Anti-trop2 antibody-drug conjugate

Abstract

To provide a method for producing an antibody-drug conjugate that exhibits an excellent therapeutic effect with superior antitumor activity and safety.SOLUTION: There is provided a method for producing an antibody-drug conjugate, where the following drug-linker structure: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX) is conjugated to an anti-TROP2 antibody via a thioether bond, comprising the steps of treating the anti-TROP2 antibody under reducing conditions and then reacting the treated antibody with the following compound: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), where -(NH-DX) represents a group represented by the following formula, and -GGFG- represents -Gly-Gly-Phe-Gly-.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for treating a tumor by binding an anti-TROP2 antibody to an antitumor drug via a linker structure. , relates to antibody-drug conjugates useful as antitumor agents. [Background technology]

[0002] Antibodies that bind to antigens that are expressed on the surface of cancer cells and can be internalized into the cells have cytotoxicity. Antibody-drug conjugates (ADCs) that bind to drugs have been shown to be effective in treating cancer. By being able to selectively deliver drugs to cancer cells, the drugs can be accumulated in cancer cells, causing cancer cells to die. It is expected that ADCs can be used to treat various diseases, such as cancers, cancers, and cancers caused by anti-CD33 antibodies (see Non-Patent Documents 1 to 3). Mylotarg (registered trademark; gemtuzumab ozogamicin) conjugated to calicheamicin was rapidly It has also been approved as a treatment for myeloid leukemia. Conjugated Adcetris® (brentuximab vedotin) in patients with Hodgkin lymphoma It has recently been approved as a treatment for differentiated large cell lymphoma (see Non-Patent Document 4). The drugs contained in the approved ADCs target DNA or tubulin.

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

[0004] [ka]

[0005] An antitumor compound (exatecan, chemical name: (1S,9S)-1-amino-9-ethyl-5-fluorouracil Oro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indo Lysino[1,2-b]quinoline-10,13(9H,15H)-dione) is a water-soluble camptothecin derivative. This compound is different from irinotecan, which is currently used clinically. The antitumor effect of irinotecan does not require enzymatic activation. It inhibits topoisomerase I more effectively than the original SN-38 and topotecan, which are also used clinically. It has strong inhibitory activity and exhibits stronger cytotoxic activity against various cancer cells in vitro. In particular, it was effective against cancer cells that exhibit resistance to SN-38 and other drugs due to the expression of P-glycoprotein. It also showed a strong antitumor effect in a mouse model in which a human tumor was subcutaneously transplanted, and clinical trials are currently underway. However, it has not yet been launched on the market (see Non-Patent Documents 5 to 10). It was unclear whether it would be effective.

[0006] DE-310 is a biodegradable carboxymethyl dextran polyalcohol polymer. It is a conjugate in which Tecan is bound via a GGF peptide spacer (Patent Document 3). By converting xatecan into a polymeric prodrug, it is possible to maintain high blood retention and By utilizing the increased permeability of tumor-forming blood vessels and tumor tissue retention, DE-310 is an enzyme-mediated cleavage of the peptide spacer, resulting in increased activity. The main component, exatecan, and exatecan with glycine bound to the amino group are continuously This results in improved pharmacokinetics. In this study, the total amount of exatecan contained in DE-310 was 100% of that contained in exatecan alone. Although the dose was lower than that of the control group, it showed higher efficacy than when administered alone. Clinical trials have been conducted on this drug, and some effective cases have been confirmed. The active substance is concentrated in tumors rather than in normal tissues. While it has been reported that DE-310 and its active substance have been confirmed to have an inhibitory effect on tumors in humans, Accumulation of the drug was not significantly different from that in normal tissues, and passive targeting was not observed in humans. As a result, DE-310 was not launched on the market. Whether exatecan will function effectively as a drug directed at such targeting was not clear.

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

[0008] Human TROP2 (TACSTD2: tumor-associated calciu m signal transducer 2,GA733-1,EGP-1,M1S1 hTROP2 is a type 1 single-pass transmembrane protein consisting of 323 amino acid residues. It is a cell membrane protein. It has been previously reported in human trophoblasts and cancer cells. It has been suggested that there exists a cell membrane protein that is involved in immune resistance, which is common to both viruses (Non-patent Document 15). However, a monoclonal antibody against a cell membrane protein of a human choriocarcinoma cell line (162-25. 3,162-46.2) have been identified and expressed on human trophoblast cells. One of the molecules was named TROP2 (Non-patent Document 16). Later, the same molecule was discovered in other research The mouse monoclonal antibody GA7 was discovered by researchers and obtained by immunizing gastric cancer cell lines. GA733-1, a tumor antigen recognized by 33 (Non-patent Document 17), and non-small cell lung cancer cells Epithelium recognized by the mouse monoclonal antibody RS7-3G11 obtained by immunization It was also called EGP-1 (non-patent document 18), but in 1995 it was discovered that the TROP2 gene The offspring were cloned and confirmed to be identical molecules (Non-Patent Document 19). The DNA and amino acid sequences of hTROP2 are publicly available in the database. For example, accession numbers such as NM_002353, NP_002344 (NCBI) It is more accessible. The hTROP2 gene is a homolog of human Trop-1 (EpCAM, EGP) with approximately 50% homology. -2, TACSTD1) constitute the TACSTD gene family (Non-Patent Document Reference 21) The hTROP2 protein contains a signal sequence consisting of 26 amino acid residues at the N-terminus, An extracellular domain consisting of 8 amino acid residues and a transmembrane domain consisting of 23 amino acid residues It consists of an intracellular domain of 26 amino acid residues. The extracellular domain contains four The apparent molecular weight is 1.0 times lower than the theoretically calculated value of 35 kilodaltons. It is known that the increase is around 0 kilodaltons (Non-Patent Document 19). To date, no physiological ligand for hTROP2 has been identified, and its molecular function has not been elucidated. Although not yet fully established, it has been shown to transmit calcium signals in tumor cells (Non-Patent Document 1). Reference 20), and Ca 2+Protein kinase C, a dependent kinase, binds to intracellular serine 3 The O3 residue is phosphorylated (Non-patent Document 18), and the intracellular domain contains a PIP2-binding site. The presence of a sequence of ribosomal proteins suggests a signal transduction function in tumor cells (Non-Patent Document twenty two). Immunohistochemical analysis of clinical specimens revealed that hTROP2 is expressed in various types of epithelial cell carcinomas. In normal tissues, it is overexpressed in epithelial cells of some tissues. It has been shown that the expression level is limited to the cytoplasm of the nucleus and is lower than that of tumor tissue (Non-Patent Documents 23-26). 27) Furthermore, hTrop2 expression is also associated with colorectal cancer (Non-patent Document 23) and gastric cancer (Non-patent Document 24). , pancreatic cancer (Non-patent Document 25), oral cancer (Non-patent Document 26), glioma (Non-patent Document 27) ) has also been reported to correlate with poor prognosis. Furthermore, a model using colon cancer cells showed that hTROP2 expression promotes anchorage-independent growth of tumor cells. It has been reported to be involved in cell proliferation and tumorigenesis in immunodeficient mice (non-patented Reference 28).

[0009] Based on information suggesting a relationship with such cancer, several anti-hTROP2 antibodies have been developed. These have been developed and their antitumor effects are being investigated. The antibody alone exhibits antitumor activity in nude mouse xenograft models (Patent Documents 5 to 8). In addition, ADCs of antibodies and anti-cellular drugs have been shown to exhibit antitumor activity (Patent Documents 9 to 12). However, their activity and range of application are still insufficient. There is an unmet medical need to therapeutically target TROP2. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Publication No. 5-59061 [Patent Document 2] Japanese Patent Application Publication No. 8-337584 [Patent Document 3] International Publication No. 1997 / 46260 [Patent Document 4] International Publication No. 2000 / 25825 [Patent Document 5] International Publication No. 2008 / 144891 [Patent Document 6] International Publication No. 2011 / 145744 [Patent Document 7] International Publication No. 2011 / 155579 [Patent Document 8] International Publication No. 2013 / 077458 [Patent Document 9] International Publication No. 2003 / 074566 [Patent Document 10] International Publication No. 2011 / 068845 [Patent Document 11] International Publication No. 2013 / 068946 [Patent Document 12] U.S. Patent No. 7,999,083 [Non-patent literature]

[0011] [Non-Patent Document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-patent document 2] Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14,529-537. [Non-patent document 3] Damle NK, Expert Opin. Biol. Ther. (2004) 4, 1445-1452. [Non-patent document 4] Senter PD, et al., Nature Biotechnology (2012) 30, 631-637.

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[0012] In tumor treatment with antibodies, even if the antibody recognizes the antigen and binds to the tumor cell, it is not effective as an anti-tumor agent. However, there are cases where the effect is insufficient, and more effective antitumor antibodies are needed. In addition, even if antitumor low molecular weight compounds have excellent antitumor effects, they may have side effects. Many of them have safety issues, such as side effects and toxicity, and there is a need to improve safety and provide better treatment. The objective of the present invention is to achieve both antitumor effects and safety. Therefore, it is an object of the present invention to obtain and provide an antitumor drug having excellent therapeutic effects. [Means for solving the problem]

[0013] The present inventors have demonstrated that anti-TROP2 antibodies are antibodies that can target tumor cells, i.e. , tumor cell recognition, tumor cell binding, or tumor cell internalization. Since it is an antibody with properties such as antitumor properties, it is possible to bind exatecan, an antitumor compound, to the antibody using a linker structure. by converting the compound into an antibody-drug conjugate in which the compound is bound to the antibody via a synthetic moiety, The antibody can be used to obtain cytotoxicity, and the antitumor compound can be used to induce tumor cell proliferation. The compound can be reliably transported to tumor cells, thereby enabling the compound to exert its antitumor effect specifically in tumor cells. Therefore, it is possible to reliably exert an antitumor effect and to control the dosage of the antitumor compound by controlling the amount of the compound itself. Furthermore, these can reduce the amount of anti-tumor activity in normal cells. These can mitigate the effects of harmful compounds, which means that they are safer. I thought it was possible to achieve this. For this purpose, the present inventors have created a linker with a specific structure, and have used this linker to bind anti-TR We succeeded in obtaining an antibody-drug conjugate combining OP2 antibody and exatecan. The present invention was completed based on the discovery that the conjugate exhibits excellent antitumor effects. That's why.

[0014] That is, the present invention is [1] The following formula [ka] and an anti-TROP2 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- The disulfide bond in the hinge region of the anti-TROP2 antibody is bonded via a linker having the structure shown in The antibody is characterized by being bound by a thioether bond formed at the bond portion. It relates to body-drug conjugates.

[0015] Here, the anti-TROP2 antibody is L 1 The antitumor compound binds to the amino acid at the 1-position. The nitrogen atom of the -(CH2)n group is used as the bonding site. 2 It bonds to the carbonyl group of the -C(=O)- moiety. In the formula, n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L 1 is -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-, where n 3 denotes an integer from 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P indicates a peptide residue consisting of 2 to 7 amino acids, L a represents -O- or a single bond, -(Succinimid-3-yl-N)- has the following formula: [ka] The structure is represented by the formula: It bonds to a methylene group in the linker structure containing it.

[0016] Furthermore, the present invention also relates to the following: [2]L P The peptide residues are phenylalanine, glycine, valine, lysine, and citrulline. , a peptide residue consisting of an amino acid selected from serine, glutamic acid, and aspartic acid The antibody-drug conjugate according to [1]. [3]L P is a peptide residue selected from the group consisting of: Drug conjugates: -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-; Here, "(D-)D" refers to D-aspartic acid. [4]L P is a peptide residue consisting of four amino acids [1] or [2] Antibody-drug conjugates. [5]L P is a tetrapeptide residue -GGFG- according to any one of [1] to [4]. Antibody-drug conjugates.

[0017] [6]n 3 is an integer from 2 to 5, and L 2 Any one of [1] to [5] where is a single bond The antibody-drug conjugate according to claim 1. [7]n 3 is an integer from 2 to 5, and L 2 -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4 but The antibody-drug conjugate according to any one of [1] to [5], which is 2 or 4. [8]-NH-(CH2)n 1 -La -(CH2)n 2 -C(=O)- is a moiety with a chain length of 4 to 7 atoms [1] The antibody-drug conjugate according to any one of [1] to [7]. [9]-NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is a moiety having a chain length of 5 or 6 atoms [1] The antibody-drug conjugate according to any one of [1] to [7].

[10] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is -NH-CH2CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or [9] The antibody-drug complex according to any one of [1] to [9], wherein -NH-CH2CH2-O-CH2-C(=O)-. Indugegate.

[11] -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- is -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)- The antibody-drug conjugate according to any one of [1] to [9],

[0018]

[12] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2Drug-linker structure in which a drug is bound to -C(=O)- Any of [1] to [9], wherein the structural portion is a drug-linker structure selected from the following group: The antibody-drug conjugate of any one of claims 1 to 4, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX) , -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-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).

[0019] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the formula: It bonds to a methylene group in the linker structure containing it. -(NH-DX) is represented by the following formula: [ka] This represents a group in which the nitrogen atom of the amino group at position 1 is the binding site. -GGFG- indicates the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[0020]

[13] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 Drug-linker structure in which a drug is bound to -C(=O)- Any of [1] to [9], wherein the structural portion is one drug-linker structure selected from the following group: The antibody-drug conjugate of any one of claims 1 to 4, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX).

[0021] Here, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- are as defined above.

[0022]

[14] The following formula [ka] and an anti-TROP2 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- The disulfide bond in the hinge region of the anti-TROP2 antibody is bonded via a linker having the structure shown in The antibody is characterized in that it is bound via a thioether bond formed at the bond portion. Body-drug conjugates. Here, the anti-TROP2 antibody is L 1 The antitumor compound binds to the -(CH2)n 2 -C(= O)-moiety. In the formula, n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L 1 is -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-, where n 3 denotes an integer from 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P represents the tetrapeptide residue of -GGFG-, L a represents -O- or a single bond, -(Succinimid-3-yl-N)- has the following formula: [ka] The structure is represented by the formula: It bonds to a methylene group in the linker structure containing it.

[0023]

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

[0014] , wherein is -O-.

[16] n 3 is 2 or 5, and L 2 the antibody according to

[14] or

[15] , wherein Drug conjugates.

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

[14] or

[15] , wherein R is 4.

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

[14] to

[17] ,

[0024]

[19] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 Drug-linker structure in which a drug is bound to -C(=O)- Any of

[14] to

[18] , wherein the structural portion is a drug-linker structure selected from the following group: The antibody-drug conjugate of any one of claims 1 to 4, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX) 、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-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);

[0025] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the formula: It bonds to a methylene group in the linker structure containing it. -(NH-DX) is represented by the following formula: [ka] This represents a group in which the nitrogen atom of the amino group at position 1 is the binding site. -GGFG- indicates the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[0026]

[20] -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 Drug-linker structure in which a drug is bound to -C(=O)- The structural part is one drug-linker structure selected from the following group

[14] to

[18] . The antibody-drug conjugate of any one of claims 1 to 4, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX). Here, -(Succinimid-3-yl-N)-, -(NH-DX) and -GGFG- are as defined above.

[0027]

[21] The average number of conjugates per antibody for each selected drug-linker structure is between 1 and 10. The antibody-drug conjugate according to any one of [1] to

[20] , wherein the number of conjugates ranges from 1 to 20.

[22] The average number of conjugates per antibody for each selected drug-linker structure is 2 to 8. The antibody-drug conjugate according to any one of [1] to

[20] , wherein the range is

[23] The average number of conjugates per antibody for each selected drug-linker structure is 3 to 8. The antibody-drug conjugate according to any one of [1] to

[20] , wherein the range is

[0028]

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

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

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

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

[26] Lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, For use in breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck cancer, or esophageal cancer The antitumor drug and / or anticancer drug according to

[25] .

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

[23] , or a salt thereof or a hydrate thereof as an active ingredient, and a pharmaceutical composition containing a pharmaceutically acceptable formulation ingredient. Finished product.

[28] Lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, For use in breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck cancer, or esophageal cancer The pharmaceutical composition described in

[27] .

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

[23] , or a salt thereof A method for treating tumors and / or cancers, comprising administering one of the compounds listed above, or a hydrate thereof.

[0029]

[30] A compound represented by the formula: (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) The product is reacted with an anti-TROP2 antibody or a reactive derivative thereof to detect disulfide residues present in the hinge region of the antibody. The drug-linker moiety is formed by forming a thioether bond at the sulfide bond site. and binding a compound to the antibody.

[0030] In the formula, n 3 denotes the integers 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P Phenylalanine, glycine, valine, lysine, citrulline, serine, glutamine It represents a peptide residue consisting of 2 to 7 amino acids selected from the group consisting of carboxylic acid, carboxylic acid, and aspartic acid. , n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L a represents -O- or a single bond, (maleimid-N-yl)- is a compound of the formula [ka] The nitrogen atom is the bonding site. -(NH-DX) is represented by the following formula: [ka] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in

[0031]

[31] A method for attaching a drug-linker moiety to an anti-TROP2 antibody includes reducing the antibody. and converting the compound into a reactive derivative.

[0032]

[32] The average number of conjugates per antibody for each selected drug-linker structure is between 1 and 10. The manufacturing method according to

[30] or

[31] , wherein the number of the components is in the range of 100 to 150.

[33] The average number of conjugates per antibody for each selected drug-linker structure is 2 to 8. The method according to

[30] or

[31] , wherein the range is

[34] The average number of conjugates per antibody for each selected drug-linker structure is 3 to 8. The method according to

[30] or

[31] , wherein the range is

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

[30] to

[34] . ugate.

[0033]

[36] Anti-TROP2 antibody is treated under reducing conditions and then reacted with a compound selected from the following group: The antibody is characterized in that a thioether is bonded to the sulfide bond in the hinge region of the antibody. The resulting antibody-drug conjugate: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C( =O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2 -C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O- CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2-O-CH2-C(=O)-(NH-DX)、はき (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX).

[0034] Here, (maleimid-N-yl)- is a compound represented by the following formula: [ka] The nitrogen atom is the bonding site. -(NH-DX) is represented by the following formula: [ka] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in -GGFG- indicates the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[0035]

[37] Anti-TROP2 antibody is treated under reducing conditions and then reacted with a compound selected from the following group: The antibody is characterized in that a thioether bond is formed at the sulfide bond portion of the hinge region. The resulting antibody-drug conjugate: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), and (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX). Here, (maleimid-N-yl)-, -(NH-DX), and -GGFG- are as defined above.

[0036]

[38] The average number of conjugates per antibody for each selected drug-linker structure is between 1 and 10. The antibody-drug conjugate according to

[36] or

[37] , wherein the number of antibody-drug conjugates is in the range of

[37] .

[39] The average number of conjugates per antibody for each selected drug-linker structure is 2 to 8. The antibody-drug conjugate according to

[36] or

[37] , wherein the range is

[40] The average number of conjugates per antibody for each selected drug-linker structure is 3 to 8. The antibody-drug conjugate according to

[36] or

[37] , wherein the range is [Effects of the Invention]

[0037] Anti-TROP2 antibody conjugated to the antitumor compound exatecan via a linker with a specific structure Antibody-drug conjugates can achieve excellent antitumor efficacy and safety. do. [Brief explanation of the drawings]

[0038] [Figure 1] The nucleotide sequence (sequence number 7) and amino acid sequence (sequence number 8) of the cTINA1 antibody heavy chain are shown. [Figure 2] The nucleotide sequence (sequence number 9) and amino acid sequence (sequence number 10) of the cTINA1 antibody light chain are shown. [Figure 3] The nucleotide sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12) of the hTINA1-H1 heavy chain are shown. [Figure 4] The nucleotide sequence (SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 14) of the hTINA1-H2 heavy chain are shown. [Figure 5] The nucleotide sequence (SEQ ID NO: 15) and amino acid sequence (SEQ ID NO: 16) of the hTINA1-H3 heavy chain are shown. [Figure 6] The nucleotide sequence (SEQ ID NO: 17) and amino acid sequence (SEQ ID NO: 18) of the hTINA1-L1 light chain are shown. [Figure 7]The nucleotide sequence (SEQ ID NO: 19) and amino acid sequence (SEQ ID NO: 20) of the hTINA1-L2 light chain are shown. [Figure 8] The nucleotide sequence (SEQ ID NO: 21) and amino acid sequence (SEQ ID NO: 22) of the hTINA1-L3 light chain are shown. [Figure 9] The amino acid sequence of CDRH1 (sequence number 23), the amino acid sequence of CDRH2 (sequence number 24), the amino acid sequence of CDRH3 (sequence number 25), the amino acid sequence of CDRL1 (sequence number 26), the amino acid sequence of CDRL2 (sequence number 27), and the amino acid sequence of CDRL3 (sequence number 28) of the TINA1 antibody are shown. [Figure 10] The cell internalization ability of anti-CD9 antibody, anti-CD46 antibody, anti-CD55 antibody, anti-CD59 antibody, anti-CD71 antibody, anti-CD73 antibody, anti-CD147 antibody, anti-CD276 antibody, anti-EpCAM antibody, anti-EGFR antibody, and anti-TROP2 antibody (TINA1 antibody) is shown. [Figure 11] 1 shows the cellular internalization ability of anti-CD59 antibody, anti-CD71 antibody, anti-EGFR antibody, anti-EpCAM antibody, and anti-TROP2 antibody (TINA1 antibody). [Figure 12] 1 shows the cellular internalization ability of various anti-TROP2 antibodies. [Figure 13] 1 shows the antitumor effect of antibody-drug conjugates (1), (6), and (12) on BALB / c-nu / nu mice subcutaneously implanted with the human colon cancer cell line COLO205. [Figure 14] 1 shows the antitumor effect of antibody-drug conjugates (1), (6), and (12) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic adenocarcinoma cell line BxPC-3. [Figure 15] 1 shows the antitumor effect of antibody-drug conjugates (1), (6), and (12) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic adenocarcinoma cell line Capan-1. [Figure 16] 1 shows the antitumor effect of antibody-drug conjugates (2), (5), (7), and (10) on BALB / c-nu / nu mice subcutaneously implanted with the human colon cancer cell line COLO205. [Figure 17] 1 shows the antitumor effect of antibody-drug conjugates (2), (5), (7), and (10) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic cancer cell line BxPC-3. [Figure 18] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human colon cancer cell line COLO205. [Figure 19] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic cancer cell line BxPC-3. [Figure 20] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human ovarian cancer cell line NIH:OVCAR-3. [Figure 21] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human gastric cancer cell line NCI-N87. [Figure 22] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human lung cancer cell line NCI-H292. [Figure 23] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human pharyngeal carcinoma cell line FaDu. [Figure 24] 1 shows the antitumor effect of antibody-drug conjugates (3), (4), (8), and (9) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic adenocarcinoma cell line CFPAC-1. [Figure 25] 1 shows the antitumor effect of antibody-drug conjugate (8) or (13) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic adenocarcinoma cell line CFPAC-1. [Figure 26]1 shows the antitumor effect of antibody-drug conjugates (8) and (13) on BALB / c-nu / nu mice subcutaneously implanted with the human pancreatic adenocarcinoma cell line HPAC. [Figure 27] Antibody-drug conjugates (8) and (13) show antitumor effects against NOD-scid mice subcutaneously implanted with human esophageal cancer tissue. DETAILED DESCRIPTION OF THE INVENTION

[0039] Preferred embodiments of the present invention will now be described with reference to the drawings. The embodiment described below is an example of a typical embodiment of the present invention. This will not narrow the scope of the present invention.

[0040] The anti-TROP2 antibody-drug conjugate of the present invention comprises an anti-TROP2 antibody and a linker structure. It is an antitumor drug in which an antitumor compound is bound via a synthetic moiety, and will be described in detail below. .

[0041] [antibody] The anti-TROP2 antibody used in the anti-TROP2 antibody-drug conjugate of the present invention may be any The cells may be derived from any species, but preferably from humans, rats, mice, and rabbits. If derived from a species other than human, they may be chimerized or humanized using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Although it may be present, monoclonal antibodies are preferred. Anti-TROP2 antibodies are antibodies that can target tumor cells, i.e., they can recognize tumor cells. The ability to bind to tumor cells, and the ability to be taken up and internalized by tumor cells. and a compound having antitumor activity is linked to the antibody-drug complex via a linker. It can be a conjugate. The binding of the antibody to tumor cells can be confirmed using flow cytometry. Methods for confirming antibody uptake into tumor cells include: (1) a secondary antibody that binds to the therapeutic antibody; An assay to visualize intracellularly incorporated antibodies using fluorescent labeling under a fluorescence microscope (Ce ll Death and Differentiation (2008) 15, 751-761), (2) secondary binding to therapeutic antibodies Molecular assay to measure the amount of fluorescence taken up into cells using antibodies (fluorescently labeled) Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) binding to therapeutic antibodies. When the immunotoxin is taken up into cells, it releases the toxin and suppresses cell proliferation. Examples include the Mab-ZAP assay (BioTechniques 28:162-165, January 2000), which is known to be effective in preventing leukemia. As an immunotoxin, the catalytic domain and protein of diphtheria toxin can be used. A recombinant conjugated protein with InG can also be used. Antibody-drug conjugates are compounds that exert antitumor effects, so antibodies It is preferable, but not essential, that the compound itself has an antitumor effect. In order to specifically and selectively exert toxicity on tumor cells, the antibody must be internalized and then injected into the tumor. It is important and preferable that the compound has the property of being transferred into cells.

[0042] Anti-TROP2 antibodies can be prepared by isolating the antigenic polypeptide using methods commonly used in the art. It can be obtained by immunizing an animal with the virus and collecting and purifying the antibodies produced in the body. The origin of the antigen is not limited to humans, and antigens derived from animals other than humans, such as mice and rats, can also be used. The heterologous antigen can also be used to immunize animals. In this case, the resulting antibodies that bind to the heterologous antigen can be used. Antibodies applicable to human diseases can be selected by testing cross-reactivity with human antigens. . Also, known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Ken net, R.ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)) By fusing antibody-producing cells that produce antibodies against antigens with myeloma cells, It is also possible to establish hybridomas and obtain monoclonal antibodies. The antigen is produced by genetically manipulating the gene encoding the antigen protein in the host cell. Specifically, a vector capable of expressing an antigen gene is prepared. This can then be introduced into host cells to express the gene, and the expressed antigen can be purified. Immunizing animals with the antigen-expressing cells or cell lines expressing the antigens obtained by the above genetic manipulation. Antibodies can also be obtained by using the method. Anti-TROP2 antibodies can be obtained by known means.

[0043] The anti-TROP2 antibodies that can be used in the present invention are not particularly limited, and include, for example, those listed in the sequence listing of the present application. Those specified by the amino acid sequences shown can be preferably used. The TROP2 antibody to be used preferably has the following properties: (1) An antibody characterized by the following properties: (a) Specific binding to TROP2 (b) It has the ability to be internalized into TROP2-expressing cells by binding to TROP2. R (2) The antibody according to (1) above, wherein TROP2 is human TROP2. (3) a C having the amino acid sequence set forth in SEQ ID NO: 23 as a complementarity-determining region in the heavy chain; DRH1, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 25 CDRH3 having the amino acid sequence shown in the table below, and the complementarity determining region in the light chain having the sequence CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, and the amino acid sequence set forth in SEQ ID NO: 27 and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 28. The antibody according to (1) or (2) above. (4) The antibody according to any one of (1) to (3) above, wherein the constant region is a human-derived constant region. . (5) The antibody according to any one of (1) to (4) above, which is humanized. (6) (a) an amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 12; (b) an amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 14; (c) (d)(a) an amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 16; an amino acid sequence having at least 95% or more homology to the sequence of (c) or (d), and e) One or several amino acids are deleted, substituted or added in the sequences of (a) to (c) a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of the amino acid sequences of ( (f) the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 18; (g) the sequence (h) the amino acid sequence set forth in amino acid numbers 21 to 129 in sequence number 20; 22, the amino acid sequence of amino acid numbers 21 to 129, (i) (f) to (h) ) an amino acid sequence having at least 95% homology to the sequence of (f ) to (h) in which one or several amino acids are deleted, substituted or added and a light chain variable region consisting of an amino acid sequence selected from the group consisting of the above (5 ) The antibody described in (7) The amino acid sequence of SEQ ID NO: 12, consisting of amino acid numbers 20 to 140 The variable region of the heavy chain and amino acids 21 to 129 in SEQ ID NO: 18 A light chain variable region consisting of the sequence set forth in amino acid numbers 20 to 140 of SEQ ID NO: 12. and a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 21 to 23 in SEQ ID NO: 20. 129, a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12, The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 to 140, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 22. a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129; A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in paragraph 14. and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 18. The variable region of the chain, the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 14 and a heavy chain variable region consisting of amino acid numbers 21 to 129 in SEQ ID NO: 20. A light chain variable region consisting of the amino acid sequence: amino acid numbers 20 to 14 in SEQ ID NO: 14 0 and a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 22, a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 16; A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 and SEQ ID NO: 1 8, a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129; A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 16. The variable region and the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 20 a light chain variable region comprising: and a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 21 to 22 in SEQ ID NO: 22. a heavy chain variable region selected from the group consisting of a light chain variable region consisting of the amino acid sequence set forth in 129; The antibody according to (6) above, having a variable region and a light chain variable region. (8) The amino acid sequence of SEQ ID NO: 12, consisting of amino acid numbers 20 to 140 The variable region of the heavy chain and amino acids 21 to 129 in SEQ ID NO: 18 A light chain variable region consisting of the sequence set forth in amino acid numbers 20 to 140 of SEQ ID NO: 14. and a heavy chain variable region consisting of the amino acid sequence of amino acid numbers 21 to 23 in SEQ ID NO: 18. 129, a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 14, The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 to 140, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20. a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129; The heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in sequence number 16 is From the variable region and the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 22 and a light chain variable region selected from the group consisting of a heavy chain variable region and a light chain variable region The antibody according to (7) above. (9) The amino acid sequence of SEQ ID NO: 12, consisting of amino acid numbers 20 to 470 a heavy chain and an amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18; a light chain comprising the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 12; and the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 20. a light chain comprising the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 12; and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 22. a light chain comprising the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 18. a light chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; a heavy chain consisting of an amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 20; a light chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; a heavy chain consisting of amino acids 21 to 234 in SEQ ID NO: 22; a light chain consisting of the sequence: amino acids 20 to 470 in SEQ ID NO: 16; and a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18. a light chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 16; a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 20; a light chain consisting of the amino acid sequence set forth in amino acids 20 to 470 of SEQ ID NO: 16; and a heavy chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 in SEQ ID NO: 22. The above (6) comprising a heavy chain and a light chain selected from the group consisting of a light chain having the amino acid sequence listed above. ) or (7). (10) A heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and an amino acid sequence set forth in SEQ ID NO: 18 a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 12; a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2; a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 0; and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12. and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 22, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 14. a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 14, a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 21; A heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 and a a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16; a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18; a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16; a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 17; a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 22. The antibody according to (6) or (7) above, which consists of a heavy chain and a light chain. (11) The amino acid sequence of SEQ ID NO: 12, consisting of amino acid numbers 20 to 470 and the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18. a light chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18. a light chain comprising the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 20. and a light chain consisting of amino acids 20 to 470 in SEQ ID NO: 16. and a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 22. The heavy chain and the light chain are selected from the group consisting of a heavy chain and a light chain consisting of a sequence of antibodies. (12) Any of the above (1) to (11) in which the lysine residue at the carboxyl terminal of the heavy chain is deleted. The antibody according to any one of the preceding claims. (13) A polynucleotide encoding the antibody according to any one of (1) to (12) above. and culturing a host cell transformed with an expression vector containing The antibody is obtained by a method for producing the antibody, which includes a step of collecting the antibody from the culture obtained. Antibodies.

[0044] The anti-TROP2 antibodies used in the present invention are described below. In this specification, the terms "cancer" and "tumor" are used interchangeably. As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA, and A and its cRNA are also included. As used herein, the term "polynucleotide" is used synonymously with nucleic acid, and Also included are NA, RNA, probes, oligonucleotides, and primers. In this specification, the terms "polypeptide" and "protein" are used interchangeably. As used herein, the term "cells" includes cells within an animal body and cultured cells. As used herein, "TROP2" is used synonymously with TROP2 protein. As used herein, "CDR" refers to a complementarity determining region (CDR). The heavy and light chains of an antibody molecule have Each CDR is known to have three CDRs. They are also called variable domains and are found in the variable regions of the heavy and light chains of antibodies. The primary structure of the heavy and light polypeptide chains is highly variable. In this specification, the CDRs of an antibody are The heavy chain CDRs are arranged as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence. The light chain CDRs are represented as CDRL1, CDR2, CDR3, and CDR4 from the amino-terminal end of the light chain amino acid sequence. These sites are designated DRL2 and CDRL3. These sites are close to each other in the three-dimensional structure and bind to each other. This determines the specificity for the antigen. In the present invention, "hybridizing under stringent conditions" means that a commercially available hybridization Hybridization Solution ExpressHyb Hybridization Sol Hybridization was performed at 68°C in a centrifuge tube (Clontech) or by immobilizing the DNA. The filters were hybridized at 68°C in the presence of 0.7-1.0 M NaCl. After the incubation, add 0.1-2x SSC solution (1x SSC is 150 mM NaCl, 15 mM sodium citrate) at 68°C. This refers to hybridization under conditions that allow identification by hybridization or conditions equivalent thereto.

[0045] 1.TROP2 TROP2 is a TACSTD expressed in human trophoblasts. It is a member of the family and is involved in immune resistance common to human trophoblast cells and cancer cells. It is a type 1 cell membrane protein with a single transmembrane span. The TROP2 protein used in the present invention is a protein derived from the T cells of humans and non-human mammals (rats, mice, etc.). It can be purified directly from ROP2-expressing cells or prepared as a cell membrane fraction. TROP2 can be synthesized in vitro or by genetic engineering. It can be obtained by producing it in a host cell. After inserting ROP2 cDNA into an expression vector, the enzymes and bases required for transcription and translation were introduced. synthesis in a solution containing proteins and energy materials, or in other prokaryotic or eukaryotic hosts The protein is obtained by expressing TROP2 by transforming cells. Furthermore, the above-mentioned genetically engineered TROP2-expressing cells or TROP2-expressing cells can be used. It is also possible to use a cell line expressing the TROP2 protein. The DNA and amino acid sequences of TROP2 are publicly available in public databases, e.g. For example, accession numbers such as NM_002353, NP_002344 (NCBI) It can be referenced. In addition, in the amino acid sequence of the above-mentioned TROP2, one or several amino acids are substituted, deleted, or and / or an added amino acid sequence, and has biological activity equivalent to that of the protein. is also included in TROP2. The human TROP2 protein contains a signal sequence consisting of 26 amino acid residues at the N-terminus, a 248 amino acid residue sequence, and an extracellular domain consisting of 26 amino acid residues; a transmembrane domain consisting of 23 amino acid residues; It consists of an intracellular domain made up of amino acid residues.

[0046] 2. Preparation of anti-TROP2 antibodies The antibodies against TROP2 of the present invention can be synthesized by conventional methods in the art. Immunizing an animal with any polypeptide selected from the amino acid sequence of P2 or TROP2, Antibodies can be obtained by collecting and purifying antibodies produced in the body. The biological species of ROP2 is not limited to humans, but also includes T cells derived from non-human animals such as mice and rats. ROP2 can also be used to immunize animals. In this case, the obtained heterologous TROP2 binds to the By testing the cross-reactivity of the antibody with human TROP2, it can be applied to human diseases. It is possible to select suitable antibodies. Also, known methods (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; Kennet, R. ed., Monoclo nal Antibodies, p.365-367, Plenum Press, N. According to Y. (1980), antibody-producing cells that produce antibodies against TROP2 and myelin Hybridomas are established by fusing them with human IgG cells to obtain monoclonal antibodies. It is also possible to do so. The antigen, TROP2, is expressed in host cells through genetic manipulation of the TROP2 gene. It can be obtained by making Specifically, a vector capable of expressing the TROP2 gene was created and introduced into host cells. The gene is then expressed, and the expressed TROP2 is purified. In addition, the above-mentioned genetically engineered TROP2-expressing cells or cells expressing TROP2 It is also possible to use a cell line containing TROP2 protein. We will explain how to obtain antibodies against this virus.

[0047] (1) Antigen preparation Antigens for producing anti-TROP2 antibodies include TROP2 or at least six of its amino acids. A polypeptide consisting of a continuous partial amino acid sequence of the above, or any amino acid sequence or Examples include derivatives to which a carrier is added. TROP2 can be directly purified from human tumor tissues or tumor cells. TROP2 can also be synthesized in vitro or produced in host cells by genetic engineering. It can be obtained by Specifically, the gene manipulation involves inserting the TROP2 cDNA into an expression vector. Then, they are synthesized in a solution containing the enzymes, substrates, and energy sources required for transcription and translation, or By transforming other prokaryotic or eukaryotic host cells to express TROP2. The antigen can be obtained by In addition, a fusion protein linking the extracellular domain of the membrane protein TROP2 with the constant domain of an antibody was developed. The antigen is expressed as a secreted protein by expressing the protein in an appropriate host-vector system. It is also possible to obtain For example, TROP2 cDNA is expressed in a cDNA live The library was used as a template to amplify the TROP2 cDNA using primers that specifically amplify the TROP2 cDNA. PCR (hereinafter referred to as "PCR"; Saiki, RK, et al., Science (1988) 239, pp. 487-489) It can be obtained by law. For in vitro synthesis of polypeptides, for example, the Roche Diagnostics Examples include, but are not limited to, Rapid Translation Systems (RTS). It will never be possible. Prokaryotic hosts include, for example, Escherichia coli and Bacillus subtilis. Examples of suitable strains include Bacillus subtilis. To be transformed into these host cells, a replicon from a species compatible with the host, i.e. Host cells are transformed with a plasmid vector containing the replication origin and regulatory sequences. In addition, as a vector, it is possible to confer selectability of phenotypes to transformed cells. Preferably, the sequence is such that the sequence can be Eukaryotic host cells include cells of vertebrates, insects, yeast, etc., and vertebrate cells include For example, COS cells, which are monkey cells (Gluzman, Y. Cell (1981) )23, p.175-182, ATCC CRL-1650;ATCC:America n Type Culture Collection), mouse fibroblast NIH3T 3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin,LAProc.Natl.Acad.Sci.USA(19 80)77, p.4126-4220) are often used, but are not limited to these. stomach. The transformants obtained as described above are cultured according to methods commonly used in this field. The culture can produce the desired polypeptide intracellularly or extracellularly. The medium used for the culture can be selected from various commonly used media depending on the host cells used by those skilled in the art. In the case of E. coli, for example, the following can be selected in LB medium as needed: Antibiotics such as ampicillin or IPMG may be added. The recombinant protein produced inside or outside the cells of the transformant by the above-mentioned culture is Proteins are separated and purified using various known separation procedures that utilize their physical and chemical properties. It is possible. Specific examples of such methods include treatment with a conventional protein precipitant, ultrafiltration, molecular Sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography Various liquid chromatography such as fluorography and affinity chromatography, dialysis Examples include methods and combinations thereof. In addition, by linking a six-residue histidine tag to the recombinant protein to be expressed, The expressed protein can be efficiently purified using a nickel affinity column. By linking the Fc region of IgG to the recombinant protein, it can be efficiently purified on a Protein A column. It can be manufactured. By combining the above methods, it is possible to easily obtain a large amount of the desired polypeptide in high yield and purity. Can be produced in large quantities. The above-mentioned transformant itself can also be used as an antigen. It is also possible to use a cell line expressing 2 as an antigen. Humanized lung cancer lines NCI-H322, PC14, NCIH-H2122, or LCAM1, human prostate cancer line PC3, human pancreatic cancer line BxPC-3, Capan-1, or PK-1, human Examples include the ovarian cancer line SKOV3 and the human colon cancer line COLO205. There is no limitation to these cell lines as long as they express P2.

[0048] (2) Production of anti-TROP2 monoclonal antibodies Examples of antibodies that specifically bind to TROP2 include monoclonal antibodies that specifically bind to TROP2. Examples of antibodies include monoclonal antibodies, and the method for obtaining them is as described below. The production of a monoclonal antibody generally requires the following steps: That is, (a) Purification of a biopolymer to be used as an antigen or preparation of antigen-expressing cells; (b) Immunize animals by injecting them with an antigen, then collect blood and test its antibody titer. determining the timing of spleen removal based on the results of the analysis, and then preparing antibody-producing cells; (c) preparation of myeloma cells (hereinafter referred to as "myeloma"); (d) cell fusion between antibody-producing cells and myeloma; (e) Selection of hybridomas producing the desired antibody; (f) division into single cell clones (cloning); (g) In some cases, culturing hybridomas for the large-scale production of monoclonal antibodies. breeding or rearing of animals transplanted with hybridomas; (h) Examination of the physiological activity and binding specificity of the monoclonal antibody thus produced. Study or test of properties as a labeling reagent etc. The method for producing a monoclonal antibody will be described in detail below along the above steps. Without being limited thereto, for example, antibody-producing cells other than splenocytes and myelomas may also be used. Cut.

[0049] (a) Antigen purification As an antigen, TROP2 or a part thereof prepared by the above-mentioned method can be used. can. In addition, membrane fractions prepared from recombinant TROP2-expressing cells or recombinant TROP2-expressing cells The somatic cells themselves, as well as portions of the proteins of the present invention chemically synthesized using methods well known to those skilled in the art, Peptides can also be used as antigens. Furthermore, a TROP2-expressing cell line can also be used as an antigen.

[0050] (b) Preparation of antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant, or Karimi It is mixed with an adjuvant such as alum and used as an immunogen to immunize experimental animals. There is also a method of immunizing experimental animals with the cells as an immunogen. The animals used in the production method can be used without any problems. Specifically, for example, mice, rats, etc. However, the antibody-producing animals that are isolated can be used. From the viewpoint of the availability of myeloma cells to be fused with the cells, mice or rats were used as immunized Preferably it is an animal. There are no particular restrictions on the strains of mice and rats that can be used. For example, each strain A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C 57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, 129, etc., and in the case of rats, for example, Wis tar, Low, Lewis, Sprague, Dawley, ACI, BN, Fisc Her etc. can be used. These mice and rats are available from, for example, CLEA Japan Co., Ltd. and Charles River Japan Co., Ltd. They can be obtained from companies and other laboratory animal breeding and sales outlets. Considering the compatibility of fusion with myeloma cells described below, mice are the most suitable animals to be immunized. The BALB / c strain is particularly preferred, and in rats, the Wistar and Low strains are particularly preferred. In addition, taking into account the homology of antigens between humans and mice, the immune system that removes autoantibodies is weakened. It is also preferable to use mice that have been bred, that is, mice with autoimmune diseases. The age of these mice or rats at the time of immunization is preferably 5 to 12 weeks, and more preferably 12 to 12 weeks. Preferably, the age is 6 to 8 weeks. To immunize animals with TROP2 or a recombinant form thereof, see, for example, Weir, DM .,Handbook of Experimental Immunology Vo lIII.III.,Blackwell Scientific Publica tions, Oxford (1987); Kabat, E. A. and Mayer, M. .M.,Experimental Immunochemistry,Charles C Thomas Publisher Springfield, Illinois (1964) and other known methods can be used. Among these immunization methods, the preferred method in the present invention is specifically as follows: It is. That is, first, the membrane protein fraction, which is the antigen, or cells expressing the antigen is injected intradermally or intradermally into an animal. However, in order to increase the efficiency of immunity, it is preferable to use both in combination. Intradermal administration followed by intraperitoneal administration in the latter or final dose can particularly enhance immune efficiency. can. The antigen administration schedule varies depending on the type of animal to be immunized, individual differences, etc., but generally follows the following schedule: The antigen is preferably administered 3 to 6 times at an interval of 2 to 6 weeks, and the antigen is preferably administered 3 to 4 times at an interval of 2 to 6 weeks. Two to four weeks is even more preferable. The dose of the antigen varies depending on the type of animal, individual differences, etc., but is generally between 0.05 and 100 mg / kg. 5 mg, preferably about 0.1 to 0.5 mg. Booster immunization is performed 1 to 6 weeks, preferably 1 to 4 weeks, after the above-described antigen administration. Preferably, the immunogen is cultured at 1 to 3 weeks after the first incubation. When the immunogen is cells, the immunogen is cultured at 1 × 10 6 〜1×10 7 pieces Use cells. The amount of antigen administered during booster immunization varies depending on the type and size of the animal. Generally, for example, in the case of a mouse, the amount is 0.05 to 5 mg, preferably 0.1 to 0.5 mg, and more preferably When the immunogen is a cell, the amount is preferably about 0.1 to 0.2 mg. 6 ~ 1×10 7 Use cells. 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days after the booster immunization First, spleen cells or lymphocytes containing antibody-producing cells are aseptically extracted from the immunized animal. If the antibody titer is measured and animals with a sufficiently high antibody titer are used as a source of antibody-producing cells, , the efficiency of subsequent operations can be improved. The antibody titer measurement method used here includes, for example, the RIA method or the ELISA method. The antibody titer of the present invention can be measured by, but is not limited to, the following methods. The ELISA method can be carried out by the following procedure. First, purified or partially purified antigen is adsorbed onto a solid phase surface such as a 96-well ELISA plate. Furthermore, the solid phase surface to which the antigen is not adsorbed is exposed to a protein unrelated to the antigen, such as bovine serum albumin. After washing the surface, a serially diluted sample (e.g., BSA) was applied as the first antibody. The sample is then contacted with a sample of, for example, mouse serum, to allow the antibody in the sample to bind to the antigen. Furthermore, an antibody against the enzyme-labeled mouse antibody was added as a second antibody to bind to the mouse antibody. After washing, the substrate of the enzyme is added and the change in absorbance due to color development caused by substrate decomposition is measured. The antibody titer is calculated by the above procedure. Antibody-producing cells can be isolated from spleen cells or lymphocytes of immunized animals by known methods (e.g., , Kohler et al., Nature (1975) 256, p. 495; ler et al.,Eur.J.Immunol.(1977)6,p.511;M ilstein et al., Nature (1977), 266, p. 550; Wa This can be done according to the method described in Nature, (1977) 266, p. 495. For example, in the case of spleen cells, the spleen is cut into small pieces and the cells are filtered through a stainless steel mesh. The standard method of isolating antibody-producing cells by suspending them in Eagle's minimum essential medium (MEM) was used. It is possible.

[0051] (c) Preparation of myeloma cells (hereinafter referred to as "myeloma"). There are no particular limitations on the myeloma cells used for cell fusion, and they can be appropriately selected from known cell lines. However, in consideration of the convenience of selecting hybridomas from fused cells, Considering this, the selection procedure for HGPRT (Hypoxanthine-guanine receptor tetracycline) is well established. The use of a mutant lacking ine phosphoribosyl transferase (NPTP) is preferable. That is, X63-Ag8 (X63), NS1-ANS / 1 (NS1), derived from mouse P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653), SP2 / 0-Ag14(SP2 / 0), MPC11-45.6TG1.7(45.6TG ), FO, S149 / 5XXO, BU.1, etc., 210.RSY3.Ag.1 derived from rat .2.3(Y3), etc., human-derived U266AR(SKO-007), GM1500·GT G-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2 ), 8226AR / NIP4-1 (NP41), etc. For example, it can be obtained from ATCC. These cell lines are cultured in an appropriate medium, such as 8-azaguanine medium (RPMI-1640 medium). The base was supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter referred to as a medium supplemented with 8-azaguanine (FBS-supplemented medium), Iscove's modified Darbec Iscove's Modified Dulbecco's Medium IMDM), or Dulbecco's Modified Eagle's Medium (Dulbec ... The cells were subcultured in DMEM (Ed Eagle Medium; DMEM) for 3 to 4 days after cell fusion. Normal medium (e.g., ASF104 medium (Ajinomoto Co., Inc.) containing 10% FCS) On the day of fusion, the cells were subcultured at 2 × 10 7 Ensure that the above number of cells is maintained.

[0052] (d) Cell fusion The fusion of antibody-producing cells with myeloma cells was carried out by a known method (Weir, DM, Han dbookof Experimental Immunology Vol.I.II .III.,Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Ex experimental Immunochemistry,Charles C Tho mas Publisher Springfield,Illinois(1964) In accordance with the above, the method can be carried out appropriately under conditions that do not excessively reduce the viability of the cells. do. As such a method, for example, an anti- Chemical methods for mixing somatic cells with myeloma cells, and physical methods using electrical stimulation Among these, specific examples of the above chemical methods are as follows: . That is, when polyethylene glycol is used as a high-concentration polymer solution, In a polyethylene glycol solution of 1500 to 6000, preferably 2000 to 4000 The antibody-producing cells and myeloma cells are incubated at a temperature of 30 to 40°C, preferably 35 to 38°C. Mixing is carried out for 1 to 10 minutes, preferably 5 to 8 minutes.

[0053] (e) Selection of hybridoma population The method for selecting hybridomas obtained by the above cell fusion is not particularly limited. AT (hypoxanthine-aminopterin-thymidine) selection method (Kohler et al. .,Nature(1975)256,p.495;Milstein et al., Nature (1977) 266, p. 550) is used. This method uses HGPRT-deficient myeloma cells that cannot survive on aminopterin. This is effective when obtaining hybridomas by the above method. By culturing in HAT medium, hives that are resistant to aminopterin were isolated. Only the lysoma can be selectively allowed to survive and grow.

[0054] (f) Division into single cell clones (cloning) Hybridoma cloning methods include, for example, the methylcellulose method, soft agarose method, and the like. Known methods such as the limiting dilution method and the limiting dilution method can be used (see, for example, Barbara, BM .and Stanley,MS:Selected Methods in Ce llular Immunology,WHFreeman and Compan (See, e.g., San Francisco (1980)). Among these methods, methyl Three-dimensional culture methods such as the cellulose method are suitable. For example, The bryozoan group was cultured in ClonaCell-HY Selection Medium D (S Methylcellulose such as temCell Technologies #03804 The hybridomas are suspended in a medium and cultured, and the resulting hybridoma colonies are collected to produce monoclonal antibodies. Each of the collected hybridoma colonies is cultured, and The hybridoma culture supernatants that showed stable antibody titers were identified as TROP2 monoclonal antibodies. A clonal antibody-producing hybridoma line is selected.

[0055] An example of a hybridoma line established in this manner is the TROP2 hybridoma T In the present specification, the term "TROP2 hybridoma" refers to a human INA1. The antibody produced by TINA1 is referred to as a "TINA1 antibody" or simply as "TINA1." . The heavy chain variable region of the TINA1 antibody has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The light chain variable region of the TINA1 antibody has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. It has columns.

[0056] (g) Preparation of monoclonal antibodies by hybridoma culture The hybridomas selected in this way can be cultured to produce monoclonal antibodies. However, prior to culturing, the desired monoclonal antibody can be obtained efficiently. It is desirable to screen for hybridomas that produce the antibody. For this screening, a method known per se can be used. The antibody titer in the present invention can be measured, for example, by the ELISA method described in the above item (b). This can be done. The hybridomas obtained by the above method are stored in liquid nitrogen or in a freezer at -80°C or below. It can be stored frozen. Once cloning is complete, the hybridomas are cultured in a normal medium instead of HT medium. will be done. Mass culture is carried out by rotary culture using large culture bottles or spinner culture. The supernatant is purified by gel filtration or other methods well known to those skilled in the art. Monoclonal antibodies that specifically bind to the proteins of the present invention can be obtained. In addition, the peritoneal cavity of mice of the same strain (e.g., the above-mentioned BALB / c) or Nu / Nu mice The hybridomas of the present invention are then injected into the mouse and allowed to grow. Ascites containing large amounts of monoclonal antibodies can be obtained. When administering intraperitoneally, administer 2,6,10,14-tetramethyl- Pentadecane (2,6,10,14-tetramethylpentadecane; Administration of mineral oils such as pristane results in a larger amount of ascites. For example, an immunosuppressant is injected into the abdominal cavity of a mouse of the same strain as the hybridoma, and T cells are After inactivation, 10 6 ~10 7 Hybridoma clone cells, serum The cells are suspended in a medium containing 0.5 ml of PEG and administered intraperitoneally. This usually results in abdominal distension and ascites. When the ascites has accumulated, the ascites is collected from the mouse. A monoclonal antibody concentration of 100 times or more can be obtained. The monoclonal antibodies obtained by the above method can be used in the methods described, for example, in Weir, DM: Handb ook of Experimental Immunology,Vol.I,II, III, Blackwell Scientific Publications, Ox It can be purified by the method described in Ford (1978). The monoclonal antibody thus obtained has high antigen specificity for TROP2. .

[0057] (h) Monoclonal antibody assay The isotype and subclass of the monoclonal antibody thus obtained were determined as follows: can be done. First, the identification methods are the Ouchterlony method, ELISA method, Another example is the RIA method. Although the Ouchterlony method is simple, it requires a concentration procedure when the concentration of monoclonal antibodies is low. It is necessary to create On the other hand, when using the ELISA or RIA method, the culture supernatant is directly used as the antigen-adsorbing solid phase. Then, secondary antibodies against various immunoglobulin isotypes and subclasses were used. By using antibodies corresponding to the monoclonal antibody, the isotype and subclass of the monoclonal antibody can be identified. It is possible to determine Furthermore, as a simpler method, a commercially available identification kit (e.g., Mouse Typer Kit) can be used. It is also possible to use a fluoropolymer (e.g., PEG-2000; manufactured by Bio-Rad Laboratories). Furthermore, protein quantification was performed using the Folin-Lowry method and absorbance at 280 nm (1. 4) (OD280 = immunoglobulin 1 mg / ml) can be done. Furthermore, steps (a) to (h) of (2) are carried out again to obtain a separate and independent monoclonal antibody. Even if a specific antibody is obtained, it is necessary to obtain an antibody with cytotoxic activity equivalent to that of the TINA1 antibody. An example of such an antibody is an antibody having the same epitope as the TINA1 antibody. Newly produced monoclonal antibodies can be used to detect T If the monoclonal antibody binds to the partial peptide or partial three-dimensional structure to which the INA1 antibody binds, It can be determined that the antibody binds to the same epitope as the TINA1 antibody. The monoclonal antibody competes with the INA1 antibody for binding to TROP2 (i.e. and confirm that the monoclonal antibody inhibits the binding of TINA1 antibody to TROP2. By doing so, even if the sequence or structure of the specific epitope has not been determined, the monoclonal antibody can be identified. It can be determined that the clonal antibody binds to the same epitope as the anti-TROP2 antibody. If the epitope is confirmed to be identical, the monoclonal antibody is a TINA1 antibody. It is strongly expected that the antibody will have antigen-binding ability or biological activity equivalent to that of the antibody.

[0058] (3) Other antibodies The antibodies of the present invention include monoclonal antibodies against human TROP2 as well as the above-mentioned monoclonal antibodies against human TROP2. Genetically modified recombinant antibodies that have been artificially modified to reduce heterologous antigenicity, for example For example, chimeric antibodies, humanized antibodies, human antibodies, etc. These antibodies can be produced using known methods. Chimeric antibodies include antibodies in which the variable and constant regions of the antibody are heterologous, such as mouse antibodies. Examples of such antibodies include chimeric antibodies in which the variable regions of a mouse or rat-derived antibody are joined to constant regions of human origin. (Proc. Natl. Acad. Sci. USA, 81, 6851- 6855, (1984)). Humanized antibodies are antibodies in which only the complementarity-determining regions (CDRs) are incorporated into a human-derived antibody. (See Nature (1986) 321, p. 522-525) and CDR grafting method. In addition to the CDR sequences, some of the framework amino acid residues were also transplanted into a human antibody. Examples of such compounds include those disclosed in International Publication No. 90 / 07861. However, the humanized antibody derived from the TINA1 antibody contains all six CDRs of the TINA1 antibody. There is no limitation to a specific humanized antibody as long as the sequence is maintained. The heavy chain variable region of the present invention is CDRH1 ( TAGMQ), CDRH2 (WINTHS) consisting of the amino acid sequence shown in SEQ ID NO: 24 GVPKYAEDFKG), and CDRH consisting of the amino acid sequence shown in SEQ ID NO: 25 3 (SGFGSSYWYFDV). The light chain variable region of the TINA1 antibody is a CDRL1 (KASQDVS) consisting of the amino acid sequence shown in SEQ ID NO: 26 in the Sequence Listing. TAVA), CDRL2 (SASYRYT) consisting of the amino acid sequence shown in SEQ ID NO: 27 ), and CDRL3 (QQHYITPLT ) is held.

[0059] Examples of humanized antibodies of the mouse antibody TINA1 include: (1) SEQ ID NO: 12, 1 (2) an amino acid sequence consisting of the 20th to 140th amino acid residues of the above ( an amino acid sequence having at least 95% homology to the amino acid sequence of 1), and (3) The amino acid sequence of (1) above in which one or several amino acids are deleted, substituted, or added. (4) a heavy chain comprising a heavy chain variable region consisting of any one of the amino acid sequences set forth in SEQ ID NO: an amino acid sequence consisting of the 21st to 129th amino acid residues of No. 18, 20, or 22; 5) Amino acids having at least 95% homology to the amino acid sequence of (4) above. (6) the amino acid sequence of (4) above, in which one or several amino acids are deleted or substituted or any combination of light chains containing a light chain variable region consisting of any one of the added amino acid sequences. A combination can be mentioned. In this specification, "several" means 1 to 10, 1 to 9, 1 to 8, 1 or more means up to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 do.

[0060] In addition, conservative amino acid substitutions are preferred as amino acid substitutions in this specification. Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related in their amino acid side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid Amino acid; basic group = lysine, arginine, histidine; nonpolar group = arylene leucine, valine, leucine, isoleucine, proline, phenylalanine, methionine, triglyceride uncharged polar family = glycine, asparagine, glutamine, cysteine , serine, threonine, tyrosine. Other preferred amino acid groups are: fat Aromatic hydroxy groups = serine and threonine; amide-containing groups = asparagine and Glutamine; Aliphatic group = Alanine, Valine, Leucine and Isoleucine; and aromatic Aromatic group = phenylalanine, tryptophan and tyrosine. Such amino acid substitutions are It is preferable to carry out the modification within a range that does not deteriorate the properties of the substance having the original amino acid sequence.

[0061] The preferred combination of heavy and light chains of the antibody is the one having the amino acid number 1 in SEQ ID NO: 12. a heavy chain having a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 20 to 140; a light chain having a variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in an antibody consisting of a chain, an amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 12; a heavy chain having a variable region consisting of amino acids 21 to 129 in SEQ ID NO: 20; an antibody comprising a light chain having a variable region comprising the amino acid sequence set forth in SEQ ID NO: 12; a heavy chain having a variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140; A variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 22 an antibody comprising a light chain having a region set forth in amino acids 20 to 140 of SEQ ID NO: 14; and a heavy chain having a variable region consisting of the amino acid sequence of SEQ ID NO: 18, an antibody comprising a light chain having a variable region comprising an amino acid sequence set forth in any one of SEQ ID NOs. 1 to 129; No. 14, which has a variable region consisting of the amino acid sequence of amino acid numbers 20 to 140. and the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 20. an antibody comprising a light chain having a variable region comprising amino acid numbers 20 to 14 in SEQ ID NO: 14; a heavy chain having a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 22; A light chain having a variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129. an antibody consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 16; A heavy chain having a variable region and an amino acid sequence set forth in amino acid numbers 21 to 129 of SEQ ID NO: 18. an antibody having a light chain with a variable region consisting of the amino acid sequence of SEQ ID NO: 16; A heavy chain having a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 2 0, and having a variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 an antibody comprising a light chain, and an antibody comprising an amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 16; and a heavy chain having a variable region consisting of amino acid sequence 21-23 in SEQ ID NO: 22. 129 to 140. An antibody having a light chain with a variable region consisting of an amino acid sequence set forth in any one of claims 1 to 129. can be done.

[0062] Further preferred combinations of antibodies include those having amino acid numbers 20 to 47 in SEQ ID NO: 12. 0 and a heavy chain consisting of amino acid sequences of amino acid numbers 21 to 22 in SEQ ID NO: 18. 34, an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 to 470; an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 21 to 234; a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 and a an antibody having a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14, and and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18. an antibody comprising the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; a heavy chain consisting of amino acids 21 to 234 in SEQ ID NO: 20; an antibody having a light chain consisting of the sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14; and a heavy chain consisting of the amino acid sequence shown in amino acid numbers 21 to 234 in SEQ ID NO: 22. an antibody having a light chain consisting of the amino acid sequence of SEQ ID NO: 16; a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, an antibody having a light chain consisting of the amino acid sequence of amino acids 1 to 234 in SEQ ID NO: 16; a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 to 470; an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NOs: 21 to 234, No. 16, a heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 470 and a sequence 22, comprising a light chain having the amino acid sequence set forth in amino acid numbers 21 to 234. Examples of antibodies include:

[0063] An excellent and preferred combination is the amino acid sequence from amino acid number 20 to 140 in SEQ ID NO: 12. a heavy chain having a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 18; an antibody comprising a light chain having a variable region consisting of an amino acid sequence set forth in any of Nos. 21 to 129; A variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 14 a heavy chain having a region and amino acids set forth in amino acids 21 to 129 of SEQ ID NO: 18; an antibody comprising a light chain having a variable region consisting of the sequence: 0 to 140, and a heavy chain having a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 20. a light chain having a variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129; and an antibody comprising the amino acid sequence set forth in amino acid numbers 20 to 140 of SEQ ID NO: 16. a heavy chain having a variable region consisting of the sequence: 9. An example of an antibody is an antibody having a light chain with a variable region consisting of the amino acid sequence of do.

[0064] Another preferred combination is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18; and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 20. a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 22; an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 14; an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18; an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 21; A heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 and an amino acid sequence set forth in SEQ ID NO: 22 an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16; No. 18, an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16, an antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 16 and the amino acid sequence set forth in SEQ ID NO: 22 Examples include antibodies having a light chain consisting of the sequence

[0065] An excellent and preferred combination is the amino acid sequence from amino acid number 20 to 470 in SEQ ID NO: 12. and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, amino acid numbers 21 to 23. 4, an antibody having a light chain consisting of the amino acid sequence of SEQ ID NO: 14, a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, and an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 14; a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 and a an antibody having a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 16; and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 22. Examples include antibodies consisting of chains.

[0066] A more excellent and preferred combination is a combination of amino acids 20 to 46 in SEQ ID NO: 12. 9 and a heavy chain consisting of amino acid sequences of amino acid numbers 21 to 22 in SEQ ID NO: 18. an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 14; a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 to 469 and a an antibody having a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 21 to 234; a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 469 and a an antibody having a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in and a duplicate consisting of the amino acid sequence set forth in amino acid numbers 20 to 469 in SEQ ID NO: 16. and the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 22. Examples include antibodies consisting of light chains.

[0067] Combining sequences that show high homology with the above heavy chain amino acid sequences and light chain amino acid sequences By doing so, it is possible to select antibodies having biological activity equivalent to that of the above antibodies. Such homology is generally 80% or more, preferably 90% or more. The homology is preferably 95% or more, and more preferably 99% or more. In addition, one or several amino acid residues are substituted or deleted in the amino acid sequence of the heavy chain or light chain. Combining deleted or added amino acid sequences can also produce antibodies with activity equivalent to that of the above antibodies. It is possible to select antibodies with biological activity.

[0068] The homology between two amino acid sequences was calculated using the Blast algorithm version n 2.2.2(Altschul, Stephen F.,Thomas L.Ma dden, Alejandro A. Schaeffer, Jinghui Zhan g, Zheng Zhang, Webb Miller, and David J. .Lipman (1997), “Gapped BLAST and PSI-BLAS T:a new generation of protein databases ``arch programs'', Nucleic Acids Res.25:338 9-3402) can be determined by using the default parameters The Blast algorithm can be found on the Internet at www.ncbi.nlm.nih.gov / blast It can also be used by accessing

[0069] In addition, in the heavy chain amino acid sequence shown in SEQ ID NO: 12, 14, or 16 in the sequence listing, The amino acid sequence consisting of the first 19th amino acid residues is a signal sequence, and the amino acid sequence consisting of the 20th to 140th amino acid residues is a signal sequence. The amino acid sequence consisting of amino acid residues 141 to 470 is a variable region. The amino acid sequence of SEQ ID NO: 12 is shown in FIG. The sequence of No. 14 is shown in FIG. 4, and the sequence of SEQ ID NO: 16 is shown in FIG. In addition, in the light chain amino acid sequence shown in SEQ ID NO: 18, 20, or 22 in the sequence listing, The amino acid sequence consisting of the first 20 amino acid residues is a signal sequence, and the amino acid sequence consisting of the 21st to 129th amino acid residues is a signal sequence. The amino acid sequence consisting of amino acid residues 130 to 234 is a variable region. The amino acid sequence of SEQ ID NO: 18 is shown in FIG. 6, and the sequence The sequence of No. 20 is shown in FIG. 7, and the sequence of SEQ ID NO: 22 is shown in FIG.

[0070] The antibodies of the present invention can further include human antibodies that bind to TROP2. Human anti-TROP2 antibodies are human antibodies that contain only the gene sequence of antibodies derived from human chromosomes. The anti-TROP2 human antibody is a human antibody containing the heavy and light chain genes of human antibodies. A method using human antibody-producing mice carrying antibody fragments (Tomizuka, K. et al. ,Nature Genetics(1997)16,p.133-143;Kuroi wa, Y. et. al., Nucl. Acids Res. (1998) 26, p. 34 47-3448;Yoshida,H.et.al.,Animal Cell Tec hnology:Basic and Applied Aspects vol.10 , p.69-73 (Kitagawa, Y., Matsuda, T. and Iijim a, S. eds.), Kluwer Academic Publishers, 199 9.;Tomizuka,K.et.al.,Proc.Natl.Acad.Sci. See USA (2000) 97, pp. 722-727, etc. do.

[0071] Such human antibody-producing mice specifically contain endogenous immunoglobulin heavy and light chain genes. The gene locus was destroyed and replaced by a yeast artificial chromosome. Genetic engineering of human immunoglobulin heavy and light chains via chromosome (YAC) vectors. As genetically modified animals into which the gene locus has been introduced, knockout animals and transgenic animals They can be created by creating animals and crossbreeding these animals. Furthermore, a gene encoding each of the heavy and light chains of such a human antibody can be produced by genetic recombination technology. A eukaryotic cell is transformed with a cDNA, preferably a vector containing the cDNA, and genetically This is achieved by culturing transformed cells that produce recombinant human monoclonal antibodies. The antibody can also be obtained from the culture supernatant. Here, examples of the host include eukaryotic cells, preferably CHO cells, lymphocytes, and myeloma cells. Mammalian cells such as the above can be used.

[0072] In addition, we have obtained human antibodies derived from phage display selected from a human antibody library. How to (Wormstone, IMet.al, Investigative O phthalmology & Visual Science.(2002)43(7 ),p.2301-2308;Carmen,S.et.al.,Briefings in Functional Genomics and Proteomics(20 02),1(2),p.189-203;Siriwardena,D.et.al., See Ophthalmology (2002) 109(3), p.427-431, etc. ) is also known. For example, the variable region of a human antibody is expressed on the surface of a phage as a single chain antibody (scFv). The phage display method (Nature Bio technology (2005), 23, (9), p. 1105-1116) It is possible. By analyzing the genes of the phages selected by binding to the antigen, The DNA sequence encoding the variable region of the corresponding human antibody can be determined. Once the DNA sequence of the scFv that binds to the antigen is determined, an expression vector containing the sequence can be prepared. Human antibodies can be obtained by creating a human antibody vector and expressing it in a suitable host. (International Publication Nos. 92 / 01047, 92 / 20791, 93 / 06213, 93 / 11236, 93 / 19172, 95 / 01438, 95 / 153 No.88;Annu.Rev.Immunol(1994)12,p.433-455;N ature Biotechnology(2005)23(9),p.1105-11 16). The newly created human antibody is a partial peptide or partial three-dimensional structure that binds to the TINA1 antibody. If the human antibody binds to the structure, it is determined that the human antibody binds to the same epitope as the TINA1 antibody. Furthermore, the human antibody can compete with the binding of the TINA1 antibody to TROP2. (i.e., the human antibody interferes with the binding of the TINA1 antibody to TROP2). By confirming this, it is possible to identify the relevant epitope even if the sequence or structure of the specific epitope has not been determined. It can be determined that the human antibody binds to the same epitope as the TINA1 antibody. If the same topography is confirmed, the human antibody will have the same biological activity as the TINA1 antibody. It is strongly expected that the The chimeric, humanized, or human antibodies obtained by the above methods can be purified by known methods, etc. By this method, the binding ability to the antigen can be evaluated and a suitable antibody can be selected.

[0073] Another index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is a thermal analysis that provides a good indicator of the relative structural stability of proteins. This is an instrument that can measure the denaturation midpoint (Tm) quickly and accurately. By measuring the Tm value and comparing the values, differences in thermal stability can be compared. It is known that the storage stability of antibodies correlates to some extent with their thermal stability. (Lori Burton,et.al.,Pharmaceutical Development opment and Technology(2007)12,p.265-273) Therefore, suitable antibodies can be selected using thermostability as an index. The targets are high yield in suitable host cells and low aggregation in aqueous solution. For example, the antibody with the highest yield does not necessarily exhibit the highest thermostability. Therefore, based on the above mentioned indicators, a comprehensive judgment is made to select the most suitable antibody for administration to humans. You need to select the body.

[0074] The antibody of the present invention also includes modified antibodies. Such modified antibodies are those that have been modified chemically or by other methods. means that the amino acid skeleton has been biologically modified. and chemical modifications of N-linked or O-linked carbohydrate chains. Examples of potential modifications include post-translational modifications (e.g., N-linked or O-linked glycosylation, N-terminal or C-terminal glycosylation, etc.). End-processing, deamidation, isomerization of aspartic acid, oxidation of methionine) When expressed in prokaryotic host cells, a methionine residue is added to the N-terminus. Also included are those prepared by using a target to enable detection or isolation of the antibody or antigen of the present invention. Such modified compounds, such as enzyme-labeled compounds, fluorescent-labeled compounds, and affinity-labeled compounds, are also included. Such modified antibodies of the present invention are intended to improve the stability and blood retention of the antibodies. These are useful for reducing antigenicity, detecting or isolating antibodies or antigens, etc.

[0075] In addition, it is possible to regulate sugar chain modification (glycosylation, defucosylation) of the antibody of the present invention. Antibody-dependent cellular cytotoxicity can be enhanced by glycosylation of antibodies. Techniques for adjusting decorations are described in International Publication Nos. 1999 / 54342 and 2000 / 61739. , 2002 / 31140, etc. are known, but are not limited to these. The antibodies of the invention also include antibodies in which the glycosylation has been regulated. When the antibody gene is isolated and then introduced into a suitable host to produce the antibody, A combination of a host and an expression vector can be used. Specific examples of antibody genes include , a gene encoding the heavy chain sequence of the antibody described herein, and a gene encoding the light chain sequence When transforming a host cell, the heavy chain The sequence gene and the light chain sequence gene can be inserted into the same expression vector. Alternatively, they may be inserted into separate expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, or eukaryotic microorganisms can be used. In particular, mammalian cells, such as monkey cells, are used. Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CR L-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) Folate reductase-deficient strain (Urlaub, G. and Chasin, LA Proc. Natl.Acad.Sci.USA(1980)77,p.4126-4220 ) can be mentioned. When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis. The desired antibody gene is introduced into these cells by transformation, and the transformed cells Antibodies are obtained by culturing the antibody in vitro. The yield may vary depending on the antibody, and antibodies with equivalent binding activity are selected as pharmaceuticals based on the yield. Therefore, the antibodies of the present invention can be selected based on the above-mentioned structure. a step of culturing the transformed host cells, and extracting the desired antibody or a method for producing said antibody, characterized in that it includes a step of collecting a functional fragment of said antibody. The term also includes antibodies obtained by the above method.

[0076] In addition, the carboxyl-terminal lysine residue of the heavy chain of the antibody produced in mammalian cultured cells is deleted. It is known that (Journal of Chromatography A, 705:129-134(1995)), and also glycine at the carboxyl terminus of the heavy chain. Two lysine amino acid residues are deleted, and a new proline residue is added at the carboxyl terminal. It is known that amidation occurs (Analytical Biochemistry , 360:75-83(2007)). However, these deletions and modifications of the heavy chain sequence have not been shown to be effective against the antibody. The body's antigen-binding ability and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) Therefore, the antibody according to the present invention includes the antibody having the modification and the antibody having the modification. Functional fragments of antibodies are also included, which lack one or two amino acids at the carboxyl terminus of the heavy chain. and amidated deletions (e.g., proline residues at the carboxyl terminal). However, the present invention also includes heavy chains in which the antigen-binding ability and effector function are maintained. As long as the deletion of the carboxyl terminal of the heavy chain of the antibody of the present invention is not limited to the above types, The two heavy chains constituting the antibody of the present invention may be full-length or the above-mentioned deleted heavy chains. The heavy chain may be any one of the heavy chains selected from the group consisting of: The quantitative ratio of each deletion may be determined depending on the species of cultured mammalian cells that produce the antibody of the present invention. Although the antibody of the present invention may be affected by the type and culture conditions, the main component of the antibody of the present invention is a pair of heavy chains. On the other hand, there are cases where one amino acid residue at the carboxyl terminus is deleted. .

[0077] The isotype of the antibody of the present invention may be, for example, IgG (IgG1, IgG2, IgG3 IgG1 or IgG2 is preferred. can be done.

[0078] The biological activity of an antibody generally includes antigen binding activity, which is the ability of the antibody to bind to an antigen. The ability to be internalized into cells expressing antigens, neutralize the activity of antigens, and enhance the activity of antigens. activity, antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity and Examples of the phagocytosis that the antibody of the present invention possesses include phagocytosis-dependent cell-mediated phagocytosis (ADCP). The function of the compound is its binding activity to TROP2, and preferably its binding to TROP2. Therefore, the antibody of the present invention has the activity of being internalized into TROP2-expressing cells. In addition to the cytotoxic activity, the antibody may also have ADCC activity, CDC activity, and / or ADCP activity. good.

[0079] The obtained antibodies can be purified to homogeneity. Separation and purification of antibodies are performed in the same way as conventional protein purification. For example, column chromatography, Filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric By appropriately selecting and combining techniques such as spot electrophoresis, antibodies can be separated and purified (Str ategies for Protein Purification and Cha racterization:A Laboratory Course Manual ,Daniel R.Marshak et al.eds.,Cold Spring Harbor Laboratory Press(1996);Antibodie s:A Laboratory Manual.Ed Harlow and Davi d Lane,Cold Spring Harbor Laboratory(198 8)) but is not limited to these. Chromatography includes affinity chromatography, ion exchange chromatography, chromatograph, hydrophobic chromatography, gel filtration chromatography, reversed phase chromatography Examples of suitable methods include chromatography, adsorption chromatography, and the like. These chromatography methods use liquid chromatography such as HPLC and FPLC. This can be done. Columns used for affinity chromatography include Protein A columns, Protein B columns, and Protein C columns. For example, a column using a protein A column can be used. Hyper D, POROS, Sepharose FF (Pharmacia), etc. Some examples include: Antibodies can also be purified by using antigen-immobilized carriers and utilizing their binding to antigens. It is possible.

[0080] [Anti-tumor compound] Antitumor compounds conjugated to the anti-TROP2 antibody-drug conjugates of the present invention The antitumor compounds used in the present invention are compounds that have antitumor effects. There are no particular limitations on the type of the linker structure as long as it has a substituent or partial structure that can be bonded to the linker structure. The antitumor compound is formed by cleaving a part or all of the linker in the tumor cell to form the antitumor compound portion. When the linker is cleaved at the site where it binds to the drug, the antitumor effect is achieved. The antitumor compound is released in its unmodified form, allowing its inherent antitumor effect to be exerted. The antitumor compound used in the present invention is exatecan, a camptothecin derivative. ((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; formula:)

[0081] [ka]

[0082] This exatecan has excellent antitumor activity. However, it has not yet been commercially available as an antitumor drug. The amino group at position 1 can be suitably used as a binding site to a linker structure. In addition, exatecan may be released within tumor cells with part of the linker still attached. However, even with this structure, it is an excellent compound that exhibits excellent antitumor effects. Since exatecan has a camptothecin structure, it is effective in acidic aqueous media (e.g., pH 3). In aqueous media (e.g., HCl), the equilibrium shifts to the structure in which the lactone ring is formed (closed ring isomer), while in aqueous media (e.g., HCl), the equilibrium shifts to the structure in which the lactone ring is formed (closed ring isomer). It is known that at pH values ​​of about 10, the equilibrium shifts toward the structure in which the lactone ring is opened (open ring isomer). Drug conjugates incorporating exatecan residues corresponding to such closed and open ring structures are The same antitumor effect is expected regardless of the gate, and both states are within the scope of the present invention. It goes without saying that it is included.

[0083] Other antitumor compounds include, for example, doxorubicin, daunorubicin, and mitomycin. C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate platinum-based antitumor agents (cisplatin or its derivatives), taxol or its derivatives, Other camptothecins or derivatives thereof (antitumor agents described in JP-A-6-87746), etc. Examples include:

[0084] In antibody-drug conjugates, the number of drugs bound to one antibody molecule determines its efficacy and safety. The production of antibody-drug conjugates is an important factor affecting the safety of the antibody-drug conjugates. The reaction conditions, such as the amounts of raw materials and reagents used, are specified to ensure a certain number of reactions. Unlike chemical reactions involving small molecules, antibody-drug conjugates can bind different numbers of drugs. The number of drugs bound to one antibody molecule is usually an average of 100,000. That is, it is specified and expressed as the average number of bound drugs. As long as the number of drugs bound is different, that is, the specific The average number of drugs conjugated is shown, except where antibody-drug conjugates with a constant number of drugs are shown. The number of exatecan molecules bound to an antibody molecule can be controlled, and The average number of exatecan molecules bound per molecule is about 1 to 10. However, the number is preferably 2 to 8, and more preferably 3 to 8. If there is a need for such a method, it is possible to design a reaction for binding a required number of drugs to an antibody based on the description of the examples of this application. This allows us to obtain antibody-drug conjugates with controlled binding of exatecan. This can be done.

[0085] [Linker structure] In the anti-TROP2 antibody-drug conjugate of the present invention, the antitumor compound is The linker structure for binding to the antibody is described below. -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- The antibody has the structure L 1 The end of (L 2 The antibody binds to the opposite end of the antibody (the end opposite to the end to which the antibody binds) and The tumorigenic compound is -L a -(CH2)n 2 It bonds through the carbonyl group of the -C(=O)- moiety. n 1 represents an integer of 0 to 6, preferably an integer of 1 to 5, more preferably 1 So it is 3.

[0086] 1.L 1 L 1 teeth, -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)- It is shown in the structure: where n 3 is an integer from 2 to 8, and "-(Succinimid-3-yl-N)-" is a compound represented by the following formula:

[0087] [ka]

[0088] The 3rd position in this partial structure is the binding site for the anti-TROP2 antibody. The binding to the antibody at position 3 is characterized by forming a thioether bond. The nitrogen atom at position 1 of this structural part is methylated to the methyl group present in the linker containing this structure. It bonds to the carbon atom of the hydroxy group, i.e., -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-L 2 - is expressed by the following formula: The structure shown is (where "Antibody-S-" is derived from the antibody).

[0089] [ka]

[0090] In the formula, n 3 is an integer from 2 to 8, preferably from 2 to 5.

[0091] L 1 Specific examples include: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)- The following can be mentioned:

[0092] 2.L 2 L 2 teeth, -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- The structure is shown by L 2 may not exist, in which case L 2 is a single bond. n 4 is an integer from 1 to 6, preferably from 2 to 4. 2 is the terminal amino group and L 1 In conclusion The carbonyl group at the opposite end of the P and combine.

[0093] L 2 Specific examples include: -NH-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH 2- O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH 2- O-CH2CH2-O-CH2CH2-C(=O)- The following can be mentioned:

[0094] 3.L P L P is a peptide residue consisting of 2 to 7 amino acids. It is composed of oligopeptide residues in which amino acids are peptide-bonded. P is the N-terminus In L 2 and at the C-terminus, the linker -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- part It binds to amino groups.

[0095] L P The amino acids constituting the nucleotide sequence are not particularly limited, but may be, for example, L- or D-amino acids. In addition to α-amino acids, β-alanine, ε-amino acids, and L-amino acids are also preferred. It may be an amino acid having a structure such as caproic acid or γ-aminobutyric acid, and further, for example, N-methyl It may also be a non-naturally occurring amino acid such as a methylated amino acid. L P The amino acid sequence of the present invention is not particularly limited, but may include phenylalanine as a constituent amino acid. Phe (F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), and arachidonic acid Ala (A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser ;S), glutamic acid (Glu;E), aspartic acid (Asp;D), etc. Among these, phenylalanine, glycine, valine, lysine, and citrulline are preferred. Examples of amino acids include thiamin, serine, glutamic acid, and aspartic acid. The number of amino acids ranges from 2 to 7. One piece is fine.

[0096] L P As a concrete example, -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, -GGFGGGF- The above "(D-)D" means D-aspartic acid. Particularly preferred L-drug conjugates P An example of such a peptide is the tetrapeptide residue -GGFG-. This can be done.

[0097] 4.L a -(CH2)n 2 -C(=O)- L a -(CH2)n 2 L in -C(=O)- a is the structure of -O- or a single bond. 2 Is it 0? It is an integer from 0 to 5, preferably 0 to 3, and more preferably 0 or 1. L a -(CH2)n 2 Examples of -C(=O)- include the following structures: -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, -O-CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2CH2-C(=O)-. Among these, -O-CH2-C(=O)-, -O-CH2CH2-C(=O)- If so, La is a single bond and n 2 It is preferred if is 0.

[0098] Linker -NH-(CH2)n 1 -L a -(CH2)n 2 Specific examples of the structure represented by -C(=O)- include: -NH-CH2-C(=O)-, -NH-CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-OC(=O)-, -NH-CH2CH2-O-CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-, The following can be mentioned:

[0099] More preferably, -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-OC(=O)- is.

[0100] Linker -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- has a chain length of 4 to 7 atoms Those having a chain length of 5 or 6 atoms are preferred, more preferably those having a chain length of 5 or 6 atoms.

[0101] The anti-TROP2 antibody-drug conjugate of the present invention translocates into tumor cells and then binds to phosphorylated The Kerr moiety is cut off, and NH2-(CH2)n 1 -L a -(CH2)n 2 Drug derivatives with the structure -C(=O)-(NH-DX) The antibody-drug conjugate of the present invention is believed to release the antibody and exert an antitumor effect. The antitumor derivatives that exhibit antitumor effects when released from the linker -NH- (CH2)n 1 -L a -(CH2)n 2 The structure has an amino group at the end of the -C(=O)- structure. Antitumor derivatives include, but are particularly preferred as follows: NH2-CH2CH2-C(=O)-(NH-DX), NH2-CH2CH2CH2-C(=O)-(NH-DX), NH2-CH2-O-CH2-C(=O)-(NH-DX), NH2-CH2CH2-O-CH2-C(=O)-(NH-DX). In the case of NH2-CH2-O-CH2-C(=O)-(NH-DX), the aminal structure in the molecule is unstable. Therefore, it further self-decomposes HO-CH2-C(=O)-(NH-DX) These compounds were confirmed to be the compounds of the antibody-drug conjugates of the present invention. It can also be suitably used as a production intermediate.

[0102] In the antibody-drug conjugate of the present invention in which the drug is exatecan, The drug-linker structural portion [-L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX)] antibody These drug-linker structural moieties are preferably linked to an average of The number of bonds may be 1 to 10, preferably 2 to 8, and more preferably Usually it is 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)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). Among these, the following are more preferred: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). Furthermore, the following is preferable: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX).

[0103] In the antibody-drug conjugate of the present invention, a linker that binds the anti-TROP2 antibody to the drug is The linker structure is a structure that connects the preferred structures shown in each linker portion described above. A preferred linker can be constructed by the above. The left end of the structure is the binding site to the antibody, The right end is the drug binding site. -(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)-, -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。 Among these, the following are more preferred. -(N-Succinimid-3-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-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-CH2C H2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-. Furthermore, the following can be preferably mentioned: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-.

[0104] [Manufacturing method] Next, a representative method for producing the antibody-drug conjugate of the present invention or its production intermediate will be described. In the following, in order to indicate the compounds, the compounds shown in each reaction scheme are In other words, they are referred to as "compound of formula (1)", "compound (1)", etc. Compounds with other numbers will be described in the same manner.

[0105] 1. Manufacturing method 1 The antibody and drug-linker structure are linked via a thioether, as shown in formula (1). The antibody-drug conjugate can be produced, for example, by the following method.

[0106] [ka]

[0107] [Wherein, AB represents an antibody having a sulfhydryl group, and L 1 'L 1 The linker structure shown in In this structure, the linker end is a maleimidyl group (following formula)

[0108] [ka]

[0109] The structure shown here (where the nitrogen atom is the binding site) is as follows: 1 of Our -(Succinimid-3-yl-N)-(CH2)n 3 In -C(=O)-, the -(Succinimid-3-yl-N)- moiety is male. represents a group that has become an imidyl group. Also, -(NH-DX) represents the following formula:

[0110] [ka]

[0111] The structure is represented by the formula: ]

[0112] In the above reaction scheme, in the compound of formula (1), the structure from the drug to the linker terminal is Although one structural moiety can be interpreted as a structure bound to one antibody, this is for the sake of explanation. This is a convenient description, and in reality, multiple such structural moieties bind to one antibody molecule. This situation is the same in the following description of the manufacturing method.

[0113] Compound (2), which can be obtained by the method described below, and antibody (3), which has a sulfhydryl group, are used. a) to produce an antibody-drug conjugate (1). do. The antibody (3a) having a sulfhydryl group can be obtained by a method well known to those skilled in the art (Her et al. manson, GT, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press(1996) For example, Traut's reagent acts on the amino groups of antibodies; N-succinimidyl S-acetylthioalkanoates react with the amino groups of the antibody, and then hydroxyl groups are N-succinimidyl 3-(pyridyldithio)propionate was prepared by the action of amine. After using the solution, a reducing agent is applied; dithiothreitol, 2-mercaptoethanol, A reducing agent such as thi(2-carboxyethyl)phosphine hydrochloride (TCEP) is applied to the antibody. and reducing disulfide bonds in the hinge region of the antibody to generate sulfhydryl groups; Methods include, but are not limited to, these. Specifically, TCEP was used as a reducing agent, and the following was used for each disulfide in the hinge region of the antibody: and reacting it with an antibody in a buffer containing a chelating agent, It is possible to obtain antibodies in which the intracellular hinge disulfides have been partially or completely reduced. Examples of rate agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine These are used at a concentration of 1 mM to 20 mM. Buffer solutions include sodium phosphate, sodium borate, and sodium acetate solutions. Specifically, the antibody is incubated with TCEP at 4°C to 37°C for 1 to 4 hours. The antibody (3a) having partially or completely reduced sulfhydryl groups is reacted with You can get it. Here, a reaction is carried out to add a sulfhydryl group to the drug-linker moiety, thereby forming a thio The drug-linker moiety can be attached by an ether bond. 2 to 20 molar equivalents of compound (2) per antibody (3a) having a sulfhydryl group ) to prepare antibody-drug conjugates with 2 to 8 drugs bound to each antibody. Specifically, antibody (3a) having a sulfhydryl group can be produced by A solution of compound (2) is added to a buffer solution containing the compound (2) and reacted. The liquid may be a sodium acetate solution, sodium phosphate, sodium borate, etc. The pH during the reaction is 5 to 9, and more preferably around pH 7. The solvent for dissolving the substance (2) is dimethyl sulfoxide (DMSO), dimethylformamide (DMSO), or the like. Dimethyl acetamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (N Organic solvents such as methyl methylpropional (MP) can be used. The organic solvent solution in which the compound (2) was dissolved was mixed with the antibody (3a) having a sulfhydryl group. The reaction can be carried out by adding 1 to 20% v / v to a buffer solution containing the enzyme. 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 reactivity of unreacted compound (2) is terminated by quenching with a thiol-containing reagent. The thiol-containing reagent can be, for example, cysteine ​​or N-acetyl-L-cysteine ​​( More specifically, NAC is used in an amount of 1 to 2 moles per mole of compound (2). The reaction can be terminated by adding 100 equivalents of ATP and incubating at room temperature for 10 to 30 minutes. The produced antibody-drug conjugate (1) was concentrated and buffered by the following common procedure. antibody-drug interaction analysis. Identification of the product conjugate (1) can be performed.

[0114] Common Procedure A: Concentration of an Aqueous Solution of an Antibody or Antibody-Drug Conjugate Amicon Ultra(50,000 MWCO, Millipore Corp The antibody or antibody-drug conjugate solution was placed in a container in the oration and centrifuged (A Centrifugation using a llegra X-15R (Beckman Coulter, Inc.) The antibody or antibody-drug complex was centrifuged at 2000 to 3800 G for 5 to 20 minutes. The diluate solution was concentrated. Common Procedure B: Antibody concentration measurement UV measurement device (Nanodrop 1000, Thermo Fisher Scien Antibody concentrations were measured using a ELISA kit (Protein Immunosorbent Assay Kit, Inc.) according to the manufacturer's instructions. At that time, the 280 nm extinction coefficient (1.3 mL mg) differs for each antibody. -1 cm -1 ~1.8 mLmg -1 cm -1 ) was used. Common Procedure C-1: Buffer Exchange of Antibody NAP-25 column (Cat. No. 17) using Sephadex G-25 support -0852-02,GE Healthcare Japan Corporation ) was diluted with sodium chloride (137 mM) and ethylenediamine Phosphate buffer (10 mM, pH 6.0; herein referred to as P) containing EDTA (5 mM) The column was equilibrated with PBS 6.0 / EDTA. After applying 2.5 mL of antibody solution, the fraction was eluted with 3.5 mL of PBS 6.0 / EDTA. This fraction (3.5 mL) was collected. This fraction was concentrated using common procedure A and purified using common procedure B. After measuring the antibody concentration, the antibody was diluted to 10 mg / mL using PBS6.0 / EDTA. The concentration was adjusted. Common Procedure C-2: Buffer Exchange of Antibodies NAP-25 column (Cat. No. 17) using Sephadex G-25 support -0852-02,GE Healthcare Japan Corporation ) was diluted with sodium chloride (50 mM) and EDTA (2 mM) according to the manufacturer's instructions. Phosphate buffer solution (50 mM, pH 6.5; referred to herein as PBS6.5 / EDTA) containing For each NAP-25 column, 2.5 mL of antibody solution was added. After that, the fraction (3.5 mL) eluted with PBS 6.5 / EDTA 3.5 mL was collected. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B. The antibody concentration was then adjusted to 20 mg / mL using PBS6.5 / EDTA. Common Procedure D: Purification of Antibody-Drug Conjugates Commercially available phosphate buffer solution (PBS7.4, Cat. No. 10010-023, Invitrogen) rogen), sodium phosphate buffer (10 mM) containing sodium chloride (137 mM) , pH 6.0; referred to herein as PBS 6.0) or containing Sorbitol (5%) acetate buffer (10 mM, pH 5.5; herein referred to as ABS) The NAP-25 column was equilibrated. The antibody-drug conjugate was loaded onto the NAP-25 column. The reaction solution (approximately 1.5 mL) was placed on the column and eluted with the amount of buffer specified by the manufacturer. The antibody fraction was then collected. This collected fraction was again loaded onto the NAP-25 column and eluted with buffer. By repeating this gel filtration purification procedure two or three times, unbound drug linkers and low molecular weight compounds were removed. Compound (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl- Antibody-drug conjugates excluding L-cysteine ​​(NAC, dimethyl sulfoxide) obtained. Common Procedure E: Antibody Concentration in Antibody-Drug Conjugates and Average Drug Per Antibody Molecule Measurement of the average number of bonds (1) The bound drug concentration in the antibody-drug conjugate is determined by the aqueous solution of the antibody-drug conjugate. After measuring the UV absorbance at two wavelengths of 280 nm and 370 nm, perform the following calculation: This allows the calculation. The total absorbance at a given wavelength is equal to the sum of the absorbances of all absorbing species present in the system ( Because of the additivity of absorbance, the difference between the antibody and drug before and after conjugation is Assuming there is no change in the molar extinction coefficient of the compound, the antibody concentration in the antibody-drug conjugate The intensity and drug concentration are expressed by the following relationship: A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Formula (I) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Formula (II) where A 280 indicates the absorbance of the antibody-drug conjugate aqueous solution at 280 nm. , A 370indicates the absorbance of the antibody-drug conjugate aqueous solution at 370 nm, and A A,280 indicates the absorbance of the antibody at 280 nm, and A A,370 is the absorbance of the antibody at 370 nm indicates A D,280 denotes the absorbance of the conjugate precursor at 280 nm, and A D,370 teeth indicates the absorbance of the conjugate precursor at 370 nm, and ε A,280 is at 280 nm is the molar extinction coefficient of the antibody, and ε A,370 is the molar extinction coefficient of the antibody at 370 nm , ε D,280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D,370 teeth C denotes the molar extinction coefficient of the conjugate precursor at 370 nm. A antibody-drug conjugate indicates the antibody concentration in the adjugate, C D is the drug concentration in the antibody-drug conjugate Shows. where ε A,280 , ε A,370 , ε D,280 , ε D,370 is a value prepared in advance (calculated estimated value or The actual value obtained from UV measurement of the compound is used. For example, ε A,280 is the antibody's amino acid sequence. From the amino acid sequence, the nucleotide sequence was calculated by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). It can be estimated. A,370 is usually zero. D,280 and ε D,370 is used The run was performed by measuring the absorbance of a solution in which the conjugate precursor was dissolved at a certain molar concentration. According to Beer-Beert's law (absorbance = molar concentration × molar extinction coefficient × cell path length), A of the antibody-drug conjugate aqueous solution 280and A 370 Measure these values By substituting into equations (I) and (II) and solving the simultaneous equations, C A and C D Ask for Furthermore, C D C A The average number of drugs bound per antibody can be calculated by dividing by This can be done.

[0115] Common Procedure F: Measurement of the average number of drugs bound per antibody molecule in antibody-drug conjugates fixed(2) The average number of drugs bound per antibody molecule in antibody-drug conjugates is In addition to procedure E, high performance liquid chromatography (HPLC) analysis was performed using the following method: It can also be sought. [F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was dissolved in dithiothreitol ( The mixture was mixed with an aqueous solution of DTT (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes. By bating the antibody, the disulfide bond between the L and H chains of the antibody-drug conjugate is cleaved. The digested sample is used for HPLC analysis. [F-2.HPLC analysis] The HPLC analysis is carried out under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent T technologies) Detector: ultraviolet spectrophotometer (measurement wavelength: 280 nm) Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Agilent T echnologies, P / N PL1912-1802) Column temperature: 80℃ Mobile phase A: 0.04% trifluoroacetic acid (TFA) in water Mobile phase B: Acetonitrile solution containing 0.04% TFA Gradient program: 29%-36% (0 min-12.5 min), 36%-42% (1 2.5-15 minutes), 42%-29% (15 minutes-15.1 minutes), 29%-29% (15.1 minutes - 25 minutes) Sample injection volume: 15 μL [F-3. Data Analysis] [F-3-1] To the L chain (L0) and H chain (H0) of an antibody to which no drug is bound, The drug-bound L chain (L chain with one drug bound: L1) and the H chain (H chain with one drug bound: L2) H chain with two drugs bound: H1, H chain with two drugs bound: H2, H chain with three drugs bound: H3) The retention time increases with the number of drugs added, which increases hydrophobicity. The peaks detected by comparing the retention times with L0 and H0 are can be assigned to either L0, L1, H0, H1, H2, or H3. [F-3-2] Because the drug linker has UV absorption, depending on the number of drug linkers bonded, Correct the peak area value using the molar extinction coefficients of the L chain, H chain, and drug linker according to the following formula: conduct.

[0116]

number

[0117]

number

[0118] Here, the molar extinction coefficients (280 nm) of the L chain and H chain of each antibody were calculated using a known method. The L and H chains of each antibody were identified by the method (Protein Science, 1995, vol. 4, 2411-2423). In the case of hTINA, the amino acid sequence can be used. According to the column, the molar extinction coefficient of the L chain is 34690 and the molar extinction coefficient of the H chain is 950. The molar extinction coefficient (280 nm) of the drug linker was estimated as 0.00. The linker was reacted with mercaptoethanol or N-acetylcysteine ​​to form a maleimide Measured molar extinction coefficient (280 nm) of the compound in which the group was converted to a succinimide thioether was used. [F-3-3] The peak area ratio (%) of each chain to the total corrected peak area is calculated according to the following formula: Calculate.

[0119]

number

[0120] [F-3-4] Average number of drugs bound per antibody molecule in antibody-drug conjugates is calculated according to the following formula: Average number of drug bonds = (L0 peak area ratio × 0 + L0 peak area ratio × 1 + H0 peak area ratio ×0 + H1 peak area ratio ×1 + H2 peak area ratio ×2 + H3 peak area ratio ×3) / 100 ×2

[0121] The compound represented by formula (2) in production method 1 is represented by the following formula: (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) It is a compound represented by the formula: During the ceremony, n 3 denotes the integers 2 to 8, L 2is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P Phenylalanine, glycine, valine, lysine, citrulline, serine, glutamine It represents a peptide residue consisting of 2 to 7 amino acids selected from the group consisting of carboxylic acid, carboxylic acid, and aspartic acid. , n 1 represents an integer from 0 to 6, n 2 represents an integer from 0 to 5, L a represents -O- or a single bond, (maleimid-N-yl)- is a compound of the formula

[0122] [ka]

[0123] A maleimidyl group (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by the formula: a group in which an atom is the bonding site, -(NH-DX) is represented by the following formula:

[0124] [ka]

[0125] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in

[0126] L 2 is a single bond or -NH-(CH2CH2-O)n 4 n when -CH2CH2-C(=O)- 4 is an integer 2 to 4 are preferred as production intermediates. L PThe peptide residues are phenylalanine, glycine, valine, lysine, citrulline, Peptide residues consisting of amino acids selected from the group consisting of sucralose, serine, glutamic acid, and aspartic acid Among such peptide residues, compounds represented by the formula: L are preferred as production intermediates. P There are four Compounds that are peptide residues composed of amino acids are preferred as production intermediates. L P A compound in which is the tetrapeptide residue of -GGFG- is preferred as a production intermediate.

[0127] Also, -NH-(CH2)n 1 -L a -(CH2)n 2 - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2 A compound in which the compound is CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are preferred as production intermediates, and more preferably -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or It is a compound where the formula is -NH-CH2CH2-O-CH2.

[0128] Furthermore, the compound represented by formula (2) is 3 is an integer between 2 and 5, and L 2 is a single bond , -NH-(CH2)n 1 -L a -(CH2)n 2 - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH The compound -CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- is a manufacturing intermediate More preferably, -NH-(CH2)n 1 -L a -(CH2)n 2-, -NH-CH2CH2-, -NH-CH2C H2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. 3 but, Compounds in which the integers are 2 or 5 are preferred.

[0129] In addition, the compound represented by formula (2) is 3 is an integer between 2 and 5, and L 2 -NH-(CH2CH2-O) n 4 -CH2CH2-C(=O)-, n 4 is an integer from 2 to 4, and -NH-(CH2)n 1 -L a -(CH2)n 2 -but,- NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-OC A compound of H2- or -NH-CH2CH2-O-CH2- is preferred as a production intermediate. , n 4 is an integer of 2 or 4. Furthermore, -NH-(CH2)n 1 -L a -(CH2)n 2 -NH-CH2CH Compounds which are 2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are preferred.

[0130] Preferred intermediates useful for producing the compounds of the present invention include the following: Examples can be given. (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0131] A drug-linker compound selected from the group of intermediate compounds for the production of an anti-TROP2 antibody or its reactive derivative to the hinge region of the anti-TROP2 antibody. The anti-TROP2 antibody of the present invention is prepared by forming a thioether bond at the disulfide bond site. -drug conjugates can be produced, in which case the reactive derivatives of the anti-TROP2 antibody It is preferable to use a derivative, and in particular, a reactive derivative obtained by reducing an anti-TROP2 antibody. A conductor is preferred.

[0132] The following compounds are more preferred as intermediates in the production: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-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-CH2CH2-C( =O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0133] Also, among the above intermediate compound groups, the following formula: (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), The compound represented by the formula: is more preferred.

[0134] In order to ensure the amount of conjugate, the average drug concentration obtained under similar conditions was Mix multiple conjugates with similar numbers (e.g., ±1) to create a new lot. In this case, the average number of drugs falls within the average number of drugs before mixing.

[0135] 2. Manufacturing method 2 The compound represented by formula (2), which is an intermediate used in the above manufacturing method, and its pharmacologically acceptable The acceptable salts can be prepared, for example, by the following method.

[0136] [ka]

[0137] [In the formula, L 1 ' indicates a terminal maleimidyl group, and P 1 , P 2 and P 3 indicates a protecting group.]

[0138] Carboxylic acid (5) is converted to an active ester, a mixed acid anhydride, an acid halide, or the like, and then a salt is obtained. In the presence of a group, the compound is reacted with NH2-DX(4) or a pharmacologically acceptable salt thereof to obtain the compound NH2-DX (4) can be used to produce exatecan (chemical name: (1S,9S)-1-acetate). 9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pi 10,13(9H,15H)-dione). This reaction can be carried out using the same reaction reagents and conditions as those normally used in peptide synthesis. There are various types of phenols, for example, phenols such as p-nitrophenol, N-hydroxyphenol, The carboxylic acid (5) was reacted with N-hydroxybenzotriazole or N-hydroxysuccinimide. N'-Dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl) It can be produced by reacting with a condensing agent such as phenyl)carbodiimide hydrochloride. The ester is a reaction product of carboxylic acid (5) and pentafluorophenyl trifluoroacetate, etc. Reaction of carboxylic acid (5) with 1-benzotriazolyloxytripyrrolidinophosphonium Reaction of carboxylic acid (5) with diethyl cyanophosphonate. Reaction (salt-addition method); Carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyl Reaction of carboxylic acid (5) with 4-(4,6-dimethoxy-1, 3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) It can also be produced by reaction with a triazine derivative such as carboxylic acid. (5) is treated with an acid halide such as thionyl chloride or oxalyl chloride in the presence of a base. The reaction can also be carried out by an acid halide method, which can be carried out by The activated ester, mixed acid anhydride, or acid halide of the carboxylic acid (5) obtained as above is reacted with compound (4) in the presence of a suitable base in an inert solvent at a reaction temperature of -78°C to 150°C. By reacting the compound (6), it is possible to produce the compound (6). The solvent is a solvent that does not interfere with the intended reaction to be carried out in the reaction in which it is employed. It means a solvent.

[0139] Specific examples of the base used in each of the above steps include sodium carbonate and potassium carbonate. , sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, water carbonates of alkali metals or alkaline earth metals, such as sodium hydride and potassium hydride; alkoxides, hydroxides, or hydrides; alkyllithium such as n-butyllithium; Organometallic salts, such as dialkylaminolithium salts like lithium diisopropylamide groups; organometallic bases of bissilylamines such as lithium bis(trimethylsilyl)amide; These include pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylenediamine, Aminomethylamine, N-methylmorpholine, diisopropylethylamine, diazabicyclo[5. 4.0]undec-7-ene (DBU) or other tertiary amines or nitrogen-containing heterocyclic compounds Examples include bases.

[0140] The inert solvent used in this reaction includes dichloromethane, chloroform, carbon tetrachloride, etc. Halogenated hydrocarbon solvents; tetrahydrofuran, 1,2-dimethoxyethane, dioxane Ether solvents such as benzene, toluene, and other aromatic hydrocarbon solvents; N,N-dimethylformamide, N,N-dimethylformamide, and other aromatic hydrocarbon solvents; dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidin-2-one, etc. In addition to these, dimethyl sulfoxide may be used. Sulfoxide solvents such as acetone and methyl ethyl ketone; Solvent: It is also possible to use alcoholic solvents such as methanol and ethanol. Furthermore, these may be used in combination.

[0141] Protecting group P of the terminal amino group of compound (6) 1 Examples include tert-butyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, benzyloxycarbonyl group, etc. Any amino protecting group commonly used in peptide synthesis can be used. Examples of the protecting group include alkanoyl groups such as acetyl groups; methoxycarbonyl groups, ethoxycarbonyl groups, and the like. alkoxycarbonyl groups such as para-methoxybenzyloxycarbonyl group, para- arylmethoxycarbonyl groups such as (or ortho)nitrobenzyloxycarbonyl groups; Arylmethyl groups such as benzyl and triphenylmethyl groups; aroyl groups such as benzoyl groups 2,4-dinitrobenzenesulfonyl group, orthonitrobenzenesulfonyl group, etc. The protecting group P 1 The property of the compound protecting the amino group The selection may be made based on the quality, etc. The protecting group P of the terminal amino group of the obtained compound (6) 1 By deprotecting the compound ( 7) can be produced. This deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. That's fine. N-terminus to P 2 The protected peptide carboxylic acid (8) is derivatized with an active ester, mixed acid anhydride, etc. and reacting the resulting compound (7) to produce compound (9). The reaction conditions for forming the peptide bond between peptide carboxylic acid (8) and compound (7) can be determined. The reagents, bases, and inert solvents are appropriately selected from those described in the synthesis of compound (6). The protecting group P 2 is appropriately selected from the protecting groups described for compound (6). The amino group protecting compound may be selected depending on the properties of the compound. As is commonly used in synthesis, the amino acids or peptides that make up the peptide carboxylic acid (8) are The compound (9) can also be produced by repeating the reaction and deprotection of the tide to elongate it. The protecting group P of the amino group of the obtained compound (9) 2 By deprotecting the compound (10 This deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. good. Carboxylic acid (11) is converted into an activated ester, a mixed acid anhydride, an acid halide, or the like, The compound (10) thus obtained can be reacted to produce the compound (2) or Reaction conditions and reagents for forming a peptide bond between carboxylic acid (11) and compound (10) The base and inert solvent may be appropriately selected from those described in the synthesis of compound (6). That's fine.

[0142] Compound (9) can also be produced, for example, by the following method. N-terminus to P 2 The protected peptide carboxylic acid (8) is derivatized with an active ester, mixed acid anhydride, etc. In the presence of a base, the carboxyl group is converted to P 3 and reacting the compound (12) with a protected amine compound (13). Compound (13) can be produced by the reaction of peptide carboxylic acid (8) with compound ( The reaction conditions, reagents, bases, and inert solvents for forming the peptide bond of 12 are as follows: It is sufficient to select and use an appropriate one from those described in the synthesis of 6). The protecting group P of the amino group of compound (13) 2 may be protected with a commonly used protecting group . Specifically, the protecting group for the hydroxyl group includes an alkoxymethyl group such as a methoxymethyl group; arylmethyl groups such as phenylmethyl, 4-methoxybenzyl, and triphenylmethyl; alkanoyl groups such as benzoyl; aroyl groups such as benzoyl; tert-butyldiphenylsilyl Examples of the carboxyl group include a methyl group, an ethyl group, a silyl group, and the like. an alkyl group such as a tert-butyl group, an allyl group, or an arylmethyl group such as a benzyl group; The amino group can be protected as an ester of the formula alkyloxycarbonyl groups such as ethoxycarbonyl, methoxycarbonyl, and ethoxycarbonyl groups ; allyloxycarbonyl group, or 9-fluorenylmethyloxycarbonyl group, benzo para-(ortho)oxycarbonyl group, para-methoxybenzyloxycarbonyl group, para-(ortho)oxycarbonyl group, Arylmethoxycarbonyl groups such as nitrobenzyloxycarbonyl groups; as well as acetyl alkanoyl groups such as benzyl groups and triphenylmethyl groups; arylmethyl groups such as benzyl groups and triphenylmethyl groups; aroyl groups such as benzoyl group; 2,4-dinitrobenzenesulfonyl group, orthonitrobenzene arylsulfonyl groups such as benzenesulfonyl groups; and the like. Carboxy protecting group P 3 As an example, in organic synthetic chemistry, especially in peptide synthesis, A protecting group that is commonly used as a protecting group for an oxy group may be used, specifically, a methyl group. , ethyl group, tert-butyl and other alkyl esters, allyl esters, benzyl esters In this case, the protecting group for the amino group is It is preferred that the protecting groups for the carboxyl group and the carboxyl group can be removed by different methods or conditions. For example, P 2 is a tert-butyloxycarbonyl group, and P 3 is a benzyl group, etc. Representative examples of these protecting groups include the amino and carboxy groups. The protecting groups may be selected from those mentioned above depending on the properties of the compound to be treated. In this case, the reagents and conditions may be selected according to the protecting group. The protecting group P of the carboxy group of the obtained compound (13)3 by deprotecting the compound (14) can be produced. This deprotection can be carried out by selecting the reagents and conditions according to the protecting group. Just do that. The obtained compound (14) is converted into an active ester, a mixed acid anhydride, an acid halide, or the like. and reacting it with compound (4) in the presence of a base to produce compound (9). This reaction can be carried out by applying the same reaction reagents and conditions as those normally used in peptide synthesis. The conditions, reagents, bases, and inert solvents are appropriately selected from those described in the synthesis of compound (6). Just use it.

[0143] Compound (2) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (13) 2 Compound (15) was prepared by deprotecting For this deprotection, reagents and conditions may be selected according to the protecting group. The carboxylic acid derivative (11) is derivatized with an activated ester, a mixed acid anhydride, or an acid halide. The compound (16) is obtained by reacting the compound (15) with the compound (16) in the presence of a base. It can be produced by forming an amide bond between peptide carboxylic acid (11) and compound (15). The reaction conditions, reagents, bases, and inert solvents used in the synthesis of compound (6) are the same as those described above. It is sufficient to select and use it appropriately. The protecting group of the carboxy group of the obtained compound (16) is deprotected to give compound ( This deprotection is the same as that of the carboxyl group in the preparation of compound (14). This can be done in the same manner as in the deprotection of the silyl group. Compound (17) is converted to an active ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with a base. Compound (2) can be produced by reacting compound (4) with a methyl group in the presence of methyl group. This reaction can be carried out by applying the reaction reagents and conditions normally used in peptide synthesis. The base and inert solvent may be appropriately selected from those described in the synthesis of compound (6). That's fine.

[0144] 3. Manufacturing method 3 The intermediate compound of formula (2) can also be prepared by the following method.

[0145] [ka]

[0146] [In the formula, L 1 ' is L with the structure in which the terminal is converted to a maleimidyl group. 1 and P 4 indicates a protecting group. ]

[0147] Compound (11) is converted to an activated ester, a mixed acid anhydride, or the like, and the C-terminus is converted to P in the presence of a base. 4 in Compound (19) is prepared by reacting with a protected peptide carboxylic acid (18). The peptide bond between the peptide carboxylic acid (18) and the compound (11) can be formed. The reaction conditions, reagents, bases, and inert solvents used are appropriately selected from those described in the synthesis of compound (6). The protecting group P of the carboxy group of compound (18) may be selected appropriately. 4 is the protection mentioned above. It is only necessary to select appropriately from the group. The protecting group of the carboxy group of the obtained compound (19) is deprotected to give compound ( This deprotection is the same as that of the carboxyl group in the preparation of compound (14). This can be done in the same manner as in the deprotection of the silyl group. The obtained compound (20) is converted into an active ester or a mixed acid anhydride, etc., to give compound (7). Compound (2) can be produced by reacting the peptide The reaction reagents and conditions normally used in synthesis can be applied. Suitable solvents may be appropriately selected from those described in the synthesis of compound (6).

[0148] 4. Manufacturing method 4 Among the intermediates (10) described in Production Method 2, n 1 =1, L a =O compound (10 The production method of the compound represented by formula (10b), a salt thereof or The solvate can be produced, for example, by the following method.

[0149] [ka]

[0150] [In the formula, L P represents the same as above, and L is an acyl group, such as an alkanoyl group, e.g., an acetyl group. X and Y are each an aroyl group such as a benzoyl group or an aroyl group, or a hydrogen atom, An oligopeptide consisting of 1 to 3 amino acids is called P 5 and P 7 represents the protecting group of the amino group, P 6 teeth represents a protecting group for a carboxy group.]

[0151] The compound represented by formula (21) can be produced by the method described in JP-A-2002-60351 or The method described in the literature (J. Org. Chem., Vol. 51, p. 3196, 1986) or an application of that method is used. The compound can be produced by removing the protecting group or converting the functional group as necessary. In addition, amino acids with protected terminal amino groups or oligopeptides with protected amino groups It can be obtained by treating the imide with an aldehyde or ketone. Compound (21) is reacted in an inert solvent in the presence of an acid or a base under a temperature condition of from cooling to room temperature. to produce compound (23) by reacting compound (22) having a hydroxyl group with It is possible. Examples of acids that can be used here include hydrofluoric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid. Inorganic acids such as acetic acid, citric acid, paratoluenesulfonic acid, methanesulfonic acid, and other organic acids Lewis tetrafluoroborate, zinc chloride, tin chloride, aluminum chloride, iron chloride, etc. Among these, sulfonic acids, particularly p-toluenesulfonic acids, Furthermore, the base may be appropriately selected from the bases already mentioned. In particular, alkali metal alkoxides such as potassium tert-butoxide; hydroxides Alkali metal hydroxides such as sodium and potassium hydroxide; sodium hydride, potassium hydride Alkali metal hydrides such as lithium; dialkylamino such as lithium diisopropylamide Organometallic bases such as lithium bis(trimethylsilyl)amide The preferred solvents for the reaction include tetrahydrofuran and tetrahydrofuran. Ether solvents such as toluene, 1,4-dioxane, etc.; aromatic hydrocarbons such as benzene, toluene, etc. Hydrogen-based solvents; etc. are used. The above solvents may be mixed with water. 5 As exemplified in The amino-protecting group to be used may be any group that is normally used for protecting an amino group, without any particular restrictions. There is no limitation to the protective group. A representative example is the amino-protecting group described in Production Method 2. However, in this reaction, P 5 In some cases, the protecting group of the amino group, for example, In this case, if necessary, the protecting group can be introduced again by reacting with an appropriate amino group protecting reagent. Just do that. Compound (24) can be prepared by removing the protecting group P of compound (23). 6 It can be produced by removing Here, P 6 The protecting group for the carboxyl group exemplified by Compounds (23) are described in the literature and can be selected appropriately from these. In this case, the amino group protecting group P 5 and a protecting group P of the carboxy group 6 can be removed in different ways or under different conditions It is preferably a protecting group. For example, P 5 is a 9-fluorenylmethyloxycarbonyl group and P 6 is a benzyl group. The protecting groups are selected depending on the properties of the compounds that protect the amino and carboxy groups. In removing these protecting groups, the reagents and conditions can be selected according to the protecting group. good. Carboxylic acid (24) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, In the presence of a base, the compound (4) or a pharmacologically acceptable salt thereof is reacted to give the compound (25) is prepared, and the protecting group P of the obtained compound (25) is 5 By removing the compound ( The reaction of compound (4) with carboxylic acid (24) and the reaction of protecting group (26) can be carried out. P 6 In the reaction for removing the compound, the same reagents and reaction conditions as those described in Production Method 2 may be used. . Compound (26) and an amino acid or an oligonucleotide having a terminal amino group protected Compound (9b) was prepared by reacting peptide (27) with 9b) Protecting group P 7 Compound (10b) can be prepared by removing P 7 The protecting group for the amino group shown in There are no particular limitations on the type of protecting group, and typical examples include the amino protecting groups described in Production Method 2. The reaction can be carried out by selecting the appropriate reagent and conditions for the removal of the protecting group. In the reaction of 26) with compound (27), the reaction reagents and conditions commonly used in peptide synthesis were used. Compound (10b) prepared by the above method can be used in the same manner as above. This can lead to the invention compound (1).

[0152] The anti-TROP2 antibody-drug conjugate of the present invention may be left in the air or recrystallized. When purified by methods such as filtration, the substance may absorb moisture, adsorbed water may adhere, or it may become a hydrate. Such compounds and salts containing water are also included in the present invention. The present invention also includes compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugate of the present invention may be doped with a non-natural atomic isotope. The atomic isotopes may also contain a natural proportion. 2 H), tritium ( 3 H), Iodine-125( 125 I) or carbon-14 ( 14 C) and the like. The substance is, for example, tritium ( 3 H), iodine-125( 125 I) or carbon-14 ( 14 C) Radiation of Radiolabeled compounds can be used as therapeutic or prophylactic agents, research experiments, or other therapeutic agents. Useful as medicines, e.g., assay reagents, and diagnostic agents, e.g., in vivo imaging agents All isotopic variants of the antibody-drug conjugates of the present invention, whether radioactive or not, may be used. However, such modifications are within the scope of the present invention.

[0153] [Pharmaceuticals] The anti-TROP2 antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells. Therefore, it can be used as a medicine, particularly as a therapeutic and / or preventive agent for cancer. can. That is, the anti-TROP2 antibody-drug conjugate of the present invention is a major therapeutic method for cancer treatment. It can be used as a selective drug for certain chemotherapy, resulting in the proliferation of cancer cells. It can slow the growth of cancer cells, inhibit their proliferation, and even destroy cancer cells. , cancer patients can be relieved from cancer-related symptoms, improve their quality of life, and save their lives. Even if the destruction of cancer cells is not achieved, the proliferation of cancer cells is prevented. By suppressing and controlling proliferation, cancer patients can achieve a higher quality of life and a longer lifespan. Survival can be achieved. The anti-TROP2 antibody-drug conjugate of the present invention can be used as a drug alone or in combination with ajuva It can also be used as a drug in combination with other therapies in clinical therapy, such as surgery and radiation therapy. It can be combined with chemotherapy, hormonal therapy, etc. Furthermore, it can be used in neoadjuvant therapy. It can also be used as a drug for drug therapy. In addition to the above therapeutic uses, the antibody's ability to bind to antigens can also be used to bind to minute metastatic cancer cells. It is expected that this will have the effect of suppressing the growth of cancer cells and even destroying them. In particular, when TROP2 expression is confirmed in primary cancer cells, the anti-TROP2 Administration of antibody-drug conjugates is expected to suppress or prevent cancer metastasis. For example, they can suppress or destroy cancer cells in body fluids during the metastasis process, or It is expected to have the effect of suppressing and destroying minute cancer cells immediately after implantation in the tissues. In particular, it is expected to have an inhibitory and preventive effect on cancer metastasis after surgical removal of cancer. It is expected to have the effect of suppressing cancer metastasis. The anti-TROP2 antibody-drug conjugate of the present invention can be used as a systemic therapy for patients. In addition to being administered systemically, it can also be administered locally to cancer tissues, with the expectation of therapeutic effects.

[0154] The types of cancer to which the anti-TROP2 antibody-drug conjugate of the present invention can be applied include lung cancer, , renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma Cancers that can be cured include tumors, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck cancer, and esophageal cancer. The protein that the antibody in the antibody-drug conjugate can recognize in the cancer cells to be treated is identified. There is no limitation to these, as long as the cancer cells express the gene.

[0155] The anti-TROP2 antibody-drug conjugate of the present invention is preferably administered to a mammal. The subject can be a mammal, but more preferably is a human.

[0156] The present invention is used in pharmaceutical compositions containing anti-TROP2 antibody-drug conjugates. The substances to be used are those that are comparable in dosage and concentration to those commonly used in this field. Additives and other additives can be appropriately selected and applied.

[0157] The anti-TROP2 antibody-drug conjugates of the present invention may contain one or more pharmaceutically compatible components. For example, the pharmaceutical composition may be administered as a pharmaceutical composition comprising: The pharmaceutical composition may contain one or more pharmaceutical carriers (e.g., sterile liquids), such as water. and oils (of petroleum, animal, vegetable, or synthetic origin). Oils include, for example, lactobacillus acidophilus. The oil may be caustic oil, soybean oil, mineral oil, sesame oil, etc. Water is used when the pharmaceutical composition is administered intravenously. Saline solution, as well as aqueous dextrose, are more typical carriers when used in Aqueous aqueous and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The compositions may also contain, if desired, If desired, the pharmaceutical carrier may contain minor amounts of wetting or emulsifying agents, or pH buffering agents. An example is "Remington's Pharmacy" by E.W. Martin. The formulation corresponds to the mode of administration.

[0158] Various delivery systems are known, and the anti-TROP2 antibody-drug conjugates of the present invention can be delivered via these systems. The methods of introduction include intradermal, intramuscular, intraperitoneal, intravenous, and The administration route can include, but is not limited to, subcutaneous administration. This may be by infusion or bolus injection. Administration of the Ligand Drug Conjugate is by injection. Parenteral administration is the preferred route of administration. It is a road.

[0159] In an exemplary embodiment, the pharmaceutical composition is a pharmaceutical composition adapted for intravenous administration to humans. The composition is formulated according to routine procedures, typically a composition for intravenous administration. is a solution in sterile isotonic aqueous buffer. If necessary, the drug may also be dissolved in water. Contains a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the ingredients are packaged in a container such as an ampoule or sachet indicating the quantity of active agent. as a dry lyophilized powder or anhydrous concentrate in a sealed container, either separately or alone. or mixed together in a dosage form. When in the form of a syringe, it may be dispensed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline. When the above medicine is administered by injection, it can be administered in a single dose using sterile water for injection or saline. An ampoule of water can, for example, be provided so that the ingredients can be mixed prior to administration.

[0160] The pharmaceutical composition of the present invention is a pharmaceutical composition containing only the anti-TROP2 antibody-drug conjugate of the present application. The composition may comprise an anti-TROP2 antibody-drug conjugate and at least one The pharmaceutical composition may also contain other cancer therapeutic agents. The conjugates can also be administered with other cancer therapeutic agents, thereby enhancing the anti-cancer effect. Other anti-cancer drugs used for this purpose are antibody-drug conjugates. The drugs may be administered to an individual simultaneously, separately, or consecutively, or the administration interval between each drug may be set. Such cancer treatment agents include abraxane, paclitaxel, and el, cisplatin, gemcitabine, irinotecan (CPT- 11), paclitaxel, pemetrexed, sorafenib, vinb lastin or a drug described in WO 2003 / 038043, and further an LH-RH antagonist. Narog (leuprorelin, goserelin, etc.), estramustine phosphate, Thrombolytic antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anas Trozole, letrozole, exemestane, etc., but antitumor activity There is no limitation as long as the drug has the above structure.

[0161] Such pharmaceutical compositions are prepared as formulations having the selected composition and required purity, such as freeze-dried formulations. Alternatively, it may be formulated as a liquid preparation. When formulating as a freeze-dried preparation, The formulation may contain suitable formulation additives used in the liquid formulation. Similarly, the present invention is also formulated as a liquid formulation containing various formulation additives used in this field. It can be made into

[0162] The composition and concentration of the pharmaceutical composition vary depending on the administration method. The anti-TROP2 antibody-drug conjugates contained in the antibody-drug conjugates are targeted to the antigen of the antibody-drug conjugate. The affinity for the antigen, i.e., the dissociation constant (Kd value) for the antigen, is high ( The lower the Kd value, the more effective the drug can be even at a smaller dose. In determining the dosage of the antibody-drug conjugate, the antibody-drug conjugate and The dosage can also be determined based on the affinity with the antigen. When the conjugate is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days. [Example]

[0163] The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto. Furthermore, these should not be interpreted in any restrictive manner. In addition, reagents, solvents, and starting materials not specifically mentioned herein were obtained from commercially available sources. are readily available from

[0164] Example 1: Immunization of mice and acquisition of hybridomas 1-1) Preparation of cells for mouse immunization 5×10 6 NCI-H322 cells (human non-small cell lung cancer cell line, ATCC CRL- 5806;ATCC:American Type Culture Collection on) to RPMI-1640(Roswell Park Memorial Inst. After culturing for 5 days in 10 ml of PBS (phosphate buffered saline), the cells were harvested and stored in PBS (phosphate buffered saline). The cells were washed twice with physiological saline and resuspended in PBS (500 μl).

[0165] 1-2) Immunization of mice BALB / c mice (6 weeks old) were immunized with NCI-H322 cells (1 × 1 0 7 The second to fifth immunizations were given intraperitoneally at weekly intervals with 1 × 10 6 NCI-H322 The cells were intraperitoneally immunized. For the sixth and final immunization, 1 × 10 NCI-H322 cells were administered intraperitoneally. 6 Thin The mice were immunized with 200 μl of PBS in the tail vein and intraperitoneally. The specimen was extracted three days after the outbreak.

[0166] 1-3) Preparation of spleen cells from immunized mice The spleens of the immunized mice were removed, mashed, and placed in RPMI 1640 with 10% FBS ( The cells were suspended in fetal bovine serum (+) medium. The cell suspension was strained through a cell strainer (100 μm, BD After passing the mixture through a microcentrifuge (Falcon), centrifuge at 1500 rpm for 5 minutes at room temperature and discard the supernatant. Tris-NH4Cl solution (20 mM Tris-HCl pH 7.5, 0.83 10 mL of RPMI 16 (% NH4Cl) was added and incubated at room temperature for 5 minutes. Add 40FBS(+) medium (10 ml), pass through a cell strainer, and then strain at 1500 rpm. The supernatant was discarded, and the spleen cells were resuspended in RPMI 1640 FBS. (-) The cells were resuspended in medium (10 ml).

[0167] 1-4) Preparation of myeloma cells P3U1 cells (mouse myeloma cell line) were harvested and heated at 1500 rpm for 5 minutes at room temperature. EDTA (0.02%) solution (10 ml) was added to the P3U1 cells and incubated at 37°C. The P3U1 cell suspension was centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cells were resuspended in RPMI 1640 FBS(-) medium (10 ml).

[0168] 1-5) Cell fusion Spleen cells and myeloma cells were mixed at a ratio of 5:1 and centrifuged (1200 rpm, After thoroughly loosening the cells in the precipitated fraction, the cells were transferred to polyethylene PEG-4000 (1 mL) was added slowly over about 1 minute. After that, RPMI medium (1 mL) was added to the cell solution several times every minute, and then RPMI medium was added. The total volume was adjusted to 50 mL. The cell suspension was centrifuged (900 rpm, 5 minutes) to obtain After gently loosening the cells in the precipitated fraction, the cells were cultured in HAT medium (10% fetal bovine serum and HAT Dissolve in 100 mL of MI1640 medium containing Media Supplement. The suspension was dispensed into a 96-well culture plate at 200 μL per well. The cells were cultured in an incubator at 37°C with 5% CO2 until they reached 50% confluence. Ta.

[0169] 1-6) Hybridoma screening with mutant adenovirus FZ33 NCI-H322 cells were cultured in a 96-well plate at 5 x 10 3 Cells / well seeded at 3 The cells were cultured at 7°C for 48 hours. The cells were washed twice with 150 μl / well of PBS and Each hybridoma culture supernatant (50 μl) was added to each well and reacted at 4°C for 1 hour. Wash twice with 1 μl / well of PBS and inoculate the adenovirus Ax3CAZ3-FZ33 (antibody-injected) β-galactosidase-expressing adenovirus with Z33 fiber engineered to bind (See U.S. Patent Application Publication No. 2012 / 0237518)) to 3 × 10 6 vp / 100 μl (1 × 10 3 in RPMI1640(-) medium to a concentration of 10 ... The diluted solution was added at 100 μl / well. After incubation at 4°C for 1 hour, 150 μl Wash the wells twice with PBS. Add 100 μl / well of RPMI1640 FBS(+) medium. The plate was then added to the wells and incubated at 37°C for 24 hours. eporter Gene Assay System (Applied Biosys NCI-H32 treated with β-Gal reporter gene assay using tems 2. Wash the cells with 200 μl / well of PBS and add 50 μl of Lysis Solution. The cell lysate (10 μL) was added to the wells of Galac ton-Plus Galacto Reaction Buffer Diluent Dilute 100-fold with 100% PBS and place in a white microwell SH 96 well plate. The mixture was added to TE (Nunc) and reacted at room temperature for 1 hour. Add 150 μl / well and use a Wallac 1420ARVO multilabel counter. Chemiluminescence was measured for 5 seconds using a chemiluminescence analyzer (Perkin Elmer) and the average value per second was measured. The value was expressed as RLU (light unit) and represented the amount of virus infected to NCI-H322 cells. In the screening of hybridoma groups conducted in the same manner, the measured values ​​(RLU) were Among the 50 First, as a primary screening, clones with a RLU of 0.000 or more were selected. Of the 960 hybridoma wells obtained, 81 positive wells were selected. Furthermore, as a confirmation screening, the data was analyzed using the same method as the primary screening. Duplicate assays were performed and both readings were 5000 RL. Wells with U or higher were considered positive, and 52 wells were selected from the 81 wells obtained in the first screening. Positive wells were selected. The selected clones were subcloned 2 to 4 times to obtain monoclonal clones. Forty-four global hybridoma cell lines were established.

[0170] Example 2: Purification of antibodies from hybridomas Pristane (2,6,10,14-tetramethylpentadecane; 0 8-10 week old mice or nude mice that had been bred for 2 weeks in advance were given intraperitoneal administration of 0.5 ml The monoclonal antibody-producing hybridoma obtained in Example 1 was then injected intraperitoneally. After 10 to 21 days, the hybridomas were allowed to develop into ascites tumors, and the ascites was then collected. After centrifuging the water to remove solids, salting out with 40-50% ammonium sulfate to obtain caprylic acid. Purification was performed using precipitation, DEAE-Sepharose column, and protein G column. The G or IgM fraction was collected and used as purified monoclonal antibody.

[0171] Example 3: Identification of antigens bound by antibodies produced by hybridomas TINA1, one of the antibodies produced by the hybridoma prepared in Example 2 The antigen was identified.

[0172] 3-1) Immunoprecipitation of biotin-labeled cell surface proteins using TINA1 antibody 5×10 6 NCI-H322 cells were harvested and washed three times with PBS. k Sulfo-NHS-Biotin (PIERCE) at a concentration of 0.1 mg / ml NCI-H322 cells were suspended in PBS. They were incubated in the biotin / PBS solution at room temperature for 30 minutes. After rotating for 1 minute, wash twice with 100 mM glycine / PBS solution (25 ml) and Afterwards, the cells were washed three times with PBS (25 ml). After washing, the cells were lysed in lysis buffer (150 mM NaCl, 50mM Tris-HCl pH7.6, 1%NP-40 + Prote ase inhibitor, Complete EDTA free (Roche) Protein G was resuspended in 100 ml of PBS (1 pellet / 50 ml; 2 ml) and treated at 4°C for 30 minutes. Sepharose(Protein G Sepharose 4 Fast Fl The buffer used in the lysing assay (GE Healthcare) was replaced with the lysis buffer. Protein G Sepharose / lysis buffer (50% slurry; 30 μl) was added to the cell lysate, rotated at 4°C for 1 hour, and then centrifuged at 4°C for 5 minutes. TINA1 antibody (3 μg) was added to the supernatant and rotated at 4°C for 1 hour. Afterwards, Protein G Sepharose / lysis buffer (50% slurry; 60 The mixture was then rotated at 4°C for 2 hours. Protein was dissolved in lysis buffer (1 ml). n G Sepharose was washed six times and then diluted with 1x SDS sample buffer / 5% 2-ME (2-mercaptoethanol) buffer (62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% (w / v) SDS, 10% glycerol and 0.0 The suspension was resuspended in 1% (w / v) phenol red. The suspension was heated at 100°C for 5 minutes. After that, the solution was collected and used for SDS-PAGE (polyacrylamide gel electrophoresis). It was simple.

[0173] 3-2) SDS-PAGE and Western blotting The SDS-PAGE sample prepared in 3-1) was loaded onto Ready Gels J 5-20 After electrophoresis at 20 mA using a % (BioRad), the gel was transferred to the membrane at 0.1 mA. / cm 2 The membrane was blotted with PBS-T (PBS(-)-0.05% T After washing with PBS (ween 20) for 5 minutes, the membrane was blocked for 1 hour. -T after three 5-minute washes with Streptavidin-horseradish p eroxidase conjugate (Amersham; 2000 in PBS-T) The membrane was then washed four times with PBS-T for 10 minutes. Afterwards, ECL western blotting detection was performed. nts (Amersham) and Hyperfilm ECL (Amersham) The target band was detected using the following method. I-H322 cells were analyzed using mass spectrometry, which has already confirmed that the antigen is TROP2. The immunoprecipitates obtained by immunoprecipitation with KCI7A3 antibody and TINA1 antibody were analyzed by D Analyzed by SDS-PAGE and Western blotting without TT or with DTT In both cases using the KCI7A3 antibody and the TINA1 antibody, In the sample with DTT added, a band with a molecular weight of 46 kDa was observed, while in the sample with DTT added, a band with a molecular weight of 37 kDa was observed. bands were detected.

[0174] 3-3) FACS analysis Based on the band pattern, it was predicted that the antigen for the TINA1 antibody was TROP2. Forced expression analysis was performed by gene transfer of cDNA without performing mass spectrometry. As a result, the TINA1 antibody showed a strong positive reaction in human TROP2-expressing CHOK1 cells. The antigen of the TINA1 antibody was shown to be TROP2. , lung cancer cell line NCI-H322, lung cancer cell line NCI-H2122, lung cancer cell line LCAM1 , lung cancer cell line LC2 / ad, pancreatic cancer cell line MIAPaCa2, pancreatic cancer cell line PK-1, prostate Cancer cell line PC3, colon cancer cell line HCT116, melanoma cell line A375, ovarian cancer cell line SKOV3, hematopoietic tumor cell line RPMI8226, hematopoietic tumor cell line K562, PBMC Similar FACS analysis was performed using human peripheral blood mononuclear cells (human peripheral blood mononuclear cells) and human platelets. All lung cancer cell lines tested positive for TROP2, while non-lung cancer cell lines tested positive for PC3, PK1, and SKOV3 was positive, while all normal blood cells were negative.

[0175] Example 4: Measurement of antibody internalization activity 4-1) Antibody internalization activity evaluation system A recombinant conjugate protein for measuring antibody internalization and immunotoxin activity. DT3C was constructed by combining the catalytic domain and protein of diphtheria toxin (DT). DT3C is a protein that combines three antibody-binding domains of G. DT3C specifically binds to the Fc portion of an antibody. Once taken up into cells, it induces cell death by inhibiting protein synthesis. By using this method, it is possible to simultaneously observe the internalization of antibodies and the cell killing effect of immunotoxins. (Yamaguchi, M., Hamada, H., et al., Biochemical and Biophysical Research C ommunications 454 (2014) 600-603).

[0176] 4-2) Evaluation of DT3C-mediated internalization and immunotoxic activity 4 μg / mL DT3C (25 μL) was added to a 96wll plate, and the Example Culture supernatant (25 μL) of 11 hybridomas obtained by the method of 1 or a method equivalent thereto was added. The mixture was added and incubated at room temperature for 30 minutes. The antigens recognized by the antibodies produced by the hybridomas other than the above are CD9, CD46, CD55, CD59, CD71, CD73, CD147, CD276, EpCAM, or EGFR I confirmed in advance that there is 2×10 4 cells / mL(20% Low IgG RP with FBS Seed NCI-H322 cells (50 μL) in MI1640 medium and incubate at room temperature for 30 minutes. After incubation, the cells were cultured in a CO2 incubator at 37°C for 3 days. Remove the medium, add 10%WST-10%FBS-RPMI1640 (100 μL), and heat at 37°C. Incubate in a CO2 incubator for 1 hour and count viable cells using a microplate reader. Leader (OD 450 ~OD 640 , infinite200, Tecan Japan Co., Ltd.) The culture supernatants of the hybridoma cells evaluated contained CD59, CD71, EGFR, Antibodies against EpCAM and TROP2 showed strong internalization and immunotoxic activity. This was confirmed (Figure 10).

[0177] 4-3) Antibodies against CD59, CD71, EGFR, EpCAM, and TROP2 Differences in internalization and immunotoxic activity DT3C (0, 0.004, 0.04, 0.4, 4, 40 μL) was added to a 96-well plate. Add 25 μL of the diluted solution (40 μg / mL) and add 25 μL of each antibody (40 μg / mL). The mixture was incubated at room temperature for 30 minutes. 4 pieces / mL (20% Low Ig NCI-H322 cells (50 μL) were seeded in RPMI 1640 medium supplemented with GFBS and incubated at room temperature. After incubation for 30 minutes, the cells were cultured in a CO2 incubator at 37°C for 3 days. After incubation, the supernatant was removed and the cells were cultured in 10% WST-1-supplemented 10% FBS-RPMI1640 (100 μL ) was added and incubated in a CO2 incubator at 37°C for 1 hour, and the viable cell count was determined. Plate reader (OD 450 ~OD 640 Among the antibodies evaluated, the most effective antibody was for TROP2. The antibody against TINA1 had the strongest internalization and immunotoxic activity (Fig. 1 1).

[0178] 4-4) Correlation between internalization and immunotoxic activity of each anti-TROP2 antibody clone Difference TINA1 obtained in Example 1 or a similar manner (immunogen: lung cancer strain NCI-H322) , KCL7A3, and KCL2D6 (immunogen: pancreatic cancer line KCL-MOH1), Pr1E1 1 and Pr8H10 (immunogen: prostate cancer line Pc-1), NY16 and NY17 (immunogen: Pancreatic cancer cell line PK-1) and commercially available anti-TROP2 antibody 77220 (R&D System) The internalization of the antibody and its immunotoxin activity were evaluated in the same manner as in Example 4-3). As a result, the TINA1 antibody had the strongest activity among the eight anti-TROP2 antibodies (Figure 12).

[0179] Example 5: Nucleotide sequence of cDNA encoding the variable region of the TINA1 antibody gene Determination of the chimeric TINA1 (cTINA1) antibody 5-1) Determination of the nucleotide sequence of the cDNA encoding the variable region of the TINA1 antibody gene fixed 5-1-1) Preparation of mRNA from TINA1 antibody-producing hybridoma To amplify cDNA containing the variable region of the TINA1 antibody, a TINA1 antibody-producing hybridoma was used. mRNA isolation kit (Roche applied s mRNA was prepared using a PCR kit (Chemistry).

[0180] 5-1-2) Synthesis of cDNA (5'-RACE-Ready cDNA) The synthesis of cDNA (5'-RACE-Ready cDNA) was performed as described in Example 5-1-1). The purified mRNA (100 ng) and SMARTer RACE cDNA Amplifi This was carried out using a cation kit (CLONTECH).

[0181] 5-1-3) cDNA containing the heavy chain variable region of TINA1 antibody by 5'-RACE PCR Amplification and sequencing of A. UPM was used as a primer to amplify the cDNA of the variable region of the heavy chain gene by PCR. (Universal Primer A Mix:SMARTer RACE cDN A Amplification Kit), and 5'-AGAGTTCCAGG TCAAGGTCACTGGCTCAGG-3' (SEQ ID NO: 33: primer mG2a The oligonucleotides used were those with the sequence of SMARTer RAC. E cDNA Amplification Kit (CLONTECH) mG2aVR2 is the sequence of the constant region of the mouse heavy chain (IgG2a) in the database. Designed from the column. This primer combination and the cDNA (5'-RA TINA1 antibody was isolated by 5'-RACE PCR using CE-Ready cDNA as a template. The cDNA containing the variable region of the heavy chain of the ribosomal protein was amplified using PCR. Using KOD-plus (TOYOBO), SMARTer RACE cDNA A Follow the instructions in the manual for the Touch Amplification Kit (CLONTECH) A down PCR program was performed. The cDNA containing the heavy chain variable region amplified by 5'-RACE PCR was then purified using MinElute After purification using PCR Purification Kit (QIAGEN), ro Blunt TOPO PCR Cloning Kit (Invitrogen The cDNA containing the heavy chain variable region was cloned using a cloned cDNA library. The sequence of the octide was analyzed. The primers mG2aVR2 and N were designed based on the sequence of the constant region of the mouse heavy chain of the genus UP(Nested Universal Primer A:SMARTer RAC E cDNA Amplification Kit) was used. Sequence analysis was performed using a gene sequence analyzer (ABI PRISM 3700 DNA Analyzer” or “Applied Biosystems 3730xl An The sequence was analyzed using the "Sequence Analyzer" (Applied Biosystems). The PCR reaction was performed using a Gene Amp 9700 (Applied Biosystems). was used. Nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the TINA1 antibody determined is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 2.

[0182] 5-1-4) cDNA containing the light chain variable region of TINA1 antibody by 5'-RACE PCR Amplification and sequencing of A. Primers for PCR amplification of the variable region cDNA of the light chain gene of the TINA1 antibody As a primer, UPM (Universal Primer A Mix: SMARTer (included in the RACE cDNA Amplification Kit) and 5'-AGTC CAACTGTTCAGGACGCCATTTTGTCG-3' (SEQ ID NO: 34: primer The oligonucleotides used were those with the sequence of SMARTe (mKVR2). r RACE cDNA Amplification Kit (CLONTECH) The mKVR2 was designed from the mouse light chain constant region sequence in the database. I calculated it. This primer combination and the cDNA (5'-RA TINA1 antibody was isolated by 5'-RACE PCR using CE-Ready cDNA as a template. The cDNA containing the variable region of the light chain of the human IgG was amplified using PCR. Using KOD-plus- (TOYOBO), SMARTer RACE cDNA Following the instructions of the Amplification Kit (CLONTECH), A step-down PCR program was performed. The cDNA containing the light chain variable region amplified by 5'-RACE PCR was then purified using MinElute After purification using PCR Purification Kit (QIAGEN), ro Blunt TOPO PCR Cloning Kit (Invitrogen The cDNA containing the cloned light chain variable region was cloned using a cloned cDNA library containing the variable region of the light chain. Sequence analysis of the octide sequence was performed. The sequence primers were designed from the mouse light chain constant region sequences in the database. The primers mKVR2 and NUP described above were used. The above-mentioned apparatus was used for the sequence analysis and the sequence reaction. Nucleotide sequence of the cDNA encoding the variable region of the light chain of the TINA1 antibody determined is shown in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 4.

[0183] 5-2) Preparation of cTINA1 antibody 5-2-1) Construction of chimeric and humanized antibody light chain expression vector pCMA-LK Restriction of the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) A fragment of approximately 5.4 kb obtained by digestion with the enzymes XbaI and PmeI and the sequence of SEQ ID NO: DNA containing the DNA sequence encoding the human κ chain secretory signal and the human κ chain constant region shown in 5 The A fragment was cloned using the In-Fusion Advantage PCR cloning kit (CL The DNA was ligated using a DNA fragment encoding the nucleotides (ONTECH) to prepare pcDNA3.3 / LK. PCR was performed using pcDNA3.3 / LK as a template and the following primer set. The approximately 3.8 kb fragment was phosphorylated and then self-ligated to CMV. Contains a signal sequence, a cloning site, and a human kappa chain constant region downstream of the promoter. A chimeric and humanized antibody light chain expression vector, pCMA-LK, was constructed. Primer set 5'-tataccgtcgacctctagctagagcttggc-3' (SEQ ID NO: No. 35: Primer 3.3-F1) 5'-gctatggcagggcctgccgccccgacgttg-3' (SEQ ID NO: No. 36: Primer 3.3-R1)

[0184] 5-2-2) Construction of chimeric and humanized antibody IgG1 type heavy chain expression vector pCMA-G1 Construction pCMA-LK was digested with XbaI and PmeI to clone the κ chain secretion signal and the human κ chain constant signal. The DNA fragment from which the region was removed, the human heavy chain signal sequence shown in SEQ ID NO: 6, and human IgG 1. A DNA fragment containing a DNA sequence encoding the amino acid sequence of the constant region was Ligation was performed using the Advantage PCR Cloning Kit (CLONTECH). The CMV promoter contains a signal sequence, a cloning site, and a human IgG1 heavy chain. pCMA-G1, an expression vector for chimeric and humanized IgG1-type heavy chains containing chain constant regions was constructed.

[0185] 5-2-3) Construction of cTINA1 antibody heavy chain expression vector The cDNA containing the heavy chain variable region of the TINA1 antibody obtained in Example 5-1-3) was used as a template. The plate used was KOD-Plus (TOYOBO) and the following primer set: A DNA fragment containing the cDNA encoding the variable region of the chain was amplified to generate chimeric and humanized IgG. The type 1 heavy chain expression vector pCMA-G1 was cleaved with the restriction enzyme BlpI and the In-F Insert using Fusion HD PCR Cloning Kit (CLONTECH) The cTINA1 antibody heavy chain expression vector was constructed by the above procedure. The plasmid was named "pCMA-G1 / cTINA1." Nucleotide sequence of the heavy chain of the cTINA1 antibody The nucleotide sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 8. The amino acid sequence of SEQ ID NO:8 is also set forth in FIG. Primer set for cTINA1 antibody heavy chain 5'-CCAGATGGGTGCTGAGCCAGATCCAGTTGGTGCAGTC TGGACCTGAG-3' (SEQ ID NO: 37: primer TINA1H-F) 5'-CTTGGTGGAGGCTGAGCTGACGGTGACCGCGGTCCCT GCGCCCCAGAC-3' (SEQ ID NO: 38: primer TINA1H-R)

[0186] 5-2-4) Construction of cTINA1 antibody light chain expression vector The cDNA containing the variable region of the light chain of the TINA1 antibody obtained in Example 5-1-4) was used as a template. The plate was KOD-Plus (TOYOBO) and the following primer set: A DNA fragment containing the cDNA encoding the variable region of the chain was amplified and used to generate chimeric and humanized antibodies. The general-purpose vector pCMA-LK was cleaved with the restriction enzyme BsiWI and then Insert using the ion HD PCR cloning kit (CLONTECH). The light chain expression vector for the cTINA1 antibody was constructed using the above procedure. The antibody was named "CMA-LK / cTINA1." Nucleotide sequence of the light chain of the cTINA1 antibody The sequence of SEQ ID NO: 9 is shown in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 10. The peptide sequence and the amino acid sequence of SEQ ID NO: 10 are also shown in FIG. Primer set for cTINA1 antibody light chain 5'-ATCTCCGGCGCGTACGGCGACATTGTGATGACCCAGT CTCACAAATTC-3' (SEQ ID NO: 39: primer TINA1L-F) 5'-GGAGGGGGCGGCCACAGCCCGTTTCAGCTCCAGCTTG GTCCCAGC-3' (SEQ ID NO: 40: primer TINA1L-R)

[0187] 5-2-5) Small-scale production of cTINA1 antibody FreeStyle 293F cells (Invitrogen) were cultured according to the manufacturer's instructions. Subculture and culture were carried out. 1 x 10 in logarithmic growth phase 7 FreeStyle 293F cells (Invitrogen) n company) and FreeStyle293 expression medium (Invi After diluting to 9.6 mL with 10 mL of 10 ... The cells were seeded in a bottle (Nalgene) and placed in an incubator at 37°C and 8% CO2. The mixture was incubated in a centrifuge at 90 rpm for 1 hour. yscience #24765;30μg) in Opti-Pro SFM (Invit (Progen; 200 μL) and then placed in a PureLink HiPure Plas Light chain expression vector (6 μg) prepared using the mid kit (Invitrogen) and heavy chain expression vector (4 μg) in Opti-Pro SFM (Invitrogen) Polyethyleneimine / Opti-Pro Add the expression vector / Opti-Pro SFM mixture (20 0 μL) and gently mix. Leave for another 5 minutes, then add FreeStyle 293 The cells were incubated at 37°C in an 8% CO2 incubator for 7 days, shaking at 90 rpm. The culture supernatant obtained by culturing was filtered through a Minisart-Plus filter (Sartor The mixture was filtered using a filter (IUS Co., Ltd.) to prepare a sample for evaluation. The combination of pCMA-G1 / cTINA1 and pCMA-LK / cTINA1 The obtained human chimeric TINA1 antibody was named "cTINA1 antibody."

[0188] Example 6: Design of a humanized mouse anti-TROP2 monoclonal antibody 6-1) Design of a humanized version of TINA1 6-1-1) Molecular modeling of the variable region of TINA1 Molecular modeling of the variable regions of TINA1 was performed using a method known as homology modeling (Me Methods in Enzymology, 203, 121-153 (1991) Implemented. Protein Data Bank (Nuc. Acid Res. 35 ,D301-D303(2007)) The sequence (3D structure derived from X-ray crystal structure is available) was determined by the previously determined TINA The results showed that 1ZEA binds to the heavy chain variable region of TINA1. Among antibodies with similar framework deletions, 3IU4 was also selected. 3IU4 had the highest sequence homology to the light chain variable region of TINA1. The three-dimensional structure of the framework region was selected to be similar to that of the heavy chain and The coordinates of 1ZEA and 3IU4 corresponding to the α- and β-light chains were combined to form the "framework model" Then, a representative conformation for each CDR was obtained. ation was incorporated into the framework model. Finally, to obtain a possible molecular model of the variable region of TINA1 in terms of energy The above procedure was then repeated for a commercially available protein. Three-dimensional structure analysis program Discovery Studio (Accelrys, Inc.) .) was used.

[0189] 6-1-2) Design of the amino acid sequence for humanized TINA1 The construction of humanized TINA1 antibody was performed using CDR grafting (Proc. Natl. Acad. Sci. USA 97:1111-1112). d. Sci. USA 86, 10029-10033 (1989)) Acceptor antibodies were selected based on amino acid homology within the framework regions. The TINA1 framework region sequences were selected based on the Kabat amino acid sequence of the antibody. All of the database (Nuc. Acid Res. 29, 205-206 (2001)) As a result, the HuPR1A3 antibody has a framework region was selected as the acceptor due to the 74% sequence homology with HuPR1. The amino acid residues in the framework region for A3 are The positions of these residues were aligned with the nucleotide sequence shown above to identify positions where different amino acids were used. The three-dimensional model of TINA1 was constructed using the TINA1 gene, and the acceptor was then analyzed. The donor residue to be rafted is selected from the group described in Queen et al., (Proc. Nat Acad.Sci.USA 86,10029-10033(1989) The donor residues were selected based on the given criteria. A humanized TINA1 sequence can be constructed as described in the Examples below by transfecting the human did.

[0190] 6-2) Humanization of TINA1 heavy chain 6-2-1) hTINA1-H1 type heavy chain: Amino acid number 21 (isoleucine) of the TINA1 heavy chain shown in SEQ ID NO: 8 in the sequence listing To valine, amino acid number 28 (proline) to alanine, amino acid number 30 (leucine) to valine, amino acid number 35 (glutamic acid) to alanine, amino acid number 36 (threonine) to threonine. onine) to serine, amino acid number 38 (arginine) to lysine, amino acid number 39 ( isoleucine) to valine, amino acid number 57 (glutamine) to arginine, amino acid Number 58 (lysine) to glutamine, amino acid number 59 (methionine) to alanine, Amino acid number 62 (lysine) to glutamine, amino acid number 65 (lysine) to glutamic acid Amino acid number 67 (isoleucine) to methionine, and amino acid number 87 (phenylalanine) to threonine, amino acid number 89 (alanine) to valine, amino acid number 88 (alanine) to threonine, (phenylalanine) to isoleucine, amino acid number 91 (leucine) to alanine, Amino acid number 92 (glutamic acid) to aspartic acid, amino acid number 95 (alanine) to threonine, amino acid number 102 (isoleucine) to leucine, amino acid number 10 4 (asparagine) to serine, amino acid number 107 (asparagine) to serine, Amino acid number 111 (threonine) to alanine, and amino acid number 112 (threonine) to vari In the α-amino acid sequence, amino acid number 114 (phenylalanine) is replaced with tyrosine, and amino acid number 132 (alanine) is replaced with tyrosine. amino acid number 135 (alanine) with leucine. The humanized TINA1 heavy chain designed with this method was named "hTINA1-H1 type heavy chain." Ta. The amino acid sequence of the hTINA1-H1 type heavy chain is set forth in SEQ ID NO: 12 of the Sequence Listing. The amino acid sequence of SEQ ID NO: 12 is a sequence consisting of amino acid residues 1 to 19, and the amino acid sequence of SEQ ID NO: 13 is a sequence consisting of amino acid residues 1 to 19, and the amino acid sequence of SEQ ID NO: 14 is a sequence consisting of amino acid residues 20 to 29, and the amino acid sequence of SEQ ID NO: 15 is a sequence consisting of amino acid residues 1 to 19, and the amino acid sequence of S a sequence consisting of amino acid residues 141 to 470; The sequences shown correspond to the signal sequence, heavy chain variable region, and heavy chain constant region, respectively. The nucleotide sequence encoding the amino acid sequence of 2 is set forth in SEQ ID NO: 11 of the Sequence Listing. A sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 11. The sequence consisting of nucleotides 58 to 420, nucleotides 421 to 1410 The sequences encoding the signal sequence, heavy chain variable region sequence, and heavy chain constant region sequence are The nucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12 are shown in FIG. It is also stated in.

[0191] 6-2-2) hTINA1-H2 type heavy chain: Amino acid number 21 (isoleucine) of the TINA1 heavy chain shown in SEQ ID NO: 8 in the sequence listing To valine, amino acid number 28 (proline) to alanine, amino acid number 30 (leucine) to valine, amino acid number 35 (glutamic acid) to alanine, amino acid number 36 (threonine) to threonine. onine) to serine, amino acid number 38 (arginine) to lysine, amino acid number 39 ( isoleucine) to valine, amino acid number 57 (glutamine) to arginine, amino acid Number 58 (lysine) to glutamine, amino acid number 59 (methionine) to alanine, Amino acid number 62 (lysine) to glutamine, amino acid number 65 (lysine) to glutamic acid Amino acid number 67 (isoleucine) to methionine, and amino acid number 87 (phenylalanine) to threonine, amino acid number 89 (alanine) to valine, amino acid number 88 (alanine) to threonine, (phenylalanine) to isoleucine, and amino acid number 92 (glutamic acid) to asparagus. amino acid number 95 (alanine) to threonine, amino acid number 102 (isothiazolinone) to guanylate leucine) to leucine, amino acid number 104 (asparagine) to serine, amino acid number 107 (asparagine) to serine, amino acid number 111 (threonine) to alanine, Amino acid number 112 (threonine) to valine, amino acid number 114 (phenylalanine) ) to tyrosine, amino acid number 132 (alanine) to glutamine, amino acid number 135 The humanized TINA1 heavy chain was designed by replacing (alanine) with leucine. The antibody was named "hTINA1-H2 type heavy chain." The amino acid sequence of the hTINA1-H2 type heavy chain is set forth in SEQ ID NO: 14 of the Sequence Listing. The amino acid sequence of SEQ ID NO: 14 is a sequence consisting of amino acid residues 1 to 19, and the amino acid sequence of SEQ ID NO: 15 is a sequence consisting of amino acid residues 20 to 29. a sequence consisting of amino acid residues 141 to 470; The sequences shown correspond to the signal sequence, heavy chain variable region, and heavy chain constant region, respectively. The nucleotide sequence encoding the amino acid sequence of 4 is set forth in SEQ ID NO: 13 of the Sequence Listing. A sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 13. The sequence consisting of nucleotides 58 to 420, nucleotides 421 to 1410 The sequences encoding the signal sequence, heavy chain variable region sequence, and heavy chain constant region sequence are The nucleotide sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 are shown in FIG. It is also stated in.

[0192] 6-2-3) hTINA1-H3 type heavy chain: Amino acid number 28 (proline) of the TINA1 heavy chain shown in SEQ ID NO: 8 in the sequence listing was amino acid number 30 (leucine) to valine, amino acid number 36 (threonine) to valine Serine, amino acid number 38 (arginine) to lysine, amino acid number 39 (isoleucine) Amino acid number 58 (lysine) to glutamine, amino acid number 65 (lysine) to valine, amino acid number 67 (isoleucine) to methionine, and amino acid number No. 87 (phenylalanine) to valine, and amino acid No. 88 (alanine) to threonine , amino acid number 92 (glutamic acid) to aspartic acid, amino acid number 95 (alanine ) to threonine, amino acid number 102 (isoleucine) to leucine, amino acid number 1 04 (asparagine) to serine, amino acid number 107 (asparagine) to serine, Amino acid number 111 (threonine) to alanine, and amino acid number 112 (threonine) to alanine. To phosphorus, amino acid number 114 (phenylalanine) to tyrosine, amino acid number 132 ( alanine) to glutamine, and amino acid number 135 (alanine) to leucine. The humanized TINA1 heavy chain designed with this in mind was named "hTINA1-H3 type heavy chain." did. The amino acid sequence of the hTINA1-H3 type heavy chain is set forth in SEQ ID NO: 16 of the Sequence Listing. The amino acid sequence of SEQ ID NO: 16 is a sequence consisting of amino acid residues 1 to 19, and the amino acid sequence of SEQ ID NO: 16 is a sequence consisting of amino acid residues 20 to 29. a sequence consisting of amino acid residues 141 to 470; The sequences shown correspond to the signal sequence, heavy chain variable region, and heavy chain constant region, respectively. The nucleotide sequence encoding the amino acid sequence of 6 is set forth in SEQ ID NO: 15 of the Sequence Listing. A sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 15. The sequence consisting of nucleotides 58 to 420, nucleotides 421 to 1410 The sequences encoding the signal sequence, heavy chain variable region sequence, and heavy chain constant region sequence are The nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16 are shown in FIG. It is also stated in.

[0193] 6-3) Humanization of TINA1 light chain 6-3-1) hTINA1-L1 type light chain: Amino acid number 23 (valine) of the TINA1 light chain shown in SEQ ID NO: 10 in the sequence listing is replaced with a group Amino acid number 28 (histidine) to proline, amino acid number 29 (lysine) to thiamine to serine, amino acid number 30 (phenylalanine) to serine, amino acid number 31 (methyl thionine) to leucine, amino acid number 33 (threonine) to alanine, amino acid number 40 (serine) to threonine, amino acid number 62 (glutamine) to lysine, amino acid Number 63 (serine) to alanine, amino acid number 80 (aspartic acid) to serine, Amino acid number 83 (threonine) to serine, amino acid number 90 (alanine) to asparagus amino acid, amino acid number 93 (phenylalanine) to leucine, amino acid number 98 (valerian) to amino acid number 100 (alanine) to proline, amino acid number 103 (leucine) to phenylalanine, amino acid number 120 (alanine) to glutamine, Amino acid number 126 (leucine) to isoleucine, and amino acid number 129 (alanine) to The humanized TINA1 light chain designed with a substitution of threonine is referred to as "hTINA1 The antibody was named "-L1 type light chain." The amino acid sequence of the hTINA1-L1 type light chain is set forth in SEQ ID NO: 18 of the Sequence Listing. The amino acid sequence of SEQ ID NO: 18 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ ID NO: 18 is a sequence consisting of amino acid residues 21 to 20. a sequence consisting of amino acid residues 130 to 234; The sequences shown correspond to the signal sequence, the light chain variable region, and the light chain constant region, respectively. The nucleotide sequence encoding the amino acid sequence of 8 is set forth in SEQ ID NO: 17 of the Sequence Listing. A sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 17. The sequence consisting of nucleotides 61 to 387, nucleotides 388 to 702 The sequences encoding the signal sequence, the light chain variable region sequence, and the light chain constant region sequence are The nucleotide sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 18 are shown in FIG. is also described.

[0194] 6-3-2) hTINA1-L2 type light chain: Amino acid number 28 (histidine) of the TINA1 light chain shown in SEQ ID NO: 10 in the sequence listing Proline, amino acid number 29 (lysine) to serine, amino acid number 30 (phenylalanine) to amino acid number 31 (methionine) to leucine, amino acid number 33 ( threonine) to alanine, amino acid number 40 (serine) to threonine, amino acid number 62 (glutamine) to lysine, amino acid number 63 (serine) to alanine, amino acid number Amino acid number 80 (aspartic acid) to serine, amino acid number 83 (threonine) to serine, Amino acid number 90 (alanine) to aspartic acid, amino acid number 93 (phenylalanine) ) to leucine, amino acid number 98 (valine) to leucine, amino acid number 100 (araline) to leucine amino acid number 103 (leucine) to phenylalanine, Number 120 (alanine) is replaced with glutamine, and amino acid number 126 (leucine) is replaced with isoleucine. The amino acid sequence was designed by replacing amino acid number 129 (alanine) with threonine. The humanized TINA1 light chain was named "hTINA1-L2 type light chain." The amino acid sequence of the hTINA1-L2 type light chain is set forth in SEQ ID NO: 20 of the Sequence Listing. The amino acid sequence of SEQ ID NO: 20 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ ID NO: 21 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ ID NO: 22 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ ID NO: 23 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ a sequence consisting of amino acid residues 130 to 234; The sequences shown correspond to the signal sequence, the light chain variable region, and the light chain constant region, respectively. The nucleotide sequence encoding the amino acid sequence of 0 is set forth in SEQ ID NO: 19 of the Sequence Listing. A sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 19, The sequence consisting of nucleotides 61 to 387, nucleotides 388 to 702 The sequences encoding the signal sequence, the light chain variable region sequence, and the light chain constant region sequence are The nucleotide sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 20 are shown in FIG. is also described.

[0195] 6-3-3) hTINA1-L3 type light chain: Amino acid number 28 (histidine) of the TINA1 light chain shown in SEQ ID NO: 10 in the sequence listing Proline, amino acid number 29 (lysine) to serine, amino acid number 30 (phenylalanine) to amino acid number 31 (methionine) to leucine, amino acid number 33 ( threonine) to alanine, amino acid number 40 (serine) to threonine, amino acid number 62 (glutamine) to lysine, amino acid number 63 (serine) to glutamine, amino acid Number 80 (aspartic acid) to serine, amino acid number 83 (threonine) to serine, Amino acid number 90 (alanine) to aspartic acid, and amino acid number 93 (phenylalanine) amino acid number 98 (valine) to leucine, amino acid number 100 (alanine) to leucine, amino acid number 103 (leucine) to phenylalanine, Amino acid number 120 (alanine) to glutamine, and amino acid number 126 (leucine) to isoleucine. The amino acid sequence was designed by replacing amino acid number 129 (alanine) with threonine. The resulting humanized TINA1 light chain was named "hTINA1-L3 type light chain." The amino acid sequence of the hTINA1-L3 type light chain is set forth in SEQ ID NO: 22 of the Sequence Listing. The amino acid sequence of SEQ ID NO: 22 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ ID NO: 21 is a sequence consisting of amino acid residues 1 to 20, and the amino acid sequence of SEQ ID NO: 22 is a sequence consisting of amino acid residues 2 ... a sequence consisting of amino acid residues 130 to 234; The sequences shown correspond to the signal sequence, the light chain variable region, and the light chain constant region, respectively. The nucleotide sequence encoding the amino acid sequence of 2 is set forth in SEQ ID NO: 21 in the sequence listing. A sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 21. The sequence consisting of nucleotides 61 to 387, nucleotides 388 to 702 The sequences encoding the signal sequence, the light chain variable region sequence, and the light chain constant region sequence are The nucleotide sequence of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22 are shown in FIG. is also described.

[0196] [Example 7: Construction of hTINA1 antibody expression vector and antibody production] 7-1) Construction of hTINA1 heavy chain expression vector 7-1-1) Construction of hTINA1-H1 expression vector Nucleotide number of the nucleotide sequence of hTINA1-H1 shown in SEQ ID NO: 11 in the Sequence Listing The DNA sequences encoding the variable regions of hTINA1-H1 shown in Nos. 36 to 437 are also included. The DNA fragment containing the nucleotide sequence was synthesized (GENEART artificial gene synthesis service). Using the A fragment as a template, KOD-Plus- (TOYOBO) and the following primers were used: The DNA fragments containing the DNA sequence encoding the variable region of hTINA1-H1 were amplified using a set of The chimeric and humanized antibody IgG1 type heavy chain expression vector pCMA-G1 was then purified using restriction enzymes The BlpI digestion site was filled with the In-Fusion HD PCR cloning kit (CL The hTINA1-H1 expression vector was constructed by inserting the The resulting expression vector was named "pCMA-G1 / hTINA1-H1." Primer set 5'-agctcccagatgggtgctgagc-3' (SEQ ID NO: 41: primer - EG-Inf-F) 5'-gggcccttggtggaggctgagc-3' (SEQ ID NO: 42: primer - EG1-Inf-R)

[0197] 7-1-2) Construction of hTINA1-H2 expression vector Nucleotide number of the nucleotide sequence of hTINA1-H2 shown in SEQ ID NO: 13 in the Sequence Listing The DNA sequences encoding the variable regions of hTINA1-H2 shown in Nos. 36 to 437 are also included. A DNA fragment containing the nucleotide sequence was synthesized (GENEART artificial gene synthesis service), and Example 7-1- The hTINA1-H2 expression vector was constructed in the same manner as in 1). The vector was named "pCMA-G1 / hTINA1-H2."

[0198] 7-1-3) Construction of hTINA1-H3 expression vector Nucleotide number of the nucleotide sequence of hTINA1-H3 shown in SEQ ID NO: 15 in the Sequence Listing The DNA sequences encoding the variable regions of hTINA1-H3 shown in Nos. 36 to 437 are also included. A DNA fragment containing the nucleotide sequence was synthesized (GENEART artificial gene synthesis service), and Example 7-1- The hTINA1-H3 expression vector was constructed in the same manner as in 1). The vector was named "pCMA-G1 / hTINA1-H3."

[0199] 7-2) Construction of hTINA1 light chain expression vector 7-2-1) Construction of hTINA1-L1 expression vector Nucleotide number of the nucleotide sequence of hTINA1-L1 shown in SEQ ID NO: 17 in the Sequence Listing The DNA sequences encoding the variable regions of hTINA1-L1 shown in Nos. 38 to 402 are included. The DNA fragment containing the nucleotide sequence was synthesized (GENEART artificial gene synthesis service). Using the A fragment as a template, KOD-Plus- (TOYOBO) and the following primers were used: The DNA fragments containing the DNA sequence encoding the variable region of hTINA1-L1 were amplified using a set of The chimeric and humanized antibody light chain expression vector pCMA-LK was digested with the restriction enzyme BsiWI. The incision was then filled with the In-Fusion HD PCR Cloning Kit (CLONTECH The hTINA1-L1 expression vector was constructed by inserting the vector into the target DNA using a recombinant DNA technology developed by the company Eppendorf. The expression vector was named "pCMA-LK / hTINA1-L1." Primer set 5'-ctgtggatctccggcgcgtacggc-3' (SEQ ID NO: 43: Pla Immer CM-LKF) 5'-ggagggggcggccaccgtacg-3' (SEQ ID NO: 44: Primer KCL-Inf-R)

[0200] 7-2-2) Construction of hTINA1-L2 expression vector Nucleotide number of the nucleotide sequence of hTINA1-L2 shown in SEQ ID NO: 19 in the Sequence Listing The DNA sequences encoding the variable regions of hTINA1-L2 shown in Nos. 38 to 402 are included. A DNA fragment containing the nucleotide sequence was synthesized (GENEART artificial gene synthesis service), and Example 7-2- The hTINA1-L2 expression vector was constructed in the same manner as in 1). The vector was named "pCMA-LK / hTINA1-L2."

[0201] 7-2-3) Construction of hTINA1-L3 expression vector Nucleotide number of the nucleotide sequence of hTINA1-L3 shown in SEQ ID NO: 21 in the Sequence Listing The DNA sequences encoding the variable regions of hTINA1-L3 shown in Nos. 38 to 402 are also included. A DNA fragment containing the nucleotide sequence was synthesized (GENEART artificial gene synthesis service), and Example 7-2- The hTINA1-L3 expression vector was constructed in the same manner as in 1). The vector was named "pCMA-LK / hTINA1-L3."

[0202] 7-3) Production and purification of hTINA1 antibody 7-3-1) Small-scale production of hTINA1 antibody It was produced in the same manner as in Example 5-2-5). Combination of pCMA-G1 / hTINA1-H1 and pCMA-LK / hTINA1-L1 The hTINA1 antibody obtained by this method was designated "hTINA1-H1L1" and pCMA-G1 / hTINA1-H2 and pCMA-LK / hTINA1-L1 The hTINA1 antibody was designated "hTINA1-H2L1" and pCMA-G1 / hTINA1- hTINA1 obtained by combining H2 with pCMA-LK / hTINA1-L2 The antibody was "hTINA1-H2L2", and pCMA-G1 / hTINA1-H3 and pCM The hTINA1 antibody obtained by the combination of A-LK / hTINA1-L3 was designated as "hT The gene was named "INA1-H3L3."

[0203] 7-3-2) Production of hTINA1 antibody hTINA1-H1L1, hTINA1-H2L1, hTINA1-H2L2, and h TINA1-H3L3 was produced in the following manner. FreeStyle 293F cells (Invitrogen) were cultured according to the manufacturer's instructions. Subculture and culture were performed. 1.2 × 10 9 FreeStyle 293 F cells (Invitrogen) were cultured in 3L Fernbach Erlenmeyer Flask (CORNING) was seeded with FreeStyle293 express Dilute with ion medium (Invitrogen) to 1.0 × 10 6 cells / ml After preparation, the mixture was shaken at 90 rpm for 1 hour in an incubator at 37°C and 8% CO2. Polyethyleneimine (Polyscience #24765 (3.6 mg) was dissolved in Opti-Pro SFM (Invitrogen; 20 ml). Then, the plasmid was purified by the PureLink HiPure Plasmid kit (Invitrogen) The light chain expression vector (0.8 mg) and heavy chain expression vector (0. 4 mg) was added to Opti-Pro SFM (Invitrogen; 20 ml). . Polyethyleneimine / Opti-Pro SFM mixture (20ml) Add the expression vector / Opti-Pro SFM mixture (20 ml) to the mixture and mix gently. After leaving it for another 5 minutes, it was added to the FreeStyle 293F cells. The culture supernatant was obtained by shaking the cells at 90 rpm in an 8% CO2 incubator for 7 days. Disposable Capsule Filter (ADVANTEC #CCS -045-E1H).

[0204] 7-3-3) Purification of hTINA1 antibody The antibody was isolated from the culture supernatant obtained in 7-3-2) above using rProtein A affinity chromatography. Two steps: chromatography (4-6°C) and ceramic hydroxyapatite (room temperature) After rProteinA affinity chromatography purification, The buffer exchange step after hydroxyapatite purification was carried out at 4-6°C. The culture supernatant was added to MabSelectSuRe (GE Healthcare) equilibrated with PBS. The culture supernatant was applied to a HiTrap column (manufactured by Bioscience). After the entire column was loaded, the column was washed with PBS at least twice the column volume. The antibody-containing fractions were collected and eluted with guanine hydrochloride solution (pH 4.0). Analysis (Thermo Scientific, Slide-A-Lyzer Dialy After replacing with PBS using a sis cassette, The antibody solution was diluted 5-fold with 5 mM MES / pH 7.0 buffer and added to 5 mM NaPi / Ceramic equilibrated with 50mM MES / 30mM NaCl / pH 7.0 buffer. hydroxyapatite column (Japan Bio-Rad, Bio-Scale CHTT The column was applied to a Type I Hydroxyapatite Column. A linear gradient elution with sodium was performed, and the fractions containing the antibody were collected. Analysis (Thermo Scientific, Slide-A-Lyzer Dialy sis Cassette) to HBS (25 mM histidine / 5% sorbitol) Finally, the solution was replaced with Centrifugal UF Filtrate (pH 6.0). er Device VIVASPIN20 (molecular weight cutoff UF10K, Sartoriu The IgG concentration was adjusted to 20 mg / ml or more to prepare a purified sample. .

[0205] [Reference Example 1: Construction of hRS7 antibody expression vector and antibody production] The hRS7 antibody comprises a light chain and a heavy chain as described in WO 2003 / 074566. It was created based on the amino acid sequence of the chain. 1-1) Construction of hRS7 antibody heavy chain expression vector Nucleotide numbers in the nucleotide sequence of the hRS7 antibody heavy chain shown in SEQ ID NO: 29 in the Sequence Listing D containing the DNA sequence encoding the variable region of the hRS7 antibody heavy chain shown in The NA fragment was synthesized (GENEART artificial gene synthesis service) and used in Example 7-1-1). The hRS7 antibody heavy chain expression vector was constructed in the same manner as described above. The amino acid sequence of the hRS7 antibody heavy chain is shown in the sequence listing. Shown in number 30.

[0206] 1-2) Construction of hRS7 antibody light chain expression vector Nucleotide numbers in the nucleotide sequence of the hRS7 antibody light chain shown in SEQ ID NO: 31 in the Sequence Listing D containing the DNA sequence encoding the variable region of the hRS7 antibody light chain shown in SEQ ID NO:38 to SEQ ID NO:402 The NA fragment was synthesized (GENEART artificial gene synthesis service) and used in Example 7-2-1). The hRS7 antibody light chain expression vector was constructed in the same manner as described above. The amino acid sequence of the heavy chain of the hRS7 antibody was shown in the sequence listing. Shown in number 32.

[0207] 1-3) Production and purification of hRS7 antibody 1-3-1) Production of hRS7 antibody The hRS7 antibody was synthesized by combining pCMA-G1 / hRS7 and pCMA-LK / hRS7. It was produced in the same manner as in Example 7-3-2).

[0208] 1-3-2) Purification of hRS7 antibody The culture supernatant obtained in 1-3-1) above was purified in the same manner as in Example 7-3-3). .

[0209] Example 8: Measurement of antigen-binding ability of hTINA1 antibody and hRS7 antibody 8-1) Measurement of antigen binding ability using small-scale produced antibodies (culture supernatant) Antibody and antigen (Recombinant Human TROP-2 Fc chimera The dissociation constants of ra) were measured using a Biacore 3000 (GE Healthcare Biosciences) The antibody was captured as a ligand on the immobilized anti-human IgG (Fab) antibody using a fluorophore (Fiberscope, Inc.). The capture method was used, in which the antibody was captured and the antigen was measured as an analyte. Human IgG (Fab) antibody (Human Fab capture kit, GE Health) Care Biosciences Co., Ltd. is using the sensor chip CM5 (BIAcore, Inc.) Approximately 2000RU was covalently bound to the reference cell using the amine coupling method. The running buffer was HBS-EP+ (10 mM HEPES p H7.4, 0.15M NaCl, 3mM EDTA, 0.05% Surfactant P20) was used. Anti-human IgG (Fab) antibody was immobilized on the chip, and the antibody was After adding the culture supernatant for approximately 80 seconds, a dilution series of antigen solutions (1-1000 nM) was added at a flow rate of 30 The regeneration solution was added at a rate of 1 μl / min for 300 seconds, followed by monitoring the dissociation phase for 600 seconds. Then, 10 mM Gly-HCl pH 1.5 containing 20% ​​DMSO was used at a flow rate of 10 μl / min. The data was analyzed using analysis software (BIAevaluation). The bivalent binding model of the NMR software (version 4.1) was used. In this case, the binding rate constant kon, the dissociation rate constant koff, and the dissociation constant (KD; KD = koff / kon) was calculated.

[0210] [Table 1]

[0211] 8-2) Measurement of antigen binding ability using purified antibodies Antibody and antigen (Recombinant Human TROP-2 Fc chimera The dissociation constants of ra) were measured using a Biacore 3000 (GE Healthcare Biosciences) The antibody was captured as a ligand on the immobilized anti-human IgG (Fab) antibody using a fluorophore (Fiberscope, Inc.). The capture method was used, in which the antibody was captured and the antigen was measured as an analyte. Human IgG (Fab) antibody (Human Fab capture kit, GE Health) Care Biosciences Co., Ltd. is using the sensor chip CM5 (BIAcore, Inc.) Approximately 2000RU was covalently bound to the reference cell using the amine coupling method. The running buffer was HBS-EP+ (10 mM HEPES p H7.4, 0.15M NaCl, 3mM EDTA, 0.05% Surfactant The antibody was applied to a chip immobilized with anti-human IgG (Fab) antibody at approximately 1 μm. After addition of the antigen solution (1-1000 nM) for 30 minutes, a dilution series of the antigen was added at a flow rate of 30 μl / min for 300 minutes. The dissociation phase was monitored for 600 seconds. 25 mM NaOH diluted with buffer was added twice at a flow rate of 100 μl / min for 3 seconds. Data analysis was performed using the method described above.

[0212] [Table 2]

[0213] [Example 9: Preparation of hTINA1-H1L1 ADC (1)]

[0214] [ka]

[0215] Step 1: tert-Butyl (4-{[(1S,9S)-9-ethyl-5-fluoro-9 -Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexyl 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl]amino}-4-oxobutyl)carbamate 4-(tert-butoxycarbonylamino)butanoic acid (0.237 g, 1.13 mm 0.1 L) in dichloromethane (10 mL) and N-hydroxysuccinimide (0.1 30g, 1.13mmol), and 1-ethyl-3-(3-dimethylaminopropyl) carboxymethyl Rubidiimide hydrochloride (0.216 g, 1.13 mmol) was added and stirred for 1 hour. Satecan mesylate (0.500 g, 0.94 mmol) and triethylamine (0 N,N-dimethylformamide solution (10 ml) containing 0.157 mL, 1.13 mmol The solvent was evaporated under reduced pressure, and the resulting residue was Column chromatography [chloroform to chloroform:methanol = 8:2 (v / v)] v)] to give the title compound (0.595 g, quantitative). 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz ),1.31(9H,s),1.58(1H,t,J=7.2Hz),1.66(2H, t,J=7.2Hz),1.89-1.82(2H,m),2.12-2.21(3H, m),2.39(3H,s),2.92(2H,t,J=6.5Hz),3.17(2H ,s),5.16(1H,d,J=19.2Hz),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=6.3Hz),7.30(1H,s),7.79(1H, d,J=11.0Hz),8.40(1H,d,J=8.6Hz). MS(APCI)m / z:621(M+H) + .

[0216] Step 2: 4-amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy] 4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl]butanamide trifluoroacetate The compound obtained in step 1 above (0.388 g, 0.61 mmol) was dissolved in dichloromethane (9 ml The mixture was dissolved in 10 mL of trifluoroacetic acid (9 mL) and stirred for 4 hours. The solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform The organic layer was purified by partitioning with ethanol:methanol:water = 7:3:1 (v / v / v) to give the title compound. (0.343 g, quantitative) was obtained. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2H z),1.79-1.92(4H,m),2.10-2.17(2H,m),2.27( 2H,t,J=7.0Hz),2.40(3H,s),2.80-2.86(2H,m) ,3.15-3.20(2H,m),5.15(1H,d,J=18.8Hz),5.2 6(1H,d,J=18.8Hz),5.42(2H,s),5.54-5.61(1H ,m),6.55(1H,s),7.32(1H,s),7.72(3H,brs),7 .82(1H,d,J=11.0Hz),8.54(1H,d,J=8.6Hz). MS(APCI)m / z:521(M+H) + .

[0217] Step 3: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanine 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]chino phosphon-1-yl]amino}-4-oxobutyl)glycinamide N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycine Dissolve cinnamic acid (0.081 g, 0.19 mmol) in dichloromethane (3 mL) and Hydroxysuccinimide (0.021 g, 0.19 mol) and 1-ethyl-3-(3 -dimethylaminopropyl)carbodiimide hydrochloride (0.036 g, 0.19 mmol) The reaction solution was added with the compound obtained in Step 2 above (0.080 g, 0.15mmol) was added dropwise to a solution of N,N-dimethylformamide (1.5mL). The mixture was stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. Purify by chromatography [chloroform to chloroform:methanol = 8:2 (v / v)]. This gave the title compound (0.106 g, 73%). 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4Hz ),1.36(9H,s),1.71(2H,m),1.86(2H,t,J=7.8H z),2.15-2.19(4H,m),2.40(3H,s),2.77(1H,dd ,J=12.7,8.8Hz),3.02(1H,dd,J=14.1,4.7Hz), 3.08-3.11(2H,m),3.16-3.19(2H,m),3.54(2H, d,J=5.9Hz),3.57-3.77(4H,m),4.46-4.48(1H, m),5.16(1H,d,J=19.2Hz),5.25(1H,d,J=18.8H z),5.42(2H,s),5.55-5.60(1H,m),6.53(1H,s) ,7.00(1H,t,J=6.3Hz),7.17-7.26(5H,m),7.31 (1H,s),7.71(1H,t,J=5.7Hz),7.80(1H,d,J=11 .0Hz),7.92(1H,t,J=5.7Hz),8.15(1H,d,J=8.2 Hz),8.27(1H,t,J=5.5Hz),8.46(1H,d,J=8.2Hz ). MS(APCI)m / z:939(M+H) + .

[0218] Step 4: Glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9 -ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3 ,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxo Butyl)glycinamide trifluoroacetate The compound obtained in step 3 above (1.97 g, 2.10 mmol) was dissolved in dichloromethane (7 mL Trifluoroacetic acid (7 mL) was added and the mixture was stirred for 1 hour. The solvent was evaporated under reduced pressure. Toluene was added to the mixture to form an azeotropic distillate, and the resulting residue was purified by silica gel column chromatography [ Partition organic layer: chloroform to chloroform:methanol:water = 7:3:1 (v / v / v) The title compound (1.97 g, 99%) was obtained. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.4H z),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, 9.4Hz),3.03-3.09(3H,m),3.18-3.19(2H,m),3 .58-3.60(2H,m),3.64(1H,d,J=5.9Hz),3.69(1 H,d,J=5.9Hz),3.72(1H,d,J=5.5Hz),3.87(1H, dd,J=16.8,5.9Hz),4.50-4.56(1H,m),5.16(1H ,d,J=19.2Hz),5.25(1H,d,J=18.8Hz),5.42(2H ,s),5.55-5.60(1H,m),7.17-7.27(5H,m),7.32 (1H,s),7.78-7.81(2H,m),7.95-7.97(3H,m),8 .33-8.35(2H,m),8.48-8.51(2H,m). MS(APCI)m / z:839(M+H) + .

[0219] Step 5: N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl} Lysylglycyl-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)glucan Lysinamide The compound obtained in step 4 above (100 mg, 0.119 mmol) of N,N-dimethylformamide To a solution of methyl amide (1.20 mL), diisopropylethylamine (20.8 μL, 0.1 19mmol), 3-(2-(2-(3-maleimidopropanamido)ethoxy)ethoxy Add N-succinimidyl (trimethoxy)propanoate (50.7 mg, 0.119 mmol) The mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. Purification by chloroform to chloroform:methanol = 5:1 (v / v) The title compound (66.5 mg, 48%) was obtained as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.85(3H,t,J=7.4Hz ),1.65-1.74(2H,m),1.77-1.90(2H,m),2.07-2 .19(4H,m),2.30(2H,t,J=7.2Hz),2.33-2.36(2 H,m),2.38(3H,s),2.76(1H,dd,J=13.7,9.8Hz) ,2.96-3.18(9H,m),3.42-3.44(4H,m),3.53-3. 76(10H,m),4.43(1H,td,J=8.6,4.7Hz),5.14(1 H,d,J=18.8Hz),5.23(1H,d,J=18.8Hz),5.38(1 H,d,J=17.2Hz),5.42(1H,d,J=17.2Hz),5.52-5 .58(1H,m),6.52(1H,s),6.98(2H,s),7.12-7.1 7(1H,m),7.18-7.25(4H,m),7.29(1H,s),7.69( 1H,t,J=5.5Hz),7.78(1H,d,J=11.3Hz),7.98-8 .03(2H,m),8.11(1H,d,J=7.8Hz),8.16(1H,t,J =5.7Hz),8.23(1H,t,J=5.9Hz),8.44(1H,d,J=9 .0Hz). MS(APCI)m / z:1149(M+H) + .

[0220] Step 6: Antibody-drug conjugate (1) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (10.0 mL) was prepared at 10 mg / mL with EDTA. The antibody was collected and diluted with 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.317 mL; 4.6 equivalents per molecule and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai T esque, Inc. (0.500 mL) was added. The pH of this solution was kept within 7.4 ± 0.1. After confirming that the antibody is present, the antibody is incubated at 37°C for 1 hour to detect the presence of the antigen. The disulfide bond in the di-part was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a room temperature water bath for 10 minutes. After the reaction, dimethyl sulfoxide (0.567 mL) was added. The compound was dissolved in 10 mM dimethyl sulfoxide (0.635 mL per antibody molecule). 9.2 equivalents) was added and the mixture was stirred using a tube rotator (MTR-103, AS ONE Corporation). The mixture was stirred at room temperature for 40 minutes to allow the drug linker to bind to the antibody. C (Sigma-Aldrich Co.LLC) aqueous solution (0.127mL; one molecule of antibody The mixture was stirred at room temperature for 20 minutes to allow the drug linker to react. It was stopped. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 35.0 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedure E(ε D,280 =4964 (measured value) 、 ε D,370 The following characteristic values ​​were obtained using a 18982 (actual measured value) sintering method. Antibody concentration: 2.70 mg / mL, antibody yield: 94.5 mg (95%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 6.6.

[0221] [Example 10: Preparation of hTINA1-H1L1 ADC (2)]

[0222] [ka]

[0223] Step 1: Antibody-drug conjugate (2) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (2.00 mL) was added to a 4 mL tube to prepare a 10 mg / mL solution. The antibody was collected and diluted with 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0690 mL; 5.0 equivalents per molecule and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai T esque, Inc.; 0.100 mL) was added. The pH of this solution was kept within 7.4 ± 0.1. After confirming that the antibody is present, the antibody is incubated at 37°C for 1 hour to detect the presence of the antigen. The disulfide bond in the di-part was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a 15°C water bath for 10 minutes. After bating, the compound obtained in Step 5 of Example 9 was dissolved in 10 mM dimethyl sulfoxide (0. Add 127 mL of PBS (9.2 equivalents per antibody molecule) and incubate in a water bath at 15°C for 1 hour. The drug linker was then attached to the antibody. Idrich Co. LLC) aqueous solution (0.0190 mL; 13.8 per antibody molecule) (equivalent) was added and stirred at room temperature for 20 minutes using a tube rotator to complete the reaction of the drug linker. The response was stopped. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 9.00 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedure E(ε D,280 =4964 (measured value) 、 ε D,370 The following characteristic values ​​were obtained using a 18982 (actual measured value) sintering method. Antibody concentration: 2.08 mg / mL, antibody yield: 18.7 mg (94%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 6.1.

[0224] [Example 11: Preparation of hTINA1-H1L1 ADC (3)]

[0225] [ka]

[0226] Step 1: Antibody-drug conjugate (3) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution was adjusted to 10 mg / mL with EDTA. 5.0 mL of this solution was placed in a 15 mL container. Then, 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.) was added under stirring. 0.0813 mL) was added, and the mixture was stirred at 37°C for 10 minutes. P (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0745 mL; 2.3 equivalents per antibody molecule) After adding the solution, check that the pH is within 7.0±0.1 and stir at 37°C for 1 hour. The disulfide bond in the hinge region of the antibody was reduced by stirring. Conjugation of antibody and drug linker: The above solution was stirred in a 15°C water bath for 10 minutes. The compound obtained in Step 5 of Example 9 was then dissolved in 10 mM dimethyl sulfoxide (0.162 mL (5.0 equivalents per antibody molecule) was slowly added dropwise and incubated in a water bath at 15°C for 1 hour. The mixture was stirred to allow the drug linker to bind to the antibody. Idrich Co. LLC) aqueous solution (0.0418 mL; 12.9 per antibody molecule) (equivalent) was added and stirred at room temperature for 20 minutes to quench the reaction of the drug linker. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 21.0 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedures E and F (ε D,280 =4964 (measured value ) 、ε D,370 The following characteristic values ​​were obtained using a 18982 (actual measured value) sintering method. Antibody concentration: 2.19 mg / mL, antibody yield: 46.0 mg (92%), using common procedure E Measured average number of drugs bound per antibody molecule (n): 3.6; measured by common procedure F The average number of drugs bound per antibody molecule (n): 3.6.

[0227] [Example 12: Preparation of hTINA1-H1L1 ADC (4)]

[0228] [ka]

[0229] Step 1: Antibody-drug conjugate (4) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (5.00 mL) was prepared at 10.0 mg / mL with EDTA. The sample was collected in a 1M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, I After adding 0.0813 mL of 10 mM ethanol, the mixture was stirred at 37°C for 10 minutes. TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.162 mL; 5.0 per antibody molecule) After adding 100 equivalents of HCl, confirm that the pH of this solution is within 7.0±0.1 and store at 37°C for 1 hour. The mixture was stirred for 1 hour to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: The above solution was stirred in a 15°C water bath for 10 minutes. The compound obtained in Step 5 of Example 9 was then dissolved in 10 mM dimethyl sulfoxide (0.389 mL (12.0 equivalents per antibody molecule) was slowly added dropwise and incubated in a water bath at 15°C for 1 hour. The mixture was stirred for 1 minute to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0418 mL; 12 mg per antibody molecule). 9 equivalents) was added and stirred at room temperature for 20 minutes to quench the reaction of the drug linker. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 21.0 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedures E and F (ε D,280 =4964 (measured value ) 、 ε D,370 The following characteristic values ​​were obtained using a 18982 (actual measured value) sintering method. Antibody concentration: 2.19 mg / mL, antibody yield: 46.0 mg (92%), using common procedure E Measured average number of drugs bound per antibody molecule (n): 7.0; measured by common procedure F The average number of drugs bound per antibody molecule (n): 7.0.

[0230] [Reference Example 13: Preparation of hRS7 ADC (5)]

[0231] [ka]

[0232] Step 1: Antibody-drug conjugate (5) Antibody reduction: hRS7 prepared in Reference Example 1 was subjected to the common procedure B (2 (80 nm extinction coefficient of 1.56) and C in PBS 6.0 / EDTA This solution (2.0 mL) was placed in a 4 mL tube and diluted to 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0690 mL; 5 per antibody molecule) 0.0 equivalent) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, In c.: 0.100 mL) was added. Confirm that the pH of this solution was within 7.4 ± 0.1. After that, the antibody was incubated at 37°C for 1 hour to remove disulfides in the hinge region. The bond was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a 15°C water bath for 10 minutes. After bating, the compound obtained in Step 5 of Example 9 was dissolved in 10 mM dimethyl sulfoxide (0. Add 127 mL of PBS (9.2 equivalents per antibody molecule) and incubate in a water bath at 15°C for 1 hour. The drug linker was then attached to the antibody. Idrich Co. LLC) aqueous solution (0.0190 mL; 13.8 per antibody molecule) (equivalent) was added and stirred at room temperature for 20 minutes using a tube rotator to complete the reaction of the drug linker. The response was stopped. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 9.00 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedure E(ε D,280 =4964 (measured value) 、 ε D,370 The following characteristic values ​​were obtained using a 18982 (actual measured value) sintering method. Antibody concentration: 2.04 mg / mL, antibody yield: 18.4 mg (92%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 6.2.

[0233] [Example 14: Preparation of hTINA1-H1L1 ADC (6)]

[0234] [ka]

[0235] Step 1: ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}a Methylaminoacetate N-9-Fluorenylmethoxycarbonylglycylglycine (4.33g, 12.2m mol), tetrahydrofuran (120 ml), and toluene (40.0 ml). The mixture was added pyridine (1.16 ml, 14.7 mmol) and lead tetraacetate (6.84 g, 14 The reaction mixture was cooled to room temperature, and the insoluble matter was removed by filtration. The residue was dissolved in ethyl acetate and diluted with water and After washing with saturated saline, the organic layer was dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure. The residue was then purified by silica gel column chromatography [hexane:ethyl acetate The title compound (3.00 g, 67%) was purified using a 200 mL (9:1 (v / v) - ethyl acetate) mixture. Obtained as a coloured solid. 1 H-NMR(400MHz,CDCl3)δ:2.07(3H,s),3.90(2H, d,J=5.1Hz),4.23(1H,t,J=7.0Hz),4.46(2H,d, J=6.6Hz),5.26(2H,d,J=7.0Hz),5.32(1H,brs) ,6.96(1H,brs),7.32(2H,t,J=7.3Hz),7.41(2H ,t,J=7.3Hz),7.59(2H,d,J=7.3Hz),7.77(2H,d ,J=7.3Hz).

[0236] Step 2: Benzyl [({N-[(9H-fluoren-9-ylmethoxy)carbonyl] Glycyl(amino)methoxy]acetate The compound obtained in step 1 above (3.68 g, 10.0 mmol) and benzyl glycolate To a solution of potassium hydroxide (4.99 g, 30.0 mmol) in tetrahydrofuran (40.0 mL), Add ammonium tert-butoxide (2.24 g, 20.0 mmol) at 0°C and add at room temperature. The mixture was stirred for 15 minutes. Ethyl acetate and water were added to the reaction solution at 0°C, and the mixture was diluted with ethyl acetate and chloroform. The organic layer was dried over sodium sulfate and filtered. The solvent was evaporated under reduced pressure. The residue was dissolved in dioxane (40.0 mL) and water (10.0 mL), and sodium bicarbonate was added. Thorium (1.01 g, 12.0 mmol), 9-fluorenylmethyl chloroformate (2. 59 g, 10.0 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. Water was added to the reaction solution, and acetic acid was added. The mixture was extracted with ethyl acetate, and the resulting organic layer was dried over sodium sulfate and filtered. The solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography [hexane:ethyl acetate=1 The title compound (1.88 g, 4 0%). 1 H-NMR(400MHz,CDCl3)δ:3.84(2H,d,J=5.5Hz), 4.24(3H,t,J=6.5Hz),4.49(2H,d,J=6.7Hz),4. 88(2H,d,J=6.7Hz),5.15-5.27(1H,m),5.19(2H ,s),6.74(1H,brs),7.31-7.39(7H,m),7.43(2H ,t,J=7.4Hz),7.61(2H,d,J=7.4Hz),7.79(2H,d ,J=7.4Hz).

[0237] Step 3: [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl} Amino)methoxy]acetic acid The compound obtained in step 2 above (1.88 g, 3.96 mmol) was dissolved in ethanol (40.0 mL) The resulting mixture was dissolved in ethyl acetate (20.0 ml) and palladium-carbon catalyst (376 mg) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. Insoluble matter was removed by filtration through Celite. The solvent was evaporated under reduced pressure to give the title compound (1.52 g, quantitative) as a colorless solid. 1 H-NMR(400MHz,DMSO-d6)δ:3.62(2H,d,J=6.3Hz ),3.97(2H,s),4.18-4.32(3H,m),4.60(2H,d,J =6.7Hz),7.29-7.46(4H,m),7.58(1H,t,J=5.9H z),7.72(2H,d,J=7.4Hz),7.90(2H,d,J=7.4Hz) ,8.71(1H,t,J=6.5Hz).

[0238] Step 4: 9H-fluoren-9-ylmethyl (2-{[(2-{[(1S,9S)-9- Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3, 9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4 ':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoe (2-Oxoethyl)carbamate Under ice cooling, exatecan mesylate (0.283 g, 0.533 mmol) and N-hydroxybenzoate were added. hydroxysuccinimide (61.4 mg, 0.533 mmol) and the compound obtained in step 3 above. The mixture (0.205 g, 0.533 mmol) was dissolved in N,N-dimethylformamide (10.0 mL) solution, N,N-diisopropylethylamine (92.9 μL, 0.533 mmol) L) and N,N'-dicyclohexylcarbodiimide (0.143 g, 0.693 mmO L) was added and stirred at room temperature for 3 days. The solvent was evaporated under reduced pressure, and the resulting residue was Column chromatography [chloroform to chloroform:methanol:water=7:3:1 The title compound (0.352 g, 82%) was purified by partitioning the organic layer (v / v / v) into pale brown precipitate. Obtained as a coloured solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.81(3H,t,J=7.4Hz ),1.73-1.87(2H,m),2.06-2.20(2H,m),2.34(3 H,s),3.01-3.23(2H,m),3.58(2H,d,J=6.7Hz), 3.98(2H,s),4.13-4.25(3H,m),4.60(2H,d,J=6 .7Hz),5.09-5.22(2H,m),5.32-5.42(2H,m),5. 50-5.59(1H,m),6.49(1H,s),7.24-7.30(3H,m) ,7.36(2H,t,J=7.4Hz),7.53(1H,t,J=6.3Hz),7 .66(2H,d,J=7.4Hz),7.75(1H,d,J=11.0Hz),7. 84(2H,d,J=7.4Hz),8.47(1H,d,J=8.6Hz),8.77 (1H,t,J=6.7Hz). MS(ESI)m / z:802(M+H) + .

[0239] Step 5: N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy -4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b ]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide N,N-dimethylformamide (0.881 g, 1.10 mmol) of the compound obtained in step 4 above To the solution of methyl amide (11.0 mL), piperidine (1.1 mL) was added and stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to give a mixture containing the title compound. This was used in the next reaction without further treatment.

[0240] Step 6: N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl -L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro -9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indole dino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycine Cinamide Under ice cooling, the mixture obtained in step 5 (0.439 mmol) and N-hydroxysuccinimide amide (0.101 g, 0.878 mmol), and N-[(9H-fluoren-9-yl) methoxy)carbonyl]glycylglycyl-L-phenylalanine (Patent Publication No. 2002-60 351; 0.440 g, 0.878 mmoL) of N,N-dimethylformamide ( 50.0 mL) solution, N,N'-dicyclohexylcarbodiimide (0.181 g, 0 The mixture was stirred at room temperature for 4 days. The solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography [chloroform to chloroform:methanol = 9:1 ​​(v / v)] to give the title compound (0.269 g, 58%) as a pale orange solid. I got it. MS(ESI)m / z:1063(M+H) + .

[0241] Step 7: Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-ox (ethoxy)methyl]glycinamide N,N-dimethylformamide (0.269 g, 0.253 mmol) of the compound obtained in step 6 above A solution of fluoromethane (4.00 mL) in piperidine (0.251 mL, 2.53 mmol) The mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to give a mixture containing the title compound. The mixture was used in the next reaction without further purification.

[0242] Step 8: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S, 9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyra no[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}- 2-Oxoethoxy)methyl]glycinamide The compound obtained in step 7 (0.253 mmoL) was dissolved in N,N-dimethylformamide (1 0.0 mL) solution, N-succinimidyl 6-maleimidohexanoate (0.156 g, The mixture was stirred at room temperature for 3 days. The solvent was evaporated under reduced pressure. The product was purified by silica gel column chromatography [chloroform to chloroform:methanol = 9:1 ​​(v / v)] to give the title compound (0.100 g, 38%) as a pale yellow solid. I got it. 1 H-NMR(400MHz,DMSO-d6)δ:0.83(3H,t,J=7.2Hz ),1.09-1.21(2H,m),1.33-1.47(4H,m),1.75-1 .90(2H,m),2.00-2.23(4H,m),2.36(3H,s),2.6 9-2.81(1H,m),2.94-3.03(1H,m),3.06-3.22(2 H,m),3.23-3.74(6H,m),3.98(2H,s),4.39-4.5 0(1H,m),4.60(2H,d,J=6.7Hz),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(1 H,d,J=11.0Hz),7.97(1H,t,J=5.7Hz),8.03(1H ,t,J=5.9Hz),8.09(1H,d,J=7.8Hz),8.27(1H,t ,J=6.5Hz),8.48(1H,d,J=9.0Hz),8.60(1H,t,J =6.5Hz). MS(ESI)m / z:1034(M+H) + .

[0243] Step 9: Antibody-drug conjugate (6) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (10.0 mL) was placed in a 50 mL tube. The antibody was then diluted with 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) in water (0.317 mL). 4.6 equivalents per molecule and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai T esque, Inc. (0.500 mL) was added. The pH of this solution was kept within 7.4 ± 0.1. After confirming that the antibody is present, the antibody is incubated at 37°C for 1 hour to detect the presence of the antigen. The disulfide bond in the di-part was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a room temperature water bath for 10 minutes. After the reaction, dimethyl sulfoxide (0.567 mL) was added. The compound was dissolved in 10 mM dimethyl sulfoxide (0.635 mL per antibody molecule). 9.2 equivalents) was added and stirred at room temperature for 40 minutes using a tube rotator to form a drug linker. Next, 100 mM NAC (Sigma-Aldrich Co.) was added to the antibody. An aqueous solution of .LLC (0.127 mL; 18.4 equivalents per antibody molecule) was added, and The mixture was stirred at room temperature for 20 minutes to terminate the reaction of the drug linker. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 35.0 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedure E(ε D,280 =5178 (measured value) 、 ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 2.70 mg / mL, antibody yield: 94.5 mg (95%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 6.4.

[0244] [Example 15: Preparation of hTINA1-H1L1 ADC (7)]

[0245] [ka]

[0246] Step 1: Antibody-drug conjugate (7) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (2.0 mL) was placed in a 4 mL tube and adjusted to 10 mg / mL with EDTA. The antibody was collected and diluted with 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0690 mL; 5.0 equivalents per molecule) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Te sque, Inc.; 0.0299 mL) was added. The pH of this solution was kept within 7.0 ± 0.1. After confirming that the antibody is present, the antibody is incubated at 37°C for 1 hour to detect the presence of the antigen. The disulfide bond in the di-part was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a 15°C water bath for 10 minutes. After the bating, the compound obtained in Step 8 of Example 14 was dissolved in 10 mM dimethyl sulfoxide (0 Add 1.127 mL of PBS (9.2 equivalents per antibody molecule) and incubate in a water bath at 15°C for 1 hour. The antibody was then incubated with 100 mM NAC (Sigma-Aldrich) to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0190 mL; 13 mg per antibody molecule). 8 equivalents) was added and stirred at room temperature for 20 minutes using a tube rotator. The reaction was stopped. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 9.00 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedure E(ε D,280 =5178 (measured value) 、 ε D,370The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 2.04 mg / mL, antibody yield: 18.4 mg (92%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 5.7.

[0247] [Example 16: Preparation of hTINA1-H1L1 ADC (8)]

[0248] [ka]

[0249] Step 1: Antibody-drug conjugate (8) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (30.0 mL) was added to a 100 mL container to prepare a 10 mg / mL solution. The sample was collected and stirred in a 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, In c.; 0.4875 mL) was added, and the mixture was stirred at 37°C for 10 minutes. CEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.9721 mL; 5.0 per antibody molecule) After adding 100 equivalents of HCl, confirm that the pH of this solution is within 7.0±0.1 and store at 37°C for 1 hour. The mixture was stirred for 1 hour to reduce the disulfide bond in the hinge region of the antibody. Conjugation of antibody and drug linker: The above solution was stirred in a 15°C water bath for 10 minutes. The compound obtained in Step 8 of Example 14 was then dissolved in 10 mM dimethyl sulfoxide (2.33 mL) (12.0 equivalents per antibody molecule) was slowly added dropwise and incubated in a water bath at 15°C for 1 hour. The mixture was stirred for 1 minute to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.251 mL; 12.9 mg / 1 antibody molecule) (equivalent) was added and stirred at room temperature for 20 minutes to quench the reaction of the drug linker. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 98.0 mL of a solution containing the compound conjugate was obtained. Concentrate the solution containing the antibody-drug conjugate by the procedure A. 5 mL was obtained. Characterization: Common procedures E and F (ε D,280 =5178(measured value ) 、 ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 14.6 mg / mL, antibody yield: 256 mg (85%), measured using common procedure E Average number of drugs bound per antibody molecule (n): 6.7; measured by standard procedure F Average number of drugs bound per antibody molecule (n): 7.0.

[0250] [Example 17: Preparation of hTINA1-H1L1 ADC (9)]

[0251] [ka]

[0252] Step 1: Antibody-drug conjugate (9) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution was adjusted to 10 mg / mL with EDTA. 5.0 mL of this solution was placed in a 15 mL container. Then, 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.) was added under stirring. 0.0813 mL) was added, and the mixture was stirred at 37°C for 10 minutes. P (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0778 mL; 2.4 equivalents per antibody molecule) After adding the solution, check that the pH is within 7.0±0.1 and stir at 37°C for 1 hour. The disulfide bond in the hinge region of the antibody was reduced by stirring. Conjugation of antibody and drug linker: The above solution was stirred in a 15°C water bath for 10 minutes. The compound obtained in Step 8 of Example 14 was then dissolved in 10 mM dimethyl sulfoxide (0.162 ml) L; 5.0 equivalents per antibody molecule) was slowly added dropwise and placed in a water bath at 15°C for 1 hour. The mixture was stirred for 1 minute to allow the drug linker to bind to the antibody. Aldrich Co. LLC) aqueous solution (0.0418 mL; 12 mg per antibody molecule). 9 equivalents) was added and stirred at room temperature for 20 minutes to quench the reaction of the drug linker. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 21.0 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedures E and F (ε D,280 =5178(measured value ) 、 ε D,370 The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 2.26 mg / mL, antibody yield: 47.5 mg (95%), using common procedure E Measured average number of drugs bound per antibody molecule (n): 3.5; measured by common procedure F The average number of drugs bound per antibody molecule (n): 3.6.

[0253] [Reference Example 18: Preparation of hRS7 ADC (10)]

[0254] [ka]

[0255] Step 1: Antibody-drug conjugate (10) Antibody reduction: hRS7 prepared in Reference Example 1 was subjected to the common procedure B (2 (80 nm extinction coefficient of 1.56) and C in PBS 6.0 / EDTA This solution (2.0 mL) was placed in a 4 mL tube and diluted to 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0690 mL; 5 per antibody molecule) 0.0 equivalent) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, In c.: 0.0299 mL) was added. Confirm that the pH of this solution was within 7.0 ± 0.1. After that, the antibody was incubated at 37°C for 1 hour to separate the disulfides in the hinge region. The amide bond was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a 15°C water bath for 10 minutes. After the bating, the compound obtained in Step 8 of Example 14 was dissolved in 10 mM dimethyl sulfoxide (0 Add 1269 mL of HCl (9.2 equivalents per antibody molecule) and incubate in a 15°C water bath for 1 hour. The antibody was then incubated with 100 mM NAC (Sigma-Aldrich) to allow the drug linker to bind to the antibody. -Aldrich Co. LLC) aqueous solution (0.0190 mL; 13 per antibody molecule) 0.8 equivalents) was added and stirred at room temperature for 20 minutes using a tube rotator to form the drug linker. The reaction was stopped. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 9.00 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedure E(ε D,280 =5178 (measured value) 、 ε D,370The following characteristic values ​​were obtained using the JIS Z 20217 (actual measured value). Antibody concentration: 2.07 mg / mL, antibody yield: 18.6 mg (93%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 5.6.

[0256] [Example 19: Preparation of hTINA1-H1L1 ADC (11)]

[0257] [ka]

[0258] Step 1: tert-butyl [2-(2-{[(1S,9S)-9-ethyl-5-fluoro 1-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,1 5-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo Lisino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl] -Bamate Exatecan mesylate (3.10 g, 5.47 mol) was added to 4-(tert-butyl) 2-[(tert-butoxycarbonyl)amino]butanoic acid instead of {2-[(tert-butoxycarbonyl) amino]ethoxy}acetic acid (J. Med. Chem., 1992, vol. 35, p. 2928; 1.55 g, 6.01 mmol) was reacted in the same manner as in Step 1 of Example 1 to give the title compound. The compound (2.56 g, 73%) was obtained as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.3Hz ),1.26(9H,s),1.81-1.91(2H,m),2.13-2.22(2 H,m),2.40(3H,s),3.08-3.26(4H,m),3.43-3.5 3(2H,m),4.00(1H,d,J=15.1Hz),4.05(1H,d,J= 15.1Hz),5.14(1H,d,J=18.7Hz),5.22(1H,d,J= 18.7Hz),5.40(1H,d,J=16.6Hz),5.44(1H,d,J= 16.6Hz),5.59-5.66(1H,m),6.53(1H,s),6.86( 1H,t,J=5.4Hz),7.31(1H,s),7.79(1H,d,J=10. 9Hz), 8.49(1H,d,J=9.1Hz). MS(APCI)m / z:637(M+H) + .

[0259] Step 2: 2-(2-aminoethoxy)-N-[(1S,9S)-9-ethyl-5-fluoro] 1-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,1 5-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indo Lisino[1,2-b]quinolin-1-yl]acetamide trifluoroacetate The compound obtained in Step 1 above (1.50 g, 2.36 mol) was treated in the same manner as in Step 2 of Example 1. The title compound (1.50 g, quantitative) was obtained as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.5Hz ),1.81-1.92(2H,m),2.15-2.23(2H,m),2.41(3 H,s),3.05(2H,t,J=5.1Hz),3.15-3.23(2H,m), 3.71(2H,t,J=5.1Hz),4.10(2H,s),5.19(1H,d, J=18.7Hz),5.24(1H,d,J=18.7Hz),5.43(2H,s) ,5.58-5.66(1H,m),6.55(1H,s),7.33(1H,s),7 .73-7.84(4H,m),8.55(1H,d,J=9.1Hz). MS(APCI)m / z:537(M+H) + .

[0260] Step 3: N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanine 1S,9S)-9-ethyl-5-fluoro-9-hydroxy-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy -4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b ]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide The compound obtained in step 2 above (554 mg, 0.85 mmol) was treated in the same manner as in step 3 of Example 1. The reaction was carried out in the same manner to obtain the title compound (775 mg, 95%). 1 H-NMR(400MHz,DMSO-d6)δ:0.85(3H,t,J=7.3Hz ),1.36(9H,s),1.78-1.89(2H,m),2.13-2.22(2 H,m),2.39(3H,s),2.71(1H,dd,J=13.4,9.8Hz) ,2.95(1H,dd,J=13.4,4.3Hz),3.09-3.23(1H,m ),3.23-3.32(2H,m),3.40-3.62(8H,m),3.73(1 H,dd,J=16.5,5.5Hz),4.03(2H,s),4.39-4.47( 1H,m),5.17(1H,d,J=18.9Hz),5.25(1H,d,J=18 .9Hz),5.41(1H,d,J=16.8Hz),5.45(1H,d,J=16 .8Hz),5.57-5.64(1H,m),6.54(1H,s),6.99(1H ,t,J=5.8Hz),7.13-7.26(5H,m),7.31(1H,s),7 .76-7.82(2H,m),7.90(1H,t,J=5.2Hz),8.13(1 H,d,J=7.9Hz),8.27(1H,t,J=5.8Hz),8.49(1H, d, J = 8.5 Hz). MS(APCI)m / z:955(M+H) + .

[0261] Step 4: 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 trifluoroacetate The compound obtained in Step 3 above (630 mg, 0.659 mmol) was subjected to the same procedure as in Step 4 of Example 1. The same reaction was carried out to obtain the title compound (588 mg, 92%). 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.3Hz ),1.79-1.90(2H,m),2.13-2.22(2H,m),2.39(3 H,s),2.71(1H,dd,J=13.4,10.1Hz),2.99(1H,d d,J=13.4,4.3Hz),3.09-3.23(1H,m),3.24-3.3 2(3H,m),3.41-3.71(7H,m),3.86(1H,dd,J=16. 8,5.8Hz),4.04(2H,s),4.52(1H,td,J=9.0,4.1 Hz),5.17(1H,d,J=18.9Hz),5.25(1H,d,J=18.9 Hz),5.41(1H,d,J=16.5Hz),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=11.0Hz),7. 87-8.01(4H,m),8.29-8.36(2H,m),8.46-8.55( 2H,m). MS(APCI)m / z:855(M+H) + .

[0262] Step 5: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )hexanoyl]glycylglycyl-L-phenylalanyl-N-[2-(2-{[(1 S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di Oxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de] Pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino {-2-oxoethoxy)ethyl]glycinamide The compound obtained in step 4 above (240 mg, 0.247 mmol) was dissolved in diisopropyl ether. Instead of methylamine, triethylamine (31.4 μL, 0.22 mmol) was used, and 3-(2 -(2-(3-maleimidopropanamido)ethoxy)ethoxy)propanoic acid N-sulfonyl Instead of succinimidyl, N-succinimidyl 6-maleimidohexanoate (95.3mM g, 0.31 mmol) was reacted in the same manner as in Step 5 of Example 1 to give the title compound (1 The yield was 62 mg (62%). 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.6Hz ),1.13-1.22(2H,m),1.40-1.51(4H,m),1.78-1 .90(2H,m),2.09(2H,t,J=7.6Hz),2.14-2.21(2 H,m),2.39(3H,s),2.74(1H,dd,J=13.6,9.7Hz) ,2.96(1H,dd,J=13.6,4.5Hz),3.08-3.24(1H,m ),3.24-3.30(1H,m),3.33-3.40(4H,m),3.47-3 .68(7H,m),3.72(1H,dd,J=16.6,5.7Hz),4.03( 2H,s),4.42(1H,td,J=8.6,4.2Hz),5.17(1H,d, J=18.7Hz),5.25(1H,d,J=18.7Hz),5.40(1H,d, J=17.2Hz),5.44(1H,d,J=17.2Hz),5.57-5.64( 1H,m),6.52(1H,s),6.99(2H,s),7.13-7.25(5H ,m),7.31(1H,s),7.74-7.81(2H,m),7.99(1H,t ,J=5.7Hz),8.03-8.11(2H,m),8.22(1H,t,J=5. 7Hz), 8.47(1H,d,J=9.1Hz). MS(APCI)m / z:1048(M+H) + .

[0263] Step 6: Antibody-drug conjugate (11) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, The solution (3.0 mL) was collected in a 15 mL container. Then, 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc.) was added under stirring. 0.0488 mL) was added, and the mixture was stirred at 37°C for 10 minutes. P (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0972 mL; 5.0 equivalents per antibody molecule) After adding the solution, check that the pH is within 7.0±0.1 and stir at 37°C for 1 hour. The disulfide bond in the hinge region of the antibody was reduced by stirring. Conjugation of antibody and drug linker: The above solution was stirred in a 15°C water bath for 10 minutes. The compound obtained in Step 8 of Example 11a was then dissolved in 10 mM dimethyl sulfoxide (0.233 3 mL; 12.0 equivalents per antibody molecule) was slowly added dropwise and the mixture was placed in a 15°C water bath. The mixture was stirred for 1 hour to allow the drug linker to bind to the antibody. ma-Aldrich Co. LLC) aqueous solution (0.0251 mL; per antibody molecule) 12.9 equivalents) was added and stirred at room temperature for 20 minutes to quench the reaction of the drug linker. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain the title antibody-drug 14 mL of a solution containing the monoconjugate was obtained. Characterization: Common procedures E and F (ε D,280 =5193 (measured value ) 、 ε D,370 The following characteristic values ​​were obtained using a tensile strength of 10000 kJ / cm2 (measured value). Antibody concentration: 1.93 mg / mL, antibody yield: 27.0 mg (90%), using common procedure E Measured average number of drugs bound per antibody molecule (n): 7.1; measured by common procedure F The average number of drugs bound per antibody molecule (n): 7.0.

[0264] [Reference Example 2: Preparation of hRS7-CL2A-SN38 (12)]

[0265] [ka]

[0266] Step 1: Antibody-drug conjugate (12) Antibody reduction: hRS7 prepared in Reference Example 1 was subjected to the common procedure B (2 (80 nm extinction coefficient of 1.54) and C in PBS 6.0 / EDTA This solution (10.0 mL) was placed in a 50 mL tube and diluted to 10 mg / mL. 0.317 mL of TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (per antibody molecule) 4.6 equivalents) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, I nc.; 0.500 mL) was added. The pH of this solution was confirmed to be within 7.4 ± 0.1. After that, the antibody was incubated at 37°C for 1 hour to separate the disulfides in the hinge region. The amide bond was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a room temperature water bath for 10 minutes. After the addition of dimethyl sulfoxide (0.567 mL), the reaction mixture was cooled to room temperature. 10 mM of CL2A-SN38 synthesized according to the specification of Patent Publication No. 2011 / 0293513 Dimethyl sulfoxide solution (0.635 mL; 9.2 equivalents per antibody molecule) was added. The mixture was stirred at room temperature for 40 minutes using a tube rotator to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0 Add 127 mL of HCl (18.4 equivalents per antibody molecule) and stir at room temperature for another 20 minutes. The drug linker reaction was stopped. Purification: The reaction solution was purified by gel filtration according to the procedure described in Production Method 1 - Common Procedure D. Repeat twice, then 25 mM trehalose containing polysorbate 80 (0.01%) The resulting solution was purified by gel filtration once using a NAP-25 column. The eluate (35 mL) was freeze-dried. Characterization: The eluate before lyophilization was evaluated using the common procedure E described in Manufacturing Method 1. The following characteristic values ​​were obtained: Antibody concentration: 2.78 mg / mL, antibody yield: 97.3 mg (97%), per antibody molecule Average number of drug bonds (n): 5.6

[0267] [Example 20: Preparation of hTINA1-H1L1 ADC (13)]

[0268] [ka]

[0269] Step 1: Antibody-drug conjugate (13) Antibody reduction: hTINA1-H1L1 prepared in Example 7 was reduced to 100 μg / ml by the method described in Production Method 1. Using common procedures B (using an extinction coefficient of 1.54 at 280 nm) and C, the medium was changed to PBS. The antibody concentration was adjusted to 10 mg / mL by replacing the EDTA with 6.0 / EDTA. ) was placed in a 250 mL polycarbonate Erlenmeyer flask and heated with a magnetic stirrer. - Add 1M dipotassium hydrogen phosphate aqueous solution (1.4 mL) at room temperature while stirring, then add 10 mM A TCEP solution (1.62 mL; 2.5 equivalents per antibody molecule) was added. After confirming that the pH was within 7.0±0.1, stop stirring and incubate at 37°C for 2 hours. By incubation, disulfide bonds in the hinge region of the antibody were reduced. Conjugation of antibody and drug linker: After cooling the above solution to 15°C, add DMSO under stirring. SO (3.24 mL) was slowly added dropwise. Then, the compound of Example 14, Step 8 was added Slowly add 1.76 mL of DMSO solution containing 10 mM of the antibody (5.0 equivalents per antibody molecule). The solution was stirred at 15°C for 1 hour to allow the drug linker to bind to the antibody. Next, with stirring, a 100 mM NAC aqueous solution (0.324 mL; 5 per antibody molecule) was added. 0.0 equivalents) and further incubated at room temperature for 20 minutes to remove any unreacted drug linkers. Responsiveness was stopped. Purification: Add 20% acetic acid (approximately 0.52 mL) and ABS (100 mL) to the above solution under stirring. The pH of this solution was adjusted to 5.5±0.1 by slowly adding HCl. This solution was filtered through a microfiltration (0.45μL). The cloudy matter was removed by filtering through a PVDF membrane (m), and about 200 mL of filtrate was obtained. On the other hand, ultrafiltration membrane (Merck Co., Ltd., Pellicon XL Cassette, U Ultracell 30KDa), tube pump (Masterflame, Cole-Parmer, USA) Pump model 77521-40, Pump head model 7518- 00) and tube (Masterflex Tube L / S16, Cole-Parmer, USA) The reaction mixture was purified by ultrafiltration using an ultrafiltration apparatus consisting of the following components: By adding ABS (total 1600 mL) dropwise as a liquid, the unbound The drug linker and other low molecular weight reagents were removed, and the buffer solution was replaced with ABS. The purified solution was then filtered (0.22 μm, PVDF membrane) was carried out to obtain 88 mL of a solution containing the title antibody-drug conjugate. Characterization: Common operation E and common operation F(ε D,280 =5178 、 ε D,370 =202 17) to obtain the following characteristic values. Antibody concentration: 9.96 mg / mL, antibody yield: 876 mg (88%), measured using common procedure E Average number of drugs bound per antibody molecule (n): 3.8; measured by standard procedure F Average number of drugs bound per antibody molecule (n): 3.8.

[0270] Example 21: Evaluation of antitumor effect of ADC 21-a) Antitumor effect of ADC (1) Mice: 5-6 week old female BALB / c-nu / nu mice (Charles River Japan strain) The mice (manufactured by the company) were acclimated under SPF conditions for 4-7 days before use. The animals were fed sterilized feed (FR-2, Funabashi Farms Co., Ltd.). They were given tap water (prepared by adding 5-15 ppm sodium hypochlorite solution). Measurements and calculations: In all studies, the major and minor diameters of the tumor were measured using electronic digital calipers. Tumor volume (m m 3 ) was calculated using the following formula: Tumor volume (mm 3 ) = 1 / 2 x major axis (mm) x [minor axis (mm)] 2

[0271] All antibody-drug conjugates were diluted with saline (Otsuka Pharmaceutical Factory, Inc.) and 10 The human colon cancer cell line COLO purchased from ATCC was administered into the tail vein at a volume of 1 mL / kg. 205 suspended in saline at 2 × 10 6 The cells were cultured in female BALB / c-nu / nu mice. The mice were subcutaneously implanted into the right flank of the mice (Day 0), and randomized to groups on Day 7. Drug conjugates (1), (6), and (12) were administered at 10 mg / day on Days 7, 14, and 21. The drug was administered intravenously at a dose of 1000 mg / kg to the tail vein. The 1L1 antibody and the hRS7 antibody were administered at a dose of 25 mg / kg. Administration of conjugates (1) and (6) reduced tumor volume. The tumor growth was significantly reduced compared to the administration of , and both showed tumor growth suppression effects (Figure 13). In the figure, the horizontal axis indicates the number of days, and the vertical axis indicates the tumor volume.

[0272] 21-b) Antitumor effects of ADC (2) The human pancreatic adenocarcinoma cell line Bx-PC3, purchased from ATCC, was cultured in female BALB / c-nu / nu The tumor fragments were transplanted into mice and further solid-cultured and placed in the right flank of female BALB / c-nu / nu mice. The mice were subcutaneously implanted into the mice (Day 0) and randomly assigned to groups on Day 16. Jugates (1), (6), and (12) were administered at 10 mg / kg on days 16, 23, and 30. The drug was administered intravenously to the tail vein at a dose of 100 mg / kg. As a negative control, the drug-free hTINA1-H1L1 The antibody and hRS7 antibody were administered at a dose of 25 mg / kg. The tumor volume was increased by administration of the antibody-drug conjugate (12). The tumor growth was significantly reduced compared to the control group, and both groups exhibited tumor growth inhibitory effects (Figure 14).

[0273] 21-c) Antitumor effects of ADC (3) The human pancreatic adenocarcinoma cell line Capan-1, purchased from ATCC, was cultured in female BALB / c-nu / n The tumor fragments were transplanted into u mice and further solid-passaged into the right flank of female BALB / c-nu / nu mice. The mice were subcutaneously implanted in the 100-well plate (Day 0) and randomly assigned to groups on Day 18. Indugates (1), (6), and (12) were administered at 10 mg / kg on days 18, 25, and 32. As a negative control, the drug-free hTINA1-H1L The antibody-drug conjugates were administered in the same manner as the hRS1 and hRS7 antibodies at a dose of 25 mg / kg. The tumor volume increased with administration of gates (1) and (6) compared with administration of antibody-drug conjugate (12). The tumor growth was significantly reduced compared to the control group, and both groups exerted tumor growth inhibitory effects (Figure 15).

[0274] 21-d) Antitumor effect of ADC (4) COLO205 was administered subcutaneously to female BALB / c-nu / nu mice in the same manner as in Example 21-a). The mice were transplanted (Day 0) and randomly assigned to groups on Day 11. Days 11, 18, and 25 The antibody-drug conjugates (2) and (5) were all administered at 10 mg / kg. Gates (7) and (10) were both administered intravenously at a dose of 3 mg / kg. Administration of jugate (2), (5), (7), and (10) all suppressed tumor growth. This was demonstrated (Figure 16).

[0275] 21-e) Antitumor effect of ADC (5) In the same manner as in Example 21-b), Bx-PC3 was subcutaneously transferred to female BALB / c-nu / nu mice. The mice were transplanted (Day 0) and randomly assigned to groups on Day 25. Antibody-drug conjugates (2), (5), (7), and (10) were administered intravenously at a dose of 3 mg / kg on days 25 and 32. The antibody-drug conjugates (2), (5), (7), and (10) were administered intravenously. Both of these compounds exerted tumor growth inhibitory effects (Fig. 17).

[0276] 21-f) Antitumor effects of ADC (6) COLO205 was administered subcutaneously to female BALB / c-nu / nu mice in the same manner as in Example 21-a). The mice were transplanted (Day 0) and randomly assigned to groups on Day 9. On Days 9 and 16, the mice were treated with antibody-drug therapy. Conjugates (3), (4), (8), and (9) were all administered intravenously at a dose of 10 mg / kg. By intravenous administration of antibody-drug conjugates (3), (4), (8), and (9), Both of them exerted tumor growth inhibitory effects (Figure 18).

[0277] 21-g) Antitumor Effect of ADC (7) In the same manner as in Example 21-b), Bx-PC3 was subcutaneously transferred to female BALB / c-nu / nu mice. On Day 21 and 28, the mice were randomly assigned to each group. Drug conjugates (3), (4), (8), and (9) were all administered intravenously at a dose of 3 mg / kg. By intravenous administration of antibody-drug conjugates (3), (4), (8), and (9), Both of them exerted tumor growth inhibitory effects (Figure 19).

[0278] 21-h) Antitumor effect of ADC (8) Human ovarian cancer cell line NIH:OVCAR-3 8 × 10 purchased from ATCC 6 cell Matrigel (Becton, Dickinson and Company) The cells were suspended in 100 ml of 10 ... Random group assignment was performed. On Day 25, the mice were treated with antibody-drug conjugates (3), (4), and ( Antibody-drug conjugates (8) and (9) were administered intravenously at a dose of 3 mg / kg. Administration of (3), (4), (8), and (9) all exerted tumor growth inhibitory effects ( Figure 20).

[0279] 21-i) Antitumor effects of ADC (9) Human gastric cancer cell line NCI-N87, purchased from ATCC, 1 × 10 7 Cells were salinated The cells were suspended in saline and subcutaneously implanted into female BALB / c-nu / nu mice (Day 0) and then transferred to the BALB / c-nu / nu mice (Day 6). On Day 6, the mice were randomly assigned to the antibody-drug conjugates (3), (4), and ( Antibody-drug conjugates (8) and (9) were administered intravenously at a dose of 3 mg / kg. Administration of (3), (4), (8), and (9) all exerted tumor growth inhibitory effects ( Figure 21).

[0280] 21-j) Antitumor effects of ADC (10) 5 × 10 human lung cancer cell line NCI-H292 purchased from ATCC 6 Physiology of cells The cells were suspended in saline and subcutaneously implanted into female BALB / c-nu / nu mice (Day 0). Randomization was performed on Day 9. Antibody-drug conjugates (3), (4), (8) and (9) were both administered intravenously at a dose of 3 mg / kg. Administration of (3), (4), (8), and (9) all exerted tumor growth inhibitory effects. (Figure 22).

[0281] Antitumor effects of 21-kADC (11) 3 × 10 human pharyngeal carcinoma cell line FaDu purchased from ATCC 6 Cells in saline The cells were suspended in 1000 μg / mL and subcutaneously implanted into female BALB / c-nu / nu mice (Day 0). Random group assignment was performed. On day 11, the mice were treated with antibody-drug conjugates (3), (4), and ( Antibody-drug conjugates (8) and (9) were administered intravenously at a dose of 3 mg / kg. Administration of (3), (4), (8), and (9) all exerted tumor growth inhibitory effects ( Figure 23).

[0282] 21-l) Antitumor effects of ADC (12) 4 × 10 human pancreatic adenocarcinoma cell line CFPAC-1 purchased from ATCC 6 cells The cells were suspended in physiological saline and subcutaneously implanted into female BALB / c-nu / nu mice (Day 0). Random group allocation was performed on day 14. On day 14, antibody-drug conjugate (3), ( 4), (8), and (9) were all administered intravenously at a dose of 3 mg / kg. Administration of drugate (3), (4), (8), and (9) all exerted tumor growth inhibitory effects. This was demonstrated (Figure 24).

[0283] Antitumor effects of 21-m) ADC (13) In the same manner as in Example 21-1, CFPAC-1 was subcutaneously injected into female BALB / c-nu / nu mice. The mice were transplanted (Day 0) and randomly assigned to groups on Day 14. Conjugates (8) and (13) were both administered intravenously at a dose of 1 mg / kg. Administration of drug conjugates (8) and (13) both exerted tumor growth inhibitory effects. (Figure 25).

[0284] Antitumor effects of 21-n) ADC (14) 3 x 10 human pancreatic adenocarcinoma cell line HPAC purchased from ATCC 6 Cells were placed in physiological saline The cells were suspended in water and subcutaneously implanted into female BALB / c-nu / nu mice (Day 0) and then transplanted into mice (Day 12). On day 12, the mice were randomly assigned to the antibody-drug conjugates (8), (13) All were administered intravenously at a dose of 3 mg / kg. 3) Administration of both compounds exerted an inhibitory effect on tumor growth (Figure 26).

[0285] 21-o) Antitumor effects of ADC (15) Human esophageal cancer tissue obtained from the Japan Health Science Foundation was transfected into NOG mice. The tumor fragments were then subcutaneously transplanted and grown at the Central Institute for Experimental Animals (Public Interest Foundation). The tumor cells were subcutaneously implanted into female NOD-scid mice (Charles River Japan) (Day On Day 27, the subjects were randomly assigned to groups. (8) and (13) were both administered intravenously at a dose of 3 mg / kg. Administration of either (8) or (13) exhibited tumor growth inhibitory effects (Figure 27).

[0286] Example 22: Evaluation of anti-cellular effects of ADCs TROP2 antigen-positive cell lines BxPC3, NCI-H292, and NIH:OVCAR -3, CFPAC-1, FaDu, human lung adenocarcinoma cell line Calu-3 (ATCC), human egg CaOV3 (ATCC) tumor cell line and TROP2 antigen-negative human lung cancer cell line The human cutaneous melanoma cell line Calu-6 (ATCC), and the human cutaneous melanoma cell line A375 (ATCC) were used for each ADC. BxPC3 and NCI-H292 were used to evaluate the anti-cellular effect. RPMI 1640 Medium (Gibco) containing NIH: OVCAR-3 was supplemented with 20% fetal bovine serum and 0.01 mg / mL insulin (Inv CFPAC-1 was cultured in RPMI 1640 medium containing 10% urea. Iscove's Modified Dulbecco's Med with Fetal Cysteine ​​Serum ium (Gibco), and FaDu, Calu-3, and Calu-6 were prepared using 10% fetal bovine serum. Eagle's Minimum Essential Medium (ATC) containing C) CaOV3 and A375 were cultured in Dulbecco's medium containing 10% fetal bovine serum. Modified Eagle Medium (Gibco) and 2.2 x 10 6 cells / mL, and 90 μL of each was seeded into a 96-well cell culture microplate. Furthermore, in RPMI1640 Medium, the concentrations were 100nM, 20nM, 4nM, 0.8nM, Antibody-drug conjugate diluted to 0.16 nM, 0.032 nM, and 0.0064 nM Add 10 μL of (4), (8), or RPMI 1640 Medium for comparison. The microplate was then incubated at 37°C under 5% CO2 for 6 days. The plate was then removed from the oven and allowed to stand at room temperature for 30 minutes. lo Luminescent Cell Viability Assay(Prom Add ega) and mix with a plate mixer for 10 minutes to lyse the cells. The amount of light emitted was measured. The cell growth inhibition rate after 6 days of culture was calculated using the following formula. Cell proliferation inhibition rate (%) = a ÷ b × 100 a: Mean value of wells with sample added after 6 days of culture - Mean value of wells without sample added at the start of culture b: Mean value of wells with medium after 6 days of culture - Mean value of wells without sample at the start of culture Also, G.I. 50 The value was calculated using the following formula: G.I. 50 (nM)=antilog((50-f)×(LOG 10 (d)-LOG 10 ( c))÷(fe)+LOG 10 (d)) c: sample concentration c d: sample concentration d e: Cell proliferation inhibition rate at sample concentration c f: Cell proliferation inhibition rate at sample concentration d c and d are the two points sandwiching a cell growth inhibition rate of 50%, where c>d.

[0287] Antibody-drug conjugates (4) and (8) were administered to the TROP2 antigen-positive cell line Bx PC3, NCI-H292, NIH:OVCAR-3, CFPAC-1, FaDu, Ca For lu-3 and CaOV3, GI 50 showed anti-cellular effects of <1 nM. No anti-cellular effect was observed against the ROP2 antigen-negative cell lines Calu-6 and A375. (>100(nM)). [Sequence List Free Text]

[0288] SEQ ID NO: 1: Nucleotide sequence of the cDNA encoding the variable region of the heavy chain of the TINA1 antibody SEQ ID NO: 2: Amino acid sequence of the variable region of the heavy chain of the TINA1 antibody SEQ ID NO: 3: Nucleotide sequence of the cDNA encoding the variable region of the light chain of the TINA1 antibody SEQ ID NO: 4: Amino acid sequence of the variable region of the light chain of the TINA1 antibody SEQ ID NO: 5: Nucleotide sequence e...

Claims

1. After treatment of the anti-TROP2 antibody under reducing conditions, the following compounds: (maleimid-N-yl)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -C(=O)-(NH-DX) comprising the step of reacting The following drug-linker structure: -(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -C(=O)-(NH-DX) A method for producing an antibody-drug conjugate in which the above is bound to an anti-TROP2 antibody via a thioether bond. (Wherein, (maleimid-N-yl)- is represented by the following formula: 【Chemistry 1】 wherein the nitrogen atom is the binding site; -(Succinimid-3-yl-N)- is a compound of the formula: 【Chemistry 2】 The structure is represented by the formula: -(NH-DX) is a compound of the formula: 【Chemistry 3】 in which the nitrogen atom of the amino group at position 1 serves as the binding site, -GGFG- represents the tetrapeptide residue -Gly-Gly-Phe-Gly-.

2. The method according to claim 1, wherein the average number of drug-linker structures bound per antibody is in the range of 2 to 8.

3. The method according to claim 1, wherein the average number of drug-linker structures bound per antibody is in the range of 3 to 8.

4. The method for producing an anti-TROP2 antibody according to any one of claims 1 to 3, wherein the anti-TROP2 antibody comprises a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO:

28.

5. The anti-TROP2 antibody A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 12, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 18; A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 18; A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 20; and A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 16, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 in SEQ ID NO: 22; The method according to any one of claims 1 to 3, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of:

6. The anti-TROP2 antibody A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 12, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18; A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 18, A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 20; and A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 16, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 22; The method of any one of claims 1 to 3, comprising a heavy chain and a light chain selected from the group consisting of:

7. The method of claim 5, wherein the anti-TROP2 antibody comprises a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 140 of SEQ ID NO: 12 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 129 of SEQ ID NO:

18.

8. The method of claim 6, wherein the anti-TROP2 antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:

18.

9. The method according to claim 6, wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-TROP2 antibody is deleted.

10. The method according to claim 8, wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-TROP2 antibody is deleted.

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