 mg (59%), and average number of conjugated drug molecules (n) per antibody molecule: 3.4. Example 41 Antibody-Drug Conjugate (41)

Process 1: tert-butyl {2-[(2-hydroxyethyl)amino]-2-oxoethyl}carbamate

[00711] N-(tert-Butoxycarbonyl)glycine (4.2 g, 24 mmol) was dissolved in dimethylformamide (40 mL). After adding aminoethanol (2.9 g, 48 mmol) and 1-hydroxybenzotriazole (3.7 g, 24 mmol) and adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.9 g, 36 mmol ), this was stirred at room temperature for 12 hours. The solvent was removed under reduced pressure, and the residue was taken up with toluene by azeotropic distillation. The obtained residue was purified by silica gel column chromatography [ethyl acetate - ethyl acetate : methanol = 10 : 1 (v/v)] to yield the title compound as a colorless oily substance (3.8 g, 72 %).

[00712] 1H-NMR (400 MHz, CDCl3) δ : 1.44 (9H, s), 1.69 (1H, brs), 3.43 (2H, td, J = 5.9, 5.1 Hz ), 3.71 (2H, t, J = 5.1 Hz), 3.79 (2H, d, J = 5.9 Hz), 5.22 (1H, brs), 6.62 (1H, brs ).

Process 2: 2-{[N-(tert-Butoxycarbonyl)glycyl]amino}ethyl4-nitrophenylcarbonate

[00713] In a THF solution (23 mL) of the compound (1.0 g, 4.59 mmol) obtained in Process 1 above, diisopropylethylamine (0.80 mL, 4.59 mmol) and bis(4- nitrophenyl) carbonate (1.32 g, 6.88 mmol) were added and stirred at room temperature for 12 hours. The solvent was removed under reduced pressure, and the obtained residue was purified by silica gel column chromatography [hexane-hexane:ethyl acetate = 1:3 (v/v)] to yield the title compound as a pale yellow solid. (1.13 g, 64%).

[00714] 1H-NMR (400 MHz, CDCl 3 ) δ : 1.44 (1H, s), 3.66 (2H, td, J = 5.1, 5.9 Hz), 3.81 (2H, d , J = 5.9 Hz), 4.36 (2H, t, J = 5.1 Hz), 5.07 (1H, s), 6.48-6.53 (1H, m), 7.38 (2H, dt, J = 9.9, 2.7 Hz), 8.27 (2H, dt, J = 9.9, 2.7 Hz).

Process 3: [(1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 2-({[(tert-Butoxycarbonyl)amino]acetyl}amino) 12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]carbamate ethyl

[00715] In methanesulfonic acid salt of exatecan (0.70 g, 1.2 mmol), the compound (0.57 g, 1.5 mmol) obtained 2 in the Process, and 1-hydroxybenzotriazole (3.7 g, 24 mmol), dimethylformamide (23 mL) were added, and diisopropylethylamine (0.43 mL, 2.5 mmol) was added and stirred at room temperature for 12 hours. The solvent was removed under reduced pressure, and the residue was taken up with toluene by azeotropic distillation. The obtained residue was purified by silica gel column chromatography [chloroform-chloroform: methanol = 10 : 1 (v/v)] to yield the title compound (0.86 g, quantitative) as a pale yellow solid.

[00716] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.4 Hz), 1.35 (9H, s), 1.78-1.94 (1H , m), 2.07-2.17 (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 = 11.0 Hz), 7.93-8.07 (2H, m).

Process 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

[00717] The compound (0.86 g, 2.1 mmol) obtained in Process 3 above was dissolved in dichloromethane (15 mL). After adding trifluoroacetic acid (15 ml), it was stirred for 1 hour. The solvent was removed under reduced pressure, and the residue was taken up with toluene by azeotropic distillation. The obtained residue was purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a pale yellow solid ( 0.86 g, 99%).

[00718] 1H-NMR (400 MHz, DMSO-d6) δ: 0.87 (3H, t, J = 7.2 Hz), 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 = 18.8 Hz), 5.46 (1H, d, J = 18.8 Hz), 6.56 (1H, s), 7.34 (1H, s), 7.65 (2H, brs), 7.79 (1H, d, J = 10.6 Hz), 7.99- 8.06 (1H, m), 8.51 (1H, t, J = 5.5 Hz).

[00719] MS (APCI) m/z: 939 (M+H)+

Process 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

[00720] N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (Japanese Patent Open to Public Inspection No. 2002-60351; 0.21 g, 0.41 mmol) was dissolved in N, N-dimethylformamide (3 ml). After adding N-hydroxysuccinimide (0.052 g, 0.45 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.086 g, 0.45 mmol), this was stirred for 1 hour. The reaction solution was added dropwise to a solution of N,N-dimethylformamide (2 mL) charged with the compound (0.24 g, 0.35 mmol) obtained in Process 4 and triethylamine (0.078 mL, 0.45 mmol), and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography [chloroform-chloroform : methanol = 8 : 2 (v/v)] to yield the title compound as a pale yellow solid (0 .24 g, 65%).

[00721] 1H-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), 3.553.79 (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 = 17.2 Hz), 5.44 (1H, d, J = 17.2 Hz), 6.53 (1H, s), 7.11-7.26 (5H, m), 7.26 -7.33 (3H, m), 7.38 (2H, t, J = 7.6 Hz), 7.57 (1H, t, J = 5.9 Hz), 7.68 (2H, d, J = 7.4 Hz), 7.77 (1H, d, J = 11.0 Hz), 7.85 (2H, d, J = 9.0 Hz), 7.91-7.97 (1H, m), 7.98-8.05 (2H, m), 8.14 (1H, d, J = 7.8 Hz), 8.31-8.26 (1H, m).

[00722] MS (APCI) m/z: 1063 (M+H)+

Process 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

[00723] The compound (0.24 g, 0.35 mmol) obtained in Process 5 above was reacted in the same manner as Process 7 of Example 26 to yield the title compound as a pale yellow solid (0.12 g, 65 %).

[00724] 1H-NMR (400MHz, DMSO-d6) δ: 0.86 (3H, t, J=7.4Hz), 1.78-1.94 (2H, m), 2.06-2 .27 (2H, 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), 4.004.21 (3H, m), 4.45-4.54 (1H, m), 5.15-5.35 (3H , m), 5.41 (1H, d, J = 17.2 Hz), 5.45 (1H, d, J = 17.2 Hz), 6.54 (1H, s), 7.11-7 .26 (6H, m), 7.32 (1H, s), 7.78 (1H, d, J = 11.0 Hz), 7.93-8.00 (1H, m), 8.03 ( 1H, d, J = 9.4 Hz), 8.06-8.13 (1H, m), 8.21-8.27 (2H, m), 8.30-8.36 (1H, m) .

[00725] MS (APCI) m/z: 841 (M+H)+

Process 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]pyran [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]carbamoyl}oxy)ethyl]glycinamide

[00726] The compound (42.0 mg, 0.0499 mmol) obtained in Process 6 was reacted in the same manner as Process 3 of Example 2 to yield the title compound as a pale yellow solid (38.3 mg, 74% ).

[00727] 1H-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 = 23.9 Hz) , 7.33 (1H, s), 7.78 (1H, d, J = 10.6 Hz), 7.91-8.16 (5H, m), 8.24-8.31 (1H, m ).

[00728] MS (ESI) m/z: 1034 (M+H)+

Process 8 : Antibody-Drug Conjugate (41)

[00729] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 7 above, the titrated antibody-drug conjugate was obtained in the same manner as Process 2 of Example 6.

[00730] Antibody concentration: 1.54 mg/mL, antibody yield: 9.2 mg (74%), and average number of conjugated drug molecules (n) per antibody molecule: 3.7. Example 42 Antibody-Drug Conjugate (42)

Process 1: Antibody-Drug Conjugate (42)

[00731] Using the trastuzumab produced in Reference Example 1 and the compound obtained 7 from Example 41 in the Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00732] Antibody concentration: 1.47 mg/ml, antibody yield: 8.8 mg (71%), and average number of conjugated drug molecules (n) per antibody molecule: 7.0. Example 43 Antibody-Drug Conjugate (43)

Process 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

[00733] The compound (53.7 mg, 50.5 µmol) obtained in Process 6 of Example 26 was dissolved in N,N-dimethylformamide (1.50 mL). After adding 1,8-diazabicyclo(5,4,0)-7-undecene (7.5 µL, 50.5 µmol), this was stirred at room temperature for 30 minutes. The reaction solution was charged with pyridinium p-toluenesulfonate (14.0 mg, 5.56 μmol), then charged with N-succinimidyl 3-(2-(2-(3-maleinimidopropanamido)ethoxy)ethoxy)propanoate ( 32.3 mg, 75.8 µmol), and stirred at room temperature for 2.25 hours. The solvent was removed under reduced pressure, and the obtained residue was purified by silica gel column chromatography [[chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a pale yellow solid (27.1 mg, 47%).

[00734] 1H-NMR (DMSO-d6) δ: 0.87 (3H, t, J = 7.0 Hz), 1.79-1.91 (2H, m), 2.18 (2H, t, J = 15.1 Hz), 2.29-2.33 (4H, m), 2.39 (3H, s), 2.76 (1H, dd, J = 13.9, 9.2 Hz), 3.02 (1H, dd, J = 13.7, 3.9 Hz), 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, m). d, J = 6.3 Hz), 5.21 (2H, s), 5.42 (2H, s), 5.60 (1H, dd, J = 13.5, 5.7 Hz), 6, 54 (1H, s), 7.00 (2H, s), 7,177.24 (6H, m), 7.31 (1H, s), 7.79 (1H, d, J = 11.0 Hz), 8.00-8.02 (2H, m), 8.13 (1H, d, J = 7.8 Hz), 8.17 (1H, t, J = 6.3 Hz), 8.52 (1H , d, J = 9.0 Hz), 8.65 (1H, t, J = 6.5 Hz).

[00735] MS (ESI) m/z = 1151 (M+H)+

Process 2: Antibody-Drug Conjugate (43)

[00736] Using the trastuzumab produced in Reference Example 1 and the compound obtained 1 in Process, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00737] Antibody concentration: 1.96 mg/mL, antibody yield: 17.6 mg (88%), and average number of conjugated drug molecules (n) per antibody molecule: 5.6. Example 44 Antibody-Drug Conjugate (44)

Process 1: tert-butyl N-[(benzyloxy)carbonyl]glycylglycyl-D-phenylalaninate

[00738] N-[(Benzyloxy)carbonyl]glycylglycine (3.00 g, 11.3 mmol) was dissolved in N,N-dimethylformamide (20.0 mL). After adding N-hydroxysuccinimide (1.43 g, 12.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.37 g, 12.4 mmol), this was stirred for 1 hour . A solution of N,N-dimethylformamide (10 mL) charged with tert-butyl D-phenylalanine (2.74 g, 12.38 mmol) and triethylamine (1.73 mL, 12.4 mmol) was added dropwise to the reaction solution and stirred at room temperature for 2 hours. The reaction solution was charged with dichloromethane and washed with water, 1N hydrochloric acid, and a saturated aqueous solution of sodium bicarbonate, and then the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue obtained was purified by column chromatography on silica gel [chloroform-chloroform : methanol = 9 : 1 (v/v)] to yield the title compound as a colorless solid (4, 21 g, 80%).

[00739] 1H-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 (1H, brs), 6.42 (1H, d, J = 10.0 Hz), 6.64-6.71 ( 1H, m), 7.11-7.15 (2H, m), 7.20-7.31 (4H, m), 7.31-7.38 (4H, m).

[00740] MS (APCI) m/z: 470 (M+H)+

Process 2: N-[(Benzyloxy)carbonyl]glycylglycyl-D-phenylalanine

[00741] The compound (4.21 g, 8.97 mmol) obtained in Process 1 was dissolved in ethyl acetate (20 mL). After adding a 4N ethyl acetate solution (20.0 mL) of hydrogen chloride, it was left overnight at room temperature. The solvent was removed under reduced pressure, then toluene was added, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a colorless solid (1 .66 g, 45%).

[00742] 1H-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).

[00743] MS (APCI) m/z: 412 (M+H) -

Process 3: N-[(Benzyloxy)carbonyl]glycylglycyl-D-phenylalanyl-N-{[2-(benzyloxy)-2-oxoethoxy]methyl}glycinamide

[00744] In a dioxane solution (25.0 mL) of the compound (1.25 g, 2.63 mmol) obtained in Process 1 of Example 32, piperidine (5.00 mL) and N,N-dimethylformamide (5 .00 ml) were added and stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure, and the residue obtained was dissolved in N,N-dimethylformamide (20.0 ml). After adding the compound (1.20 g, 2.90 mmol) from Process 2 above and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1 .03 g, 3.16 mmol) containing 16.4% water was stirred at room temperature for 2 hours. The reaction solution was charged with chloroform and washed with water, and then the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography [chloroform-chloroform : methanol = 9 : 1 (v/v)] to yield the title compound as a colorless solid (270 mg , 16%).

[00745] 1H-NMR (DMSO-d6) δ: 2.78 (1H, dd, J = 13.6, 10.0 Hz), 3.05 (1H, dd, J = 13.9, 4.2 Hz), 3.56-3.79 (6H, m), 4.15 (2H, s), 4.474.54 (1H, m), 4.63 (2H, d, J = 6.7 Hz), 5.03 (2H, s), 5.15 (2H, s), 7,147.39 (15H, m), 7.50 (1H, t, J = 5.7 Hz), 8.02 (1H, t , J = 5.4 Hz), 8.16 (1H, d, J = 7.9 Hz), 8.34 (1H, t, J = 6.0 Hz), 8.60 (1H, t, J = 7.0 Hz).

[00746] MS (APCI) m/z: 648 (M+H)+

Process 4: Glycylglycyl-D-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide

[00747] The compound (200 mg, 0.31 mmol) obtained in Process 3 above was dissolved in N,N-dimethylformamide (5.0 mL). After adding 5% palladium carbon catalyst (0.12 g), it was stirred under a hydrogen atmosphere at room temperature for 9 hours. The reaction solution was filtered through Celite, and the residue was washed with a mixed solvent of water and N,N-dimethylformamide. The filtrate and washing were combined, and the solvent was removed under reduced pressure to yield the title compound as a colorless solid (0.15 g, quantitative).

[00748] 1H-NMR (DMSO-d6) δ: 2.85 (1H, dd, J = 13.3, 9.7 Hz), 3.08 (1H, dd, J = 13.9, 5.4 Hz), 3.43-3.52 (4H, m), 3.62-3.89 (7H, m), 4.364.44 (1H, m), 4.58-4.67 (2H, m) , 7.12-7.29 (5H, m), 8.44 (1H, t, J = 5.7 Hz), 8.67 (1H, d, J = 7.3 Hz), 8.78 ( 1H, t, J = 5.4 Hz), 8.91 (1H, brs).

[00749] MS (APCI) m/z: 424 (M+H)+

Process 5: N-[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-D-phenylalanyl-N-[(carboxymethoxy)methyl]glycinamide,

[00750] The compound (0.15 g, 0.35 mmol) obtained in Process 4 above was dissolved in N,N-dimethylformamide (10 mL). After adding N-succinimidyl 6-maleimide hexanoate (0.11 g, 0.35 mmol), this was stirred at room temperature for 1 hour. The reaction solution was charged with chloroform and washed with water, and then the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the obtained residue was purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v/v)] to yield the title compound as a colorless solid (41 mg, 26%).

[00751] 1H-NMR (DMSO-d6) δ: 1.13-1.24 (2H, m), 1.42-1.53 (4H, m), 2.12 (2H, t, J = 7 .3 Hz), 2.82 (1H, dd, J = 13.9, 10.0 Hz), 3.09 (1H, dd, J = 13.9, 4.8 Hz), 3.17 (2H , d, J = 4.2 Hz), 3.47-3.89 (8H, m), 4.084.14 (1H, m), 4.41-4.49 (1H, m), 4.58- 4.69 (2H, m), 7.00 (2H, s), 7,147.27 (5H, m), 8.31 (1H, t, J = 6.0 Hz), 8.39 (1H, brs ), 8.55 (2H, brs), 8.93 (1H, brs).

[00752] MS (APCI) m/z: 615 (M-H) -

Process 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]pyran [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

[00753] In a solution of N,N-dimethylformamide (10 mL) of methanesulfonic acid salt of exatecan (22 mg, 0.388 mmol), triethylamine (5.42 μL, 0.388 mmol), the compound (29 mg, 0.466 mmol) ) obtained in Process 5 above, 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 solvent in the reaction solution was removed under reduced pressure, and then the obtained residue was purified by silica gel column chromatography [chloroform - split organic layer of chloroform : methanol : water = 7 : 3 : 1 (v/v /v)] to yield the title compound as a pale yellow solid (26 mg, 65%).

[00754] 1H-NMR (DMSO-d6) δ: 0.87 (3H, t, J = 7.3 Hz), 1.12-1.22 (2H, m), 1.40-1.51 ( 4H, m), 1.79-1.92 (2H, m), 2.09 (2H, t, J = 7.6 Hz), 2.13-2.23 (2H, m), 2.39 (3H, s), 2.78 (1H, dd, J = 13.6, 9.4 Hz), 2.983.05 (1H, m), 3.13-3.23 (2H, m), 3, 54-3.78 (8H, m), 4.02 (2H, s), 4.414.50 (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 = 10.9 Hz), 8.01 (1H, t, J = 5.4 Hz), 8.07 (1H , t, J = 5.7 Hz), 8.14 (1H, d, J = 7.9 Hz), 8.31 (1H, t, J = 5.7 Hz), 8.53 (1H, d , J = 9.1 Hz), 8.63 (1H, t, J = 6.3 Hz).

[00755] MS (APCI) m/z: 1034 (M+H)+

Process 7: Antibody-Drug Conjugate (44)

[00756] Using the trastuzumab produced in Reference Example 1 and the compound obtained in Process 6 above, the titrated antibody-drug conjugate was obtained in the same manner as Process 1 of Example 7.

[00757] Antibody concentration: 1.87 mg/mL, antibody yield: 16.8 mg (84%), and average number of conjugated drug molecules (n) per antibody molecule: 6.1. Example 45 Intermediate (45)

Process 1: (2-{[(1S, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa- tert-butyl hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethyl)carbamate

[00758] In a dichloromethane solution (3.00 mL) of N-(tert-butoxycarbonyl)-glycine (0.395 g, 2.26 mmol), N-hydroxysuccinimide (0.260 g, 2.26 mmol) and 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.433 mg, 2.26 mmol) was added and stirred at room temperature for 1 hour. This solution was added to a solution consisting of methanesulfonic acid salt of exatecan (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 and washed with 10% citric acid solution, and then the organic layer was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the obtained residue was purified by silica gel column chromatography [chloroform-chloroform : methanol = 9 : 1 (v/v)] to yield the title compound as a yellow solid (1, 16 g, 99%).

[00759] 1H-NMR (400 MHz, DMSO-d6) δ: 0.86 (3H, t, J = 7.2 Hz), 1.30 (9H, s), 1.81-1.89 (2H , m), 2.09-2.21 (2H, m), 2.38 (3H, s), 3.153.17 (2H, m), 3.55-3.56 (2H, m), 5, 15 (1H, d, J = 18.8 Hz), 5.23 (1H, d, J = 19.2 Hz), 5.41 (2H, s), 5.55-5.56 (1H, m ), 6.53 (1H, s), 6.95 (1H, t, J = 5.5 Hz), 7.28 (1H, s), 7.77 (1H, d, J = 11.0 Hz ), 8.39 (1H, d, J = 8.6 Hz).

[00760] MS (APCI) m/z: 593 (M+H)+

Process 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

[00761] The compound (0.513 g, 1.01 mmol) obtained in Process 1 above was reacted in the same manner as Process 2 of Example 1 to yield the title compound as a yellow solid (0.463 g, 93%).

[00762] 1H-NMR (400 MHz, CD3OD) δ: 0.96 (3H, t, J = 7.0 Hz), 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 = 16.0 Hz), 5.48 ( 1H, d, J = 16.0 Hz), 5.62-5.64 (1H, m), 7.40-7.45 (2H, m).

[00763] MS (APCI) m/z: 493 (M+H)+ Example 46 Antibody-Drug Conjugate (46)

Process 1: Antibody-Drug Conjugate (46)

[00764] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (50 mL) was placed in a 125 mL polycarbonate Erlenmeyer flask, charged with a 1 M aqueous solution of dipotassium hydrogen phosphate at room temperature (0.750 mL) with stirring using a magnetic stirrer, and then charged with a 10 mM aqueous solution of TCEP (1.857 mL; 5.4 equivalents per antibody molecule). After confirming that the solution had a pH of 7.0 ± 0.1, stirring was terminated, and disulfide binding to the hinge part on the antibody was reduced by incubating at 37°C for 1 hour.

[00765] Conjugation between antibody and drug ligand: After cooling the above solution to 15°C, a DMSO solution containing 10 mM of the compound of Process 8 of Example 26 (2.958 mL; 8.6 equivalents per molecule of antibody ) was gradually added thereto dropwise with stirring. At 15°C, the reaction solution was shaken for the first 30 minutes and incubated without shaking to conjugate the drug binder to the antibody for the next 1 hour. Then, an aqueous solution (0.444 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was added thereto with stirring and stirred at room temperature to terminate the reactivity of unreacted drug binders for another 20 minutes.

[00766] Purification: By gradually adding 20% aqueous acetic acid solution (about 0.25 mL) and ABS (50 mL) to the above solution with stirring, the pH of the solution was adjusted to 5.5 ± 0. 1. This solution was subjected to microfiltration (Millipore Corp., Millex-HV filter, 0.45 μm, PVDF membrane) to remove whitish matter. This solution was subjected to purification by ultrafiltration using an ultrafiltration mechanism composed of an ultrafiltration membrane (Merck Japan, Pellicon XL Cassette, Biomax 50 KDa), a tube pump (Cole-Parmer International, MasterFlex Pump model 77521-40, Pump Head model 7518-00), and a tube (Cole-Parmer International, MasterFlex Tube L/S16). Specifically, at the same time that ABS was added dropwise (a total of 800 mL) as a buffering solution for purification to the reaction solution, purification by ultrafiltration was performed to remove unconjugated drug binders and other low molecular weight reagents. , likewise replacing the buffering solution with ABS, and concentrating the solution. The purified solution obtained was subjected to microfiltration (0.22 μm (Millipore Corp., Millex-GV filter, PVDF membrane) and 0.10 μm (Millipore Corp., Millex-VV filter, PVDF membrane)) to produce 42 .5 mL of a solution containing the titrated antibody-drug conjugate.

[00767] Physicochemical Characterization: Using Common procedures E and F and (SD,28Q = 5178 (measured value), and SD,3?Q = 20217 (measured value) were used), the following characteristic values were obtained.

[00768] Antibody concentration: 10.4 mg/mL, antibody yield: 442 mg (88.5%), average number of conjugated drug molecules (n) per antibody molecule measured by procedure Common E: 6.0 , and mean number of conjugated drug molecules (n) per antibody molecule measured by the Common F procedure: 7.5. Example 47 Antibody-Drug Conjugate (47)

Process 1: Antibody-Drug Conjugate (47)

[00769] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (15 mL) was placed in a polypropylene tube and loaded with a 10 mM aqueous solution of TCEP (0.567 mL; 5.5 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (0.225 mL). After confirming that the solution had a pH of 7.0 ± 0.1, the disulfide bond to the hinge part on the antibody was reduced by incubating at 37°C for 2 hours.

[00770] Conjugation between antibody and drug ligand: After adding DMSO (0.146 mL) and a DMSO solution containing 10 mM of the Process 8 compound of Example 26 (0.928 mL; 9.0 equivalents per antibody molecule) to the above solution at room temperature, this was incubated to conjugate drug binder to antibody at 15°C for 30 minutes. Then, an aqueous solution (0.133 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was added thereto and stirred at room temperature to terminate the reactivity of unreacted drug binders for another 20 minutes.

[00771] Purification: The above solution was subjected to purification using the Common D procedure (ABS was used as a buffering solution) to yield 49 mL of a solution containing the compound of interest.

[00772] Physicochemical Characterization: Using the Common E procedure (SD,280 = 5178 and SD,3?O = 20217 were used), the following characteristic values were obtained.

[00773] Antibody concentration: 2.91 mg/mL, antibody yield: 143 mg (95%), and average number of conjugated drug molecules (n) per antibody molecule: 6.2. Example 48 Antibody-Drug Conjugate (48)

Process 1: Antibody-Drug Conjugate (48)

[00774] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (280 mL) was placed in a 1000 mL polycarbonate Erlenmeyer flask, charged with a room temperature 1 M aqueous solution of dipotassium hydrogen phosphate (4,200 mL) with stirring using a magnetic stirrer, and then charged with a 10 mM aqueous solution of TCEP (10.594 ml; 5.5 equivalents per antibody molecule). After confirming that the solution had a pH of 7.4 ± 0.1, stirring was terminated, and disulfide binding to the hinge part on the antibody was reduced by incubating at 37°C for 2 hours.

[00775] Conjugation between antibody and drug ligand: After cooling the above solution to 15°C, a DMSO solution containing 10 mM of the compound of Process 8 of Example 26 (17.335 mL; 9.0 equivalents per molecule of antibody ) was gradually added thereto dropwise with stirring. The reaction solution was stirred at 15°C to conjugate drug ligand to antibody for 30 minutes. Then, an aqueous solution (2.485 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was added thereto with stirring and stirred at room temperature to terminate the reactivity of unreacted drug binders for another 20 minutes.

[00776] Purification: By gradually adding 20% aqueous acetic acid solution (about 1.4 mL) and ABS (280 mL) to the above solution with stirring, the pH of the solution was adjusted to 5.5 ± 0. 1. This solution was subjected to microfiltration (0.45 μm, PVDF membrane) to remove whitish matter while producing about 600 mL of a filtrate. This solution was subjected to purification by ultrafiltration using an ultrafiltration mechanism composed of an ultrafiltration membrane (Merck Japan, Pellicon XL Cassette, Biomax 50 KDa), a tube pump (Cole-Parmer International, MasterFlex Pump model 77521-40, Pump Head model 7518-00), and a tube (Cole-Parmer International, MasterFlex Tube L/S16). Specifically, at the same time that ABS was added dropwise (a total of 4800 mL) as a buffering solution for purification to the reaction solution, purification by ultrafiltration was performed to remove unconjugated drug binders and other low molecular weight reagents. , likewise replacing the buffering solution with ABS, and also concentrating the solution. The purified solution obtained was subjected to microfiltration (twice with 0.22 μm and 0.10 μm, PVDF membrane) to produce 70 ml of a solution containing the titrated antibody-drug conjugate.

[00777] Physicochemical Characterization: Using the Common procedure E and (SD,28Q = 5178 and SD,3?Q = 20217 were used), the following characteristic values were obtained.

[00778] Antibody concentration: 35.96 mg/ml, antibody yield: 2517 mg (90%), and average number of conjugated drug molecules (n) per antibody molecule: 6.2. Example 49 Antibody-Drug Conjugate (49)

Process 1: Antibody-Drug Conjugate (49)

[00779] Antibody Reduction: The trastuzumab produced in Reference Example 1 was prepared to have an antibody concentration of 10 mg/mL by replacing the medium with PBS6.0/EDTA using Common procedure C-1 and Common procedure B (as absorption coefficient at 280 nm, 1.48 mLmg-1cm-1 was used). The solution (280 mL) was placed in a 1000 mL polycarbonate Erlenmeyer flask, charged with a 1 M aqueous solution of dipotassium hydrogen phosphate at room temperature (4,200 mL) with stirring using a magnetic stirrer, and then charged with a 10 mM aqueous solution of TCEP (10.594 ml; 5.5 equivalents per antibody molecule). After confirming that the solution had a pH of 7.0 ± 0.1, stirring was terminated, and disulfide binding to the hinge part on the antibody was reduced by incubating at 37°C for 2 hours.

[00780] Conjugation between antibody and drug ligand: After cooling the above solution to 15°C, a DMSO solution containing 10 mM of the compound of Process 8 of Example 26 (17.335 mL; 9.0 equivalents per molecule of antibody ) was gradually added thereto dropwise with stirring. The reaction solution was stirred at 15°C to conjugate drug ligand to antibody for 30 minutes. Then, an aqueous solution (2.485 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was added thereto with stirring and stirred at room temperature to terminate the reactivity of unreacted drug binders for another 20 minutes.

[00781] Purification: By gradually adding 20% aqueous acetic acid solution (about 1.4 mL) and ABS (280 mL) to the above solution with stirring, the pH of the solution was adjusted to 5.5 ± 0. 1. This solution was subjected to microfiltration (0.45 μm, PVDF membrane) to remove whitish matter while producing about 600 mL of a filtrate. This solution was subjected to purification by ultrafiltration using an ultrafiltration mechanism composed of an ultrafiltration membrane (Merck Japan, Pellicon XL Cassette, Ultracell 30 KDa), a tube pump (Cole-Parmer International, MasterFlex Pump model 77521-40, Pump Head model 7518-00), and a tube (Cole-Parmer International, MasterFlex Tube L/S16). Specifically, at the same time that ABS was added dropwise (a total of 4800 mL) as a buffering solution for purification to the reaction solution, purification by ultrafiltration was performed to remove unconjugated drug binders and other low molecular weight reagents. , likewise replacing the buffering solution with ABS, and also concentrating the solution. The purified solution obtained was subjected to microfiltration (twice with 0.22 μm and 0.10 μm, PVDF membrane) to produce 130 mL of a solution containing the titrated antibody-drug conjugate.

[00782] Physico-chemical characterization: Using common Procedure E (SD, 280 = 5178 and SD, 370 = 20217 were used), the following characteristic values were obtained.

[00783] Antibody concentration: 21.00 mg/mL, antibody production: 2730 mg (97.5%), and average number of conjugated drug molecules (n) per antibody molecule: 6.3.

Example 50 Antibody-Drug Conjugate (50) Process 1: Antibody-Drug Conjugate (50)

[00784] The antibody-drug conjugates (47), (48), and (49) produced in Examples 47, 48, and 49 were mixed (243 mL) and also loaded with ABS (39.75 mL) to yield 283 mL of a solution containing the titrated antibody-drug conjugate.

[00785] Physicochemical characterization: Using Common procedures E and F (sD, 280 = 5178, and sD, 370 = 20217 were used), the following characteristic values were obtained.

[00786] Antibody concentration: 20.0 mg/mL, antibody production: 5655 mg, average number of conjugated drug molecules (n) per antibody molecule measured by Common Procedure E: 6.3, and average number of drug molecules conjugates (n) per antibody molecule measured by Common Procedure F: 7.8.

Evaluation Example 1 Cell growth inhibitory effect (1) of antibody-drug conjugate

[00787] Human breast cancer line KPL-4 of HER2 antigen positive cells (Dr. Junichi Kurebayashi, Kawasaki Medical School, British Journal of Cancer, (1999) 79 (5/6). 707-717) or lineage antigen-negative cell MCF7 human breast cancer (European Collection of Cell Cultures; ECACC) was cultured in RPMI1640 (GIBCO) containing 10% fetal bovine serum (MOREGATE) (hereinafter referred to as medium). or MCF7 were prepared to have a concentration of 2.5 (10 4 cells/mL using medium, added at a concentration of 100 μL/well to a 96-well cell culture microplate, and cultured overnight.

[00788] The next day, trastuzumab or the antibody-drug conjugate diluted in 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM using medium was added in a concentration of 10 μL/well to the microplate. Medium was added at a concentration of 10 μL/well to wells not supplemented with antibody. Cells were cultured under 5% CO2 at 37oC for 5 to 7 days. After culture, the microplate was removed from the incubator and allowed to rest at room temperature for 30 minutes. The culture solution was loaded with an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) and shaken. After the microplate was allowed to stand at room temperature for 10 minutes, the luminescence intensity of each well was measured using a plate reader (PerkinElmer). The IC50 value was calculated according to the following equation: IC50 (nM) = antilog((50 - d) x (LOG10(b) - LOG10 (a)) / (d - c) + LOG10 (b)) a : Sample concentration a b: Sample concentration b c: Cell Viability of sample a d: Cell Viability of sample b

[00789] Cell viability at each concentration was calculated according to the following equation: Cell viability (%) = a / b x 100 a: Average luminescence intensity of sample wells (n = 2) b: Luminescence intensity mean of non-supplemented antibody wells (n = 10)

[00790] Antibody-drug conjugates (2), (3), (5), (7), (10), (12), (13), (16), (18), (40), and (42) exhibited a cell growth inhibitory effect of IC50 < 0.1 (nM) against KPL-4 cells.

[00791] Antibody-drug conjugates (4), (6), (9), (15), (17), (21), (22), (25), (36), (37), ( 39), (41), and (43) exhibited a cell growth inhibitory effect of 0.1 < IC50 < 1 (nM) against the cells.

[00792] Antibody-drug conjugates (20), (24), and (27) exhibited a cell growth inhibitory effect of 1 < IC50 < 100 (nM) against cells. None of the antibody-drug conjugates (19) or (26) exhibited a cell growth inhibitory effect against cells (>100 (nM)).

[00793] On the other hand, antibody-drug conjugates (5), (13), and (43) exhibited a cell growth inhibitory effect of 1 < IC50 < 100 (nM) against MCF7 cells, whereas none of antibody-drug conjugates (2), (3), (4), (6), (7), (9), (10), (12), (15), (16), (17), (18), (25), (26), (27), (39), (40), (41), (42), and (44) exhibited a cell growth inhibitory effect against cells (>100 (nM)).

[00794] Trastuzumab exhibited a cell growth inhibitory effect against neither KPL-4 cells nor MCF7 cells (>100 (nM)).

Assessment Example 2 Antitumor test (1)

[00795] Mouse: 5 to 6 week old female nude mice (Charles River Laboratories Japan, Inc.) were acclimated for 4 to 7 days under SPF conditions prior to use in the experiment. Mice were fed sterile solid food (FR-2, Funabashi Farms Co., Ltd) and given sterile tap water (prepared by adding 5 to 15 ppm sodium hypochlorite solution).

[00796] Assay and Calculus Expression: In all studies, the major axis and minor axis of a tumor were measured twice weekly using an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and the tumor volume ( mm3) was calculated. The calculation expression is as shown below. Tumor volume (mm3)= ½ x Major axis (mm) x [Minor axis (mm)]2

[00797] All antibody-drug and antibody conjugates were diluted with physiological saline (Otsuka Pharmaceutical Factory, Inc.) and used in a volume of 10mL/kg for intravenous administration into the tail vein of each mouse.

[00798] KPL-4 cells were suspended in physiological saline, and 1.5 x 10 7 cells were subcutaneously transplanted into the right side of the body of each female nude mouse (Day 0), and mice were randomly pooled on Day 15. Antibody-drug conjugates (27) or the anti-HER2 antibody trastuzumab (Reference Example 1) to a control group were intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Days 15 and 22. One untreated group was established as a control group.

[00799] The results are shown in Figure 3. Administration of trastuzumab inhibited tumor growth whereas administration of the antibody-drug conjugate (27) produced a more significant tumor growth inhibitory effect. In the drawing, the abscissa describes days after tumor inoculation, and the ordinate describes the volume of the tumor. Furthermore, mice that received trastuzumab or the antibody-drug conjugate (27) were free of notable signs such as weight loss, suggesting that the antibody-drug conjugate (27) is highly safe. In the Evaluation Examples below relating to the antitumor test, the test was conducted by the procedure used in this Evaluation Example, unless otherwise specified.

Assessment Example 3 Antitumor test (2)

[00800] NCI-N87 cells of human gastric cancer lineage acquired from ATCC (American Type Culture Collection) were suspended in physiological saline, and 1 x 10 7 cells were subcutaneously transplanted into the right side of the body of each female nude mouse (Day 0 ), and mice were randomized grouped on Day 7. Antibody-drug conjugates (8) or (28), or trastuzumab emtansine (Reference Example 2) were intravenously administered at a dose of 10 mg/kg into the tail vein. of each mouse on Day 7. An untreated group was established as a control group.

[00801] The results are shown in Figure 4. Antibody-drug conjugates (8) and (28) were confirmed to have a strong antitumor effect with tumor regression equivalent to that of trastuzumab emtansine. Furthermore, administration of antibody-drug conjugate (8) or (28), or trastuzumab emtansine was found to be free of weight loss in mice.

Assessment Example 4 Antitumor test (3)

[00802] JIMT-1 cells of human breast cancer lineage acquired from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) were suspended in physiological saline, and 3 x 10 6 cells were subcutaneously transplanted to the right side of the body of each nude mouse (Day 0), and mice were randomly pooled on Day 12. Antibody-drug conjugates (8), (29), or (30), trastuzumab, or trastuzumab emtansine were administered intravenously at a dose of 10 mg/day. kg to the tail vein of each mouse on Days 12 and 19. A physiological saline administration group was established as a control group.

[00803] Results are shown in Figure 5. Administration of trastuzumab or trastuzumab emtansine did not inhibit JIMT-1 tumor growth. On the other hand, administration of the antibody-drug conjugate (8), (29), or (30) inhibited tumor growth. Furthermore, administration of the antibody-drug conjugate (8), (29), or (30), trastuzumab, or trastuzumab emtansine was found to be free of weight loss in mice.

Evaluation Example 5 Cell growth inhibitory effect (2) of antibody-drug conjugate

[00804] Calu-3 human non-small cell lung cancer (ATCC) strain was cultured in Eagle's Minimum Essential Medium (GIBCO) containing 10% fetal bovine serum (MOREGATE) (hereinafter referred to as MEM medium).

[00805] Human gastric cancer strain NCI-N87 (ATCC) or human gastric cancer strain MKN-45 (Health Science Research Resources Bank) was cultured in RPMI1640 Medium (GIBCO) containing 10% fetal bovine serum (hereinafter referred to as as an RPMI medium).

[00806] Human breast cancer line MDA-MB-453 (ATCC) or human breast cancer line MDA-MB-468 (ATCC) was cultured in Leibovitz L-15 Medium (GIBCO) containing 10% bovine serum fetal tissue (hereinafter referred to as Leibovitz's medium).

[00807] Among these 5 types of cell lines, Calu-3, NCI-N87, and MDA-MB-453 are HER2 positive cells, and MKN-45 and MDA-MB-468 are HER2 negative cells.

[00808] Calu-3, NCI-N87, or MKN-45 was prepared to have a concentration of 4 x 10 4 cells/mL using MEM medium or RPMI medium, added at a concentration of 25 μL/well to a microplate of 96 wells for cell culture loaded with 65 μL/well of medium, and grown overnight under 5% CO2 at 37oC. Likewise, MDA-MB-453 or MDA-MB-468 was prepared to have a concentration of 4 x 104 cells/mL using Leibovitz's medium, added at a concentration of 25 μL/well in a 96 microplate. wells for cell culture loaded with 65 μL/well of medium, and cultured overnight at 37oC without fixing the CO2 concentration.

[00809] The next day, a test substance diluted in 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM using RPMI medium or Leibovitz medium, or RPMI medium or Leibovitz medium was added at a concentration of 10 μL/well to the microplate. Cells were cultured under 5% CO2 at 37oC or 37oC without fixing the CO2 concentration for 6 days.

[00810] For Calu-3, NCI-N87, and MDA-MB-468, antibody-drug conjugate (46) was added as the test substance. For the other cells, the antibody-drug conjugate (50) was added as the test substance. After culture, the microplate was removed from the incubator and allowed to rest at room temperature for 30 minutes. The culture solution was loaded with an equal amount of CellTiter-Glo Luminescent Cell Viability Assay (Promega) and shaken with a plate shaker to completely lyse the cells. After the microplate was allowed to stand at room temperature for 10 minutes, luminescence intensity was measured using a plate reader.

[00811] Cell viability was calculated according to the following equation: Cell viability (%) = a / b x 100 a: Mean luminescence intensity of wells supplemented with test substance b: Mean luminescence intensity of wells supplemented with medium

[00812] The IC50 value was calculated according to the following equation: IC50 (nM) = antilog ((50 - d) x (LOG10 (b) - LOG10 (a)) / (d - c) + LOG10 (b )) a: Test substance concentration a b: Test substance concentration b c: Viability of cell supplemented with test substance having concentration a d: Viability of cell supplemented with test substance having concentration b

[00813] Concentrations a and b establish the relationship a > b crossing 50% cell viability.

[00814] The antibody-drug conjugate (46) exhibited a cell growth inhibitory effect of IC50 < 1 (nM) against Calu-3 and NCI-N87 HER2 positive cells. On the other hand, the antibody-drug conjugate (46) did not exhibit any cell growth inhibitory effect against HER2 negative MDA-MB-468 cells (>100 (nM)).

[00815] The antibody-drug conjugate (50) exhibited a cell growth inhibitory effect of IC50 < 1 (nM) against MDA-MB-453 HER2 positive cells. On the other hand, the antibody-drug conjugate (50) did not exhibit cell growth inhibitory effect against HER2 negative MKN-45 cells (> 100 (nM)).

Assessment Example 6 Anti-tumor test (4)

[00816] Human pancreatic cancer (ATCC) lineage Capan-1 cells weakly expressing HER2 were suspended in physiological saline, and 4 x 10 7 cells were subcutaneously transplanted into the right side of the body of each female nude mouse to generate the solid tumor of Capan-1. Thereafter, this solid tumor was maintained in several transplant passages in female nude mice and used in this test. A tumor section of the solid tumor was subcutaneously transplanted into the right side of the body of each female nude mouse (Day 0), and the mice were randomly pooled on Day 20.

[00817] Antibody-drug conjugates (31), trastuzumab, or trastuzumab emtansine were intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 20. A physiological saline administration group was established as a control group.

[00818] Results are shown in Figure 6. Administration of trastuzumab or trastuzumab emtansine did not inhibit Capan-1 tumor growth. By comparison, administration of the antibody-drug conjugate (31) inhibited tumor growth, demonstrating that the antibody-drug conjugate (31) is effective even for tumors with low HER2 expression. Antibody-drug conjugates (31) do not inhibit the growth of HER2 not expressing the gastric cancer lineage GCIY tumor.

[00819] Regarding tumor HER2 expression, based on the results of immunohistochemical staining measurement described in the 3rd edition of the HER2 testing guidelines (developed by the Japanese Pathology Board for Optimal Use of Trastuzumab, The Japanese Society Pathology), the classification was performed such that the count of 3+: high expression, 2+: moderate expression, and 1+: low expression. Although the count was 0 in this measurement method, tumor found to be HER2 positive by other measurement methods such as a measurement method using a flow cytometer was classified as weakly expressing tumor.

Assessment Example 7 Anti-tumor test (5)

[00820] Human gastric cancer lineage NCI-N87 cells acquired from ATCC were suspended in physiological saline, and 1 x 10 7 cells were subcutaneously transplanted into the right side of the body of each female nude mouse (Day 0), and the mice were randomly pooled on Day 6. Antibody-drug conjugate (50) was intravenously administered at each dose of 0.3, 1, 3, or 10 mg/kg into the tail vein of each mouse on Day 6. of acetate buffering solution was established as a control group.

[00821] The results are shown in Figure 7. The antibody-drug conjugate (50) exhibited a dose-dependent antitumor effect. Furthermore, administration of the antibody-drug conjugate (50) was found to be free of weight loss in mice.

Assessment Example 8 Anti-tumor test (6)

[00822] This test was performed by the following method.

[00823] Mouse: 6 to 12 week old female nude mice (Charles River Laboratories Japan, Inc.) were subjected to the experiment.

[00824] Assay and calculation expression: Tumor major axis and minor axis were measured twice weekly using an electronic digital caliper, and the tumor volume (mm3) was calculated. The calculation expression is as shown below. Tumor volume (mm3) = 0.52 (Major axis (mm) x [Minor axis (mm)]2

[00825] The antibody-drug conjugate, trastuzumab, and trastuzumab emtansine was diluted with an acetate buffer solution and used in a volume of 10 mL/kg for intravenous administration into the tail vein of each mouse.

[00826] Tumor (ST225; South Texas Accelerated Research Therapeutics (START)) cut from a breast cancer patient and maintained at multiple transplant passages in female nude mice was used in this test. This tumor moderately expressed HER2 (which received a count of 2+ on immunohistochemical staining).

[00827] A tumor section of the solid tumor was subcutaneously transplanted into the side of the body of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of administration of acetate buffering solution was established as a control group.

[00828] The results are shown in Figure 8. The administration of trastuzumab did not inhibit the growth of the moderately HER2 expressing ST225 breast cancer tumor. By comparison, administration of trastuzumab emtansine or the antibody-drug conjugate (50) remarkably inhibited tumor growth.

Assessment Example 9 Anti-tumor test (7)

[00829] Tumor (ST910;START) cut from a breast cancer patient and maintained at multiple transplant passages in female nude mice was used in this test. This tumor weakly expressed HER2 (which received a count of 1+ on immunohistochemical staining).

[00830] A tumor section of the solid tumor was subcutaneously transplanted into the body side of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of administration of acetate buffering solution was established as a control group.

[00831] The results are shown in Figure 9. The administration of trastuzumab or trastuzumab emtansine did not inhibit the growth of the ST910 breast cancer tumor weakly expressing HER2. By comparison, administration of antibody-drug conjugate (50) remarkably inhibited tumor growth, demonstrating that antibody-drug conjugate (50) is effective for breast cancer tumor weakly expressing HER2. This Assessment Example 9 was performed by the same procedure as Assessment Example 8.

Assessment Example 10 Anti-tumor test (8)

[00832] This test was performed by the following procedure. Evaluation examples 11 to 13 were similarly performed by this procedure.

[00833] Mouse: 5- to 8-week-old female nude mice (Harlan Laboratories Ltd.) were subjected to the experiment.

[00834] Assay and calculation expression: Tumor major axis and minor axis were measured twice weekly using an electronic digital caliper, and the tumor volume (mm3) was calculated. The calculation expression is as shown below. Tumor volume (mm3) = 0.52 x Major axis (mm) x [Minor axis (mm)]2

[00835] The antibody-drug conjugate, trastuzumab, and trastuzumab emtansine were diluted with an acetate buffering solution and used in a volume of 10 mL/kg for intravenous administration into the tail vein of each mouse.

[00836] Tumor (CTG-0401; Champions Oncology Inc.) cut from a colorectal cancer patient and maintained at multiple transplant passages in female nude mice was used in this test. This tumor expressed weakly or moderately HER2 (which received a count of 1+ or 2+ on immunohistochemical staining).

[00837] A tumor section of the solid tumor was subcutaneously transplanted into the left side of the body of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of administration of acetate buffering solution was established as a control group.

[00838] The results are shown in Figure 10. Administration of trastuzumab or trastuzumab emtansine did not inhibit the growth of the CTG-0401 colorectal cancer tumor weakly or moderately expressing HER2. By comparison, administration of the antibody-drug conjugate (50) remarkably inhibited tumor growth.

Assessment Example 11 Antitumor test (9)

[00839] Tumor (CTG-0860; Champions Oncology Inc.) cut from a non-small cell lung cancer patient and maintained at multiple transplant passages in female nude mice was used in this test. This tumor moderately expressed HER2 (which received a count of 2+ on immunohistochemical staining).

[00840] A tumor section of the solid tumor was subcutaneously transplanted into the left side of the body of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of administration of acetate buffering solution was established as a control group.

[00841] The results are shown in Figure 11. Administration of trastuzumab or trastuzumab emtansine did not inhibit the growth of the moderately HER2 expressing non-small cell lung cancer CTG-0860 tumor. By comparison, administration of the antibody-drug conjugate (50) remarkably inhibited tumor growth.

Assessment Example 12 Antitumor test (10)

[00842] Tumor (CTG-0927; Champions Oncology Inc.) cut from a patient with bile duct cancer and maintained at multiple transplant passages in female nude mice was used in this test. This tumor expressed HER2 highly (which received a count of 3+ on immunohistochemical staining).

[00843] A tumor section of the solid tumor was subcutaneously transplanted into the left side of the body of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of administration of acetate buffering solution was established as a control group.

[00844] The results are shown in Figure 12. The administration of trastuzumab did not inhibit the growth of the highly HER2-expressing bile duct cancer CTG-0927 tumor. By comparison, trastuzumab emtansine administration inhibited tumor growth. Furthermore, administration of the antibody-drug conjugate (50) induced tumor regression.

Assessment Example 13 Antitumor test (11)

[00845] Tumor (CTG-0137; Champions Oncology Inc.) cut from an esophageal cancer patient and maintained at multiple transplant passages in female nude mice was used in this test. This tumor expressed HER2 highly (which received a count of 3+ on immunohistochemical staining).

[00846] A tumor section of the solid tumor was subcutaneously transplanted into the left side of the body of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of administration of acetate buffering solution was established as a control group.

[00847] The results are shown in Figure 13. The administration of trastuzumab did not inhibit the growth of the CTG-0137 tumor of highly HER2-expressing esophageal cancer. By comparison, administration of trastuzumab emtansine or the antibody-drug conjugate (50) remarkably inhibited tumor growth.

Assessment Example 14 Antitumor test (12)

[00848] Human ovarian cancer lineage SK-OV-3 cells highly expressing HER2 acquired from ATCC were suspended in physiological saline, and 4 x 10 7 cells were subcutaneously transplanted into the right side of the body of each female nude mouse to prepare the tumor solid for SK-OV-3. Thereafter, this solid tumor was maintained in several transplant passages in female nude mice and used in this test.

[00849] A tumor section of the solid tumor was subcutaneously transplanted into the right side of the body of each female nude mouse, and the mice were randomly grouped when the tumor volume reached 100 to 300 mm3. Pooling data was defined as Day 0. The antibody-drug conjugate (50), trastuzumab, or trastuzumab emtansine was intravenously administered at a dose of 10 mg/kg into the tail vein of each mouse on Day 0. A group of Physiological saline administration was established as a control group.

[00850] Results are shown in Figure 14. Administration of trastuzumab did not inhibit SK-OV-3 tumor growth. By comparison, administration of trastuzumab emtansine or the antibody-drug conjugate (50) remarkably inhibited tumor growth.

SEQUENCE LISTING FREE TEXT

[00851] SEQ ID NO: 1 - Amino acid sequence of a humanized anti-HER2 monoclonal antibody heavy chain

[00852] SEQ ID NO: 2 - Amino acid sequence of a humanized anti-HER2 monoclonal antibody light chain.

Claims (13)

1. Antibody-drug conjugate, characterized in that an antitumor compound represented by the following formula:

is conjugated to an anti-HER2 antibody as in the form of a drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O -CH2-C(=O)-(NH-DX), or -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(= O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), via a thioether bond that is formed in a disulfide-bonding moiety present in the hinge part of the anti-HER2 antibody, in which - (Succinimid-3-yl-N)- has a structure represented by the following formula:

which is attached to the anti-HER2 antibody at position 3 thereof and is attached to the methylene group in the linker structure containing this structure at the nitrogen atom at position 1, - (NH-DX) represents a group represented by the following formula:

wherein the nitrogen atom of the amino group at position 1 is the connecting position, -GGFG- represents the tetrapeptide residue of -Gly-Gly-Phe-Gly-, and the anti-HER2 antibody comprises a heavy chain consisting of a sequence of amino acids consisting of amino acid residues 1 to 449 of SEQ ID NO: 1 and a light chain consisting of an amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2, or comprises a heavy chain consisting of the sequence of amino acids represented by SEQ ID NO: 1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 2. 2. Antibody-drug conjugate, according to claim 1, characterized in that the drug-ligand structure is: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH - CH2CH2-O-CH2-C(=O)-(NH-DX). 3. Antibody-drug conjugate, according to claim 1, characterized in that the drug-ligand structure is: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2 -O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). 4. Antibody-drug conjugate according to any one of claims 1 to 6, characterized in that the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence consisting of amino acid residues 1 to 449 of SEQ ID NO : 1 and a light chain consisting of the amino acid sequence consisting of amino acid residues 1 to 214 of SEQ ID NO: 2. 5. Antibody-drug conjugate according to any one of claims 1 to 3, characterized in that the anti-HER2 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of in the amino acid sequence represented by SEQ ID NO: 2. 6. Antibody-drug conjugate according to any one of claims 1 to 5, characterized in that the average number of units of an antibody-conjugated selected drug-ligand structure is in the range 2 to 8. 7. Antibody-drug conjugate according to any one of claims 1 to 5, characterized in that the average number of units of an antibody-conjugated selected drug-ligand structure is in the range of 3 to 8. 8. Drug, characterized in that it contains the antibody-drug conjugate, as defined in any one of claims 1 to 7, a salt thereof, or a hydrate thereof. 9. Antitumor drug and/or anticancer drug, characterized in that it contains the antibody-drug conjugate, as defined in any one of claims 1 to 7, a salt thereof, or a hydrate thereof. 10. Antitumor drug and/or anticancer drug, according to claim 9, characterized in that it is for use against lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma. 11. Pharmaceutical composition, characterized in that it contains the antibody-drug conjugate, as defined in any one of claims 1 to 7, a salt thereof, or a hydrate thereof, as an active component, and a formulation component pharmaceutically acceptable. 12. Pharmaceutical composition according to claim 11, characterized in that it is for use against lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, cancer gastric cancer, gastrointestinal stromal tumor, uterine cervix cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma. 13. Use of an antibody-drug conjugate, as defined in any one of claims 1 to 7, a salt thereof, or a hydrate thereof, characterized in that it is for the preparation of a drug, as defined in claim 8, or a pharmaceutical composition, as defined in claim 11, for treating tumor and/or cancer.

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