Anti-HER3 antibody-drug conjugates

Abstract

To provide an antitumor drug having excellent therapeutic effect, which is excellent in terms of antitumor effect and safety.SOLUTION: Provided is an antibody-drug conjugate in which an antitumor compound, represented by the formula illustrated below, is conjugated to an anti-HER3 antibody via a linker having a structure represented by the formula: -L1-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O)- or -L1-L2-LP-.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a compound comprising an anti-HER3 antibody and an antitumor drug linked via a linker structure moiety. The present invention 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 (antibodies that bind to the antigens) The antibody-drug conjugate is a cytotoxic drug conjugate that binds to the antibody (which can also be internalized into cells). Antibody-drug conjugates (ADCs) can selectively deliver drugs to cancer cells. This is expected to allow the drug to accumulate in cancer cells and kill them (Non-Patent Document (See References 1 to 3.) For example, Mylotar, an anti-CD33 antibody conjugated with calicheamicin, is used as an ADC. g (registered trademark; gemtuzumab ozogamicin) was approved as a treatment for acute myeloid leukemia. In addition, Adcetris (registered trademark; Brenda) is an anti-CD30 antibody conjugated to auristatin E. ntuximab vedotin is the most effective treatment for Hodgkin's lymphoma and anaplastic large cell lymphoma Recently, ADCs have been approved (see Non-Patent Document 4). or targeting tubulin.

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

[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 this 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 accumulate in tumors, There are also reports that the accumulation in tissues is not significantly different, and passive targeting was not observed in humans. As a result, DE-310 was not launched on the market, and exatecan However, it is unclear whether it will function effectively as a drug aimed at such targeting. There wasn't.

[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 epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor protein HER1 (also known as EGFR, epidermal growth factor receptor) is a target tyrosine kinase ), HER2 and HER4 (see Non-Patent Documents 15 to 17), as well as receptor protein tyrosine It belongs to the EGFR subfamily of kinases. Like the classic epidermal growth factor receptor, it binds to the membrane The transmembrane receptor HER3 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, and It consists of a main domain, a transmembrane domain, and a carboxyl-terminal phosphorylation domain. In addition to the main domain, HER1, HER2, and HER4 have intracellular protein tyrosine kinase domains However, HER3 lacks this domain and does not have the ability to autophosphorylate. do not have. The ligand, heregulin (HRG), binds to the extracellular domain of HER3. binds to ATP and dimerizes with other human epidermal growth factor receptor (HER) family members and intracellular domains, activating receptor-mediated signaling pathways. Dimerization with HER family members enhances the signaling potential of HER3. This increases the signal strength and provides a means for signal amplification as well as signal diversification. The HER2 / HER3 heterodimer is the most important proliferation signaling molecule in the HER family. HER3 is involved in several types of cancer, including breast, gastrointestinal, and pancreatic cancers. Interestingly, HER2 / HER3 expression and the non-invasive stage A correlation has been shown between progression to the invasive stage (see Non-Patent Documents 18 to 20). Therefore, there is a demand for substances that inhibit HER3-mediated signaling. Immunoconjugates of the above have been reported in Patent Documents 5 to 10, etc. [Prior art documents] [Patent documents]

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

Patent document 4

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Patent document 10

Non-licensed literature

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

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Non-licensed literature 9

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[0011] 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]

[0012] The present inventors have demonstrated that anti-HER3 antibodies are antibodies that can target tumor cells, i.e., The ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be internalized by tumor cells, Or, since it is an antibody having cytocidal activity against tumor cells, it is an antitumor compound. An antibody-drug conjugate in which exatecan is bound to the antibody via a linker structure moiety. By converting the compound into a steroid, the antitumor compound can be transported more reliably to tumor cells, and the compound can be The antitumor effect of the compound can be specifically exerted on tumor cells, and therefore the antitumor effect It is expected that the efficacy of this drug will be enhanced and the cytotoxic effect of anti-HER3 antibodies will be enhanced. The dose of the antitumor compound can be reduced compared to when the compound is administered alone; In other words, these can mitigate the effects of antitumor compounds on normal cells. We believe that it is possible to achieve higher safety with this technology. For this purpose, the present inventors have created a linker with a specific structure, and have used this linker to bind to anti-HE. Successfully obtained an antibody-drug conjugate combining the R3 antibody and exatecan The present invention was completed based on the discovery that the conjugate exhibits excellent antitumor effects. That is why.

[0013] That is, the present invention is [1] The following formula [ka] and an anti-HER3 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- or -L 1 -L 2 -L P - The disulfide bond present in the hinge region of the anti-HER3 antibody is bonded via a linker having the structure shown in An antibody characterized by being bound by a thioether bond formed in the binding portion -Drug conjugates.

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

[0015] 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 or five amino acids [1] or [2] 1. The antibody-drug conjugate described herein. [5]L P is -GGFG- or -DGGFG-. Conjugates. [6]L P [4] The antibody-drug conjugate according to any one of [1] to [4], wherein to.

[0016] [7]n 3 is an integer from 2 to 5, and L 2 is a single bond. The antibody-drug conjugates described above. [8] The linker is -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- [1] to [7] The antibody-drug conjugate according to any one of the preceding claims. [9]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 [8], which is 2 or 4.

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

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

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

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

[12] ,

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

[15] L P The antibody-drug conjugate according to

[14] , wherein is -DGGFG-.

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

[15] , wherein Indugegate.

[0017]

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

[0018] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the 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- is a tetrapeptide residue of -Gly-Gly-Phe-Gly-, and -DGGFG- is a tetrapeptide residue of -Asp-Gly-Gly-Phe-Gly. - indicates the pentapeptide residue.

[0019]

[18] -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 antibody-drug linker according to [1], wherein the structural moiety is one drug-linker structure selected from the following group: Monoconjugates: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

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

[0021]

[19] The following formula: [ka] and an anti-HER3 antibody represented by the following formula: -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- The disulfide bond present in the hinge region of the anti-HER3 antibody is bonded via a linker having the structure shown in An antibody characterized by being bound via a thioether bond formed in the binding portion -Drug conjugates. Here, the anti-HER3 antibody is L 1The antitumor compound binds to the -(CH2)n 2 -C(=O) - binds to the carbonyl group of the 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.

[0022]

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

[0019] , wherein is -O-.

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

[19] or

[20] , wherein Drug conjugates.

[22] 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

[19] or

[20] , wherein R is 4.

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

[19] to

[22] ,

[0023]

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

[19] to

[23] , wherein the structural portion is a drug-linker structure selected from the following group: 1. The antibody-drug conjugate according to any one of claims 1 to 9, -(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-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX);

[0024] where -(Succinimid-3-yl-N)- is a compound of the formula: [ka] The structure is represented by the 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-.

[0025]

[25] -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 a drug-linker structure selected from the following group:

[19] to

[23] The antibody-drug conjugate of any one of the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX). Here, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- are as defined above.

[0026]

[26] 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

[25] , wherein the number of the antibody-drug conjugates ranges from 1 to 25.

[27] 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

[25] , wherein the range is

[28] 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

[25] , wherein the range is

[0027]

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

[28] , a salt thereof, or is a medicine containing such a hydrate.

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

[28] , a salt thereof, or are antitumor and / or anticancer drugs containing hydrates thereof.

[31] Lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, Breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, Squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer Bowel cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma or an antitumor drug and / or anticancer drug according to

[30] for application to penile cancer.

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

[28] , a salt thereof, or and a pharmaceutical composition containing the hydrate thereof as an active ingredient and a pharmaceutically acceptable formulation component. .

[33] Lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, Breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, Squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer Bowel cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma or the pharmaceutical composition according to

[32] for use in treating penile cancer.

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

[28] , a salt thereof, or and a method for treating tumors and / or cancers, which comprises administering a hydrate thereof.

[35] The pharmaceutical composition according to

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

[30] or

[0031] , or the pharmaceutical composition according to

[32] or

[33] . Or the treatment method described in

[34] .

[36] The pharmaceutical composition according to

[32] or

[33] , which also contains other drugs as active ingredients. .

[0028]

[35] A compound represented by the formula: (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) or (male imid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -(NH-DX) is reacted with an anti-HER3 antibody or a reactive derivative thereof to form a disulfide bond at the hinge region of the antibody. The drug-linker moiety is formed by forming a thioether bond at the drug-linking moiety. and binding the antibody to the drug conjugate.

[0029] In the formula, n 3 denotes the integers 2 to 8, L 2 is -NH-(CH2CH2-O)n 4 represents —CH2CH2—C(═O)— or a single bond, where n 4 represents an integer from 1 to 6, L P 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 group having the following formula: [ka] The nitrogen atom is the bonding site. -(NH-DX) is represented by the following formula: [ka] This is a group in which the nitrogen atom of the amino group at position 1 is the binding site, as shown in

[0030]

[36] A method for binding a drug-linker moiety to an anti-HER3 antibody includes reducing the antibody.

[35] , wherein the compound is converted into a reactive derivative.

[0031]

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

[35] or

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

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

[35] or

[36] , wherein the range is

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

[35] or

[36] , wherein the range is

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

[35] to

[39] ugate.

[0032]

[41] After treating the anti-HER3 antibody under reducing conditions, a compound selected from the following compound group is added: The antibody is reacted with a thioester at the disulfide bond in the hinge region. Antibody-drug conjugates obtained by forming ether bonds: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C( =O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-C H2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2 -C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O- CH2-C(=O)-(NH-DX) (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGF G-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

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

[0034]

[42] After treating the anti-HER3 antibody under reducing conditions, a compound selected from the following compound group is added: The antibody is reacted with a thioester at the disulfide bond in the hinge region. Antibody-drug conjugates obtained by forming ether bonds: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX). Here, (maleimid-N-yl)-, -(NH-DX), and -GGFG- are as defined above.

[0035]

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

[41] or

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

[41] or

[42] .

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

[41] or

[42] , wherein the range is

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

[41] or

[42] , wherein the range is

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

[40] to

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

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

[40] to

[45] , or a salt thereof. or an antitumor drug and / or an anticancer drug containing a hydrate thereof.

[48] ​​Lung cancer, kidney cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, Breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, Squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer Bowel cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma or an antitumor drug and / or anticancer drug according to

[47] for application to penile cancer.

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

[40] to

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

[50] Lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, Breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, Squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer Bowel cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma or the pharmaceutical composition according to

[49] for use in treating penile cancer.

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

[40] to

[45] , or a salt thereof. A method for treating tumors and / or cancers, comprising administering the compound or a hydrate thereof.

[52] The pharmaceutical composition according to

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

[47] or

[0048] , or the pharmaceutical composition according to

[49] or

[50] . Or the treatment method described in

[51] .

[53] The pharmaceutical composition according to

[49] or

[50] , which also contains other drugs as active ingredients. . [Effects of the Invention]

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

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

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

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

[0040] The anti-HER3 antibody-drug conjugate of the present invention comprises an anti-HER3 antibody and a linker structure portion. These are antitumor drugs in which an antitumor compound is bound via a molecule, and will be described in detail below. Within the context of the present invention, the term "scaffold protein" as used herein means a polypeptides or polypeptides with exposed surfaces that are highly tolerant of amino acid insertions, substitutions, or deletions; means a protein. Examples of scaffold proteins that can be used in accordance with the present invention are: From Staphylococcus aureus Protein A obtained from Pieris brassicae ) bilin-binding proteins or other lipocalins, ankyrin repeat proteins obtained from and human fibronectin (Binz and Pluckthun, Curr (Reviewed in Opin Biotechnol. 16, 459-69.) Protein engineering involves engineering the structural framework of a stably folded protein or This can be seen as porting or embedding affinity functionality into a general framework. Compatibility function means protein binding affinity according to the present invention. The scaffold provides binding specificity. Generally, such artificial affinity reagents are structurally separable from the amino acid sequence they provide. Proteins that appear suitable for development are identified by inference, or most commonly HER3. (either purified proteins or proteins displayed on the cell surface) In vitro displayed artificial scaffold library binders, such as panning, Combinatorial protein engineering techniques for the synthesis of ribozymes (these techniques are known in the art) (Skerra, J.Mol.Recog., 2000;Binz and Pluc Furthermore, antibodies with antibody-like binding activity can be obtained by the method of The scaffold protein can be derived from an acceptor polypeptide containing a scaffold domain. The binding characteristics of the donor polypeptide are then determined on a scaffold domain containing the acceptor polypeptide. To confer isomerism, the acceptor polypeptide must contain the binding domain of the donor polypeptide. The inserted binding domain can be, for example, an antibody, in particular an anti- The insertion may be in the complementarity determining region (CDR) of a HER3 antibody. Various forms of recombinant methods, such as polypeptide synthesis and nucleic acid synthesis of encoded amino acids, This can be achieved by a variety of methods known to those skilled in the art.

[0041] [antibody] Furthermore, as used herein, the term "antibody" or "anti-HER3 antibody" refers to Monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies (Jones et al. al., Nature 321(1986),522-525;Riechmann e t al., Nature 332(1988), 323-329; and Presta ,Curr.Op.Struct.Biol.2(1992),593-596), La antibody (Morrison et al., Proc. Natl. Acad. Sci. U. SA81(1984),6851-6855), human antibodies and fully human antibodies, (Tom izuka, K. et al., Nature Genetics (1997) 16, p. .133-143,;Kuroiwa,Y.et.al.,Nucl.Acids Re s.(1998)26,p.3447-3448;Yoshida,H.et.al., Animal Cell Technology:Basic and Applied Aspects vol.10, p.69-73(Kitagawa,Y.,Mats uda, T. and Iijima, S. eds.), Kluwer Academic Publishers,1999.;Tomizuka,K.et.al.,Proc. Natl.Acad.Sci.USA(2000)97,pp.722-727, International and multispecific antibodies formed from at least two antibodies. The term "antibody fragment" refers to an antibody (e.g., a bispecific antibody) or an antibody fragment thereof. The term includes any part of said antibodies, preferably the antigen binding or variable regions thereof. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, and the like. Hollinger et al., Proc.Natl.Acad.Sci.U .SA90(1993), 6444-6448), single chain antibody molecules (Pluckth un in:The Pharmacology of Monoclonal Antib odies 113, Rosenburg and Moore, EDS, Spring er Verlag, NY (1994), 269-315) and the desired targeting of HER3 Other fragments are included as long as they exhibit binding ability.

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

[0043] In the present invention, the term "Fv fragment" refers to the smallest antibody fragment that contains the complete antigen-recognition and binding site. These regions consist of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. The three CDRs of each variable domain are H -V L Dimer It is in this configuration that they interact to define antigen-binding sites on the surface. Together, the six CDRs confer antigen-binding specificity to the antibody. Even the main (or half of an Fv containing only three CDRs specific for an antigen) is generally , which has the ability to recognize and bind antigen, although with lower affinity than the complete binding site. The "Fab fragment" also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. "Fab fragments" contain an antibody hinge region-derived fragment at the carboxy terminus of the heavy chain CH1 domain. The fragment is called a "Fab" fragment in that it has several additional residues, including one or more cysteines. The "F(ab')2 fragment" is originally an "F(ab')2 fragment" with hinge cysteines between them. Such antibody fragments are produced as pairs of "ab' fragments." Methods for preparation are known to those skilled in the art.

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

[0045] The anti-HER3 antibody used in the anti-HER3 antibody-drug conjugate of the present invention is either The cells may be derived from any species, but preferably from humans, rats, mice, and rabbits. If derived from a species other than human, it 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 antibodies may be used, monoclonal antibodies are preferred. Anti-HER3 antibodies are antibodies that can target tumor cells, i.e., they can recognize tumor cells. The ability to bind to tumor cells, the ability to be internalized by tumor cells, and the ability to The compound has antitumor activity, such as cytotoxicity against tumor cells, and is attached via a linker. and conjugated to form an antibody-drug conjugate. The binding of the antibody to tumor cells can be confirmed using flow cytometry. Antibody uptake into the cell can be achieved by (1) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody. Cell Death and Differentiation Assay (Cell Death and Differentiation Assay) on (2008) 15, 751-761), (2) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody to detect the cells. Assay to measure the amount of fluorescence taken up into the vesicles (Molecular Biology of the Cell Vol. 1 5, 5268-5282, December 2004), or (3) using immunotoxins that bind to therapeutic antibodies. When taken up into cells, Mab-ZA releases toxins that inhibit cell proliferation. P assay (BioTechniques 28:162-165, January 2000). As an immunotoxin, the catalytic domain of diphtheria toxin A recombinant conjugate protein of the region and protein G can also be used. The antitumor activity of an antibody can be confirmed in vitro by measuring its inhibitory activity against cell proliferation. For example, a cancer cell line that overexpresses the antibody target protein is cultured, and various concentrations of the antibody are added to the culture system. The antibodies were added at various concentrations to inhibit focus formation, colony formation, and spheroid growth. In vivo, for example, tumor cells that highly express the target protein can be measured. The antibody was administered to nude mice transplanted with the cell line, and changes in the cancer (tumor) cells were measured. Therefore, antitumor activity can be confirmed. 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.

[0046] The anti-HER3 antibody can be prepared by isolating the antigenic polypeptide using a method commonly used in the art. It can be obtained by immunizing an animal with the antibody and collecting and purifying the antibodies produced in the body. The origin of the antigen is not limited to humans, but can also be antigens derived from animals other than humans, such as mice and rats. In this case, the obtained antibodies that bind to the heterologous antigen and the human By testing cross-reactivity with human antigens, antibodies applicable to human diseases can be selected. 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. The anti-HER3 antibody can be obtained by known means.

[0047] The anti-HER3 antibody that can be used in the present invention is not particularly limited, but for example, it may have the following properties: It is desirable to have something like this. (1) An anti-HER3 antibody characterized by the following properties: (a) Binds specifically to HER3. (b) It has the activity of being internalized into HER3-expressing cells by binding to HER3. (2) The antibody described in (1) above that binds to the extracellular domain of HER3. (3) The antibody according to (1) or (2) above, which is a monoclonal antibody. (4) Antibody-dependent cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity The antibody according to any one of (1) to (3) above, having the following properties: (5) Mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies Any of the above (1) to (4) which is a human or fully human (monoclonal) antibody. The antibody described in any one of the above. (6) A heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2 Any of the above (1) to (5) which is a humanized monoclonal antibody comprising a light chain consisting of The antibody according to any one of the preceding claims. (7) Any of the above (1) to (6) in which the lysine residue at the carboxyl terminal of the heavy chain is deleted. The antibody described above. (8) A heavy chain variable region represented by the amino acid sequence set forth in SEQ ID NO: 70 and a heavy chain variable region represented by the amino acid sequence set forth in SEQ ID NO: 72 The antibody according to (7) above, comprising a light chain variable region represented by the amino acid sequence listed below. (9) A method for producing a medicament comprising the steps of: (1) detecting a medicament containing a polynucleotide encoding the antibody according to any one of (1) to (8); a step of culturing a host cell transformed with an expression vector having the gene, and and a method for producing the antibody, the method comprising the step of collecting the antibody from the culture. body.

[0048] The anti-HER3 antibody used in the present invention will be described below. In this specification, the terms "cancer" and "tumor" are used interchangeably. As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA, and 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, "HER3" is used synonymously with HER3 protein. As used herein, "CDR" refers to a complementarity determining region (CDR). The heavy and light chains of an antibody molecule Each chain is known to have three CDRs. The variable domains of the heavy and light chains of an antibody are also called The primary structure of the heavy and light polypeptide chains is particularly variable. In the present specification, the CDRs of an antibody are separated into three regions. The heavy chain CDRs are CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence. The light chain CDRs are represented as CDRL1, CDRH2, and CDRH3, and the light chain CDRs are represented as CDRL1, CDRH3, and CDRH4 from the amino-terminal end of the light chain amino acid sequence. These sites are located close to each other in the three-dimensional structure and are bonded together. The specificity for the antigen to which it binds is determined. 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.

[0049] 1.HER3 HER3 is human epidermal growth factor receptor 3, also known as HER3 or ErbB3, and is a receptor protein It is a protein tyrosine kinase that, along with HER1, HER2, and HER4, binds to the receptor protein tyrosine kinase. HER3 belongs to the epidermal growth factor receptor subfamily of HER3 kinases. It consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, and a transmembrane domain. domain, intracellular protein tyrosine kinase domain (TKD) and carboxyl-terminal phosphorylase Contains an oxidative domain. HER3 is involved in several types of cancer, including breast, gastrointestinal, and pancreatic cancers. HER2-HER3 expression and progression from non-invasive to invasive stages There is a correlation between The HER3 protein used in the present invention is a HER3 protein derived from humans and non-human mammals (rats, mice, etc.). It can be directly purified from R3-expressing cells or prepared as a cell membrane fraction of the cells. HER3 can be synthesized in vitro or expressed in host cells by genetic engineering. Specifically, genetic manipulation can be used to produce HER3. After inserting the cDNA into an expression vector, the enzymes, substrates, and enzymes required for transcription and translation are introduced. They are synthesized in solutions containing energy substances or in other prokaryotic or eukaryotic host cells. The protein can be obtained by expressing HER3 through transformation. In addition, the HER3-expressing cells or cells expressing HER3 obtained by the above-mentioned genetic manipulation can be used. It is also possible to use a cell line as the HER3 protein. The RNA sequence, cDNA sequence, and amino acid sequence of HER3 are publicly available in the database. For example, AAA35979 (a signal sequence consisting of 19 amino acid residues at the amino terminal) These can be referenced by accession numbers such as M34309 (NCBI) and M34309 (precursor containing M34309). do. In addition, in the amino acid sequence of the HER3, one or several amino acids are substituted, deleted, or added. A protein consisting of an added and / or inserted amino acid sequence and having biological activity equivalent to that of the protein. White matter is also included in HER3.

[0050] 2. Preparation of anti-HER3 antibodies The antibody against HER3 of the present invention can be prepared by a method commonly used in the art. or by immunizing an animal with any polypeptide selected from the amino acid sequence of HER3, It can be obtained by collecting and purifying antibodies produced by the HER3 antigen. The biological species is not limited to humans, but also includes HER3 derived from non-human animals such as mice and rats. In this case, the obtained antibody that binds to heterologous HER3 can be used. Antibodies applicable to human diseases can be selected by testing cross-reactivity with human HER3. Cut. 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 HER3 and myelin Hybridomas are established by fusing them with human cells to produce monoclonal antibodies. It is also possible. The antigen, HER3, is expressed in host cells through genetic manipulation of the HER3 gene. This can be obtained by Specifically, a vector capable of expressing the HER3 gene was prepared and introduced into host cells. The gene is expressed using the vector, and the expressed HER3 is then purified. In addition, the above-mentioned genetically engineered HER3-expressing cells or cell lines expressing HER3 It is also possible to use the HER3 protein as the HER3 protein. Explain how to obtain the body.

[0051] (1) Antigen preparation The antigen for producing anti-HER3 antibodies is HER3 or at least six consecutive HER3 fragments. Polypeptides consisting of the partial amino acid sequence, or any amino acid sequence or carrier added to these. Examples of such derivatives include those with additions. HER3 can be directly purified from human tumor tissues or tumor cells and used. HER3 can be synthesized in vitro or produced in host cells by genetic engineering. can be obtained by Specifically, the genetic engineering involved inserting HER3 cDNA into an expression vector. Then, they are synthesized in a solution containing the enzymes, substrates, and energy sources necessary for transcription and translation, or by transforming a prokaryotic or eukaryotic host cell of the present invention to express HER3, The antigen can be obtained. In addition, a fusion protein that links the extracellular domain of the membrane protein HER3 with the constant domain of an antibody. The antigen can be obtained as a secreted protein by expressing it in an appropriate host-vector system. It is also possible to HER3 cDNA can be obtained, for example, from a cDNA library expressing HER3 cDNA. Using the HER3 cDNA as a template, a polymerase chain reaction was carried out using primers that specifically amplify HER3 cDNA. PCR (hereinafter referred to as "PCR"; Saiki, RK, et al., Science nce (1988) 239, pp. 487-489) You can get it by doing this. In vitro synthesis of polypeptides is achieved using, for example, Roche Diagnostics. One example is the Rapid Translation System (RTS) manufactured by Tix. However, this is not limited to this. 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 a method commonly used in this field. The culture allows the production of the desired polypeptide either intracellularly or extracellularly. The medium used for the culture may be any of various commonly used media depending on the host cells used. In the case of E. coli, for example, an LB medium containing an antibiotic such as ampicillin may be used. It can be used by adding biomaterials or IPMG. 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 the antigen as an antigen.

[0052] (2) Production of anti-HER3 monoclonal antibodies As an example of an antibody that specifically binds to HER3, a monoclonal antibody that specifically binds to HER3 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 draw 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.

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

[0054] (b) Preparation of antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant, or 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 HER3 or a recombinant form thereof, see, for example, Weir, D.M. Handbook of Experimental Immunology Vol. I.II.III.,Blackwell Scientific Publicati ons,Oxford(1987);Kabat,EAand Mayer,MM .,Experimental Immunochemistry,Charles C. Thomas Publisher Springfield,Illinois(1 964) 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. The surface was covered with BSA, and after washing, a stepwise dilution of the first antibody was applied. The antigen is then contacted with a purified sample (eg, 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.

[0055] (c) Preparation of myeloma cells (hereinafter referred to as "myeloma"). There are no particular limitations on the myeloma cells used for cell fusion, and 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 glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereafter referred to as a medium supplemented with 8-azaguanine (hereinafter referred to as "FBS"), Iscove's modified Darmstadt medium, Dulbecco's Medium (Iscove's Modified Dulbecco's Medium) m; IMDM), or Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium The cells are subcultured in DMEM (Diluted Eagle Medium; hereafter referred to as "DMEM"). Three to four days before fusion, the cells were cultured in normal medium (e.g., ASF104 medium containing 10% FCS ( On the day of fusion, 2 × 10 7 Ensure that the above number of cells is maintained.

[0056] (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. Such a method involves, for example, preparing antibodies in a highly concentrated polymer solution such as polyethylene glycol. Chemical methods that mix living cells with myeloma cells, and physical methods that use electrical stimulation, etc. Among these, specific examples of the chemical method 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.

[0057] (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.

[0058] (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 designated as anti-HER3 monoclonal antibodies. A clonal antibody-producing hybridoma line is selected.

[0059] (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 the formation of 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, D.M.: 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 HER3.

[0060] (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 more convenient 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 This is done by calculating OD280 (immunoglobulin 1 mg / mL). This 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 an anti-HER3 antibody. An example of such an antibody is an antibody having the same epitope as the anti-HER3 antibody. Newly produced monoclonal antibodies can be used to bind to the If the monoclonal antibody binds to the partial peptide or partial three-dimensional structure to which the HER3 antibody binds, It can be determined that the antibody binds to the same epitope as the anti-HER3 antibody. The monoclonal antibody competes with the HER3 antibody for binding to HER3 (i.e., (2) Confirm that the monoclonal antibody inhibits the binding of the anti-HER3 antibody to HER3. By this, even if the sequence or structure of the specific epitope has not been determined, the monoclonal antibody It can be determined that the native antibody binds to the same epitope as the anti-HER3 antibody. If the same topope is confirmed, the monoclonal antibody is considered to be the same as the anti-HER3 antibody. It is strongly expected that the antibody will have antigen-binding ability or biological activity such as those listed above.

[0061] (3) Other antibodies The antibodies of the present invention include monoclonal antibodies against HER3 as well as allogeneic antibodies against humans. Recombinant antibodies that have been artificially modified to reduce species antigenicity, 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)). As for humanized antibodies, the complementarity determining regions (CDRs) An antibody in which only the terminating region is incorporated into a human-derived antibody (Nat (See ure (1986) 321, pp. 522-525), and by CDR grafting method, In addition to the sequence of the antibody, some of the framework amino acid residues were also transplanted into a human antibody (International Publication No. Patent No. 90 / 07861). In this specification, "several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. Taste.

[0062] In the present invention, the amino acid sequence of the binding protein of the present invention is composed of 20 common amino acids. It should be understood that the present invention is not limited to the above. s(2 nd Edition, ESGolub and DRGren, Eds. , Sinauer Associates, Sunderland, Mass.(19 91) (hereby incorporated by reference). For example, amino The acids include stereoisomers of the 20 common amino acids (e.g., D-amino acids), α,α-disubstituted unnatural amino acids such as substituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids Unconventional amino acids may also be suitable components for the binding proteins of the present invention. Examples of acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N- Trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methyl arginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline ) is included.

[0063] 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 and uncharged polar family = glycine, asparagine, glutamine, cysteine Other suitable amino acid groups are: Aliphatic hydroxy groups = serine and threonine; amide-containing groups = asparagine and glutamine; aliphatic group = alanine, valine, leucine and isoleucine; and Aromatic groups = phenylalanine, tryptophan and tyrosine. It is preferable that the substitution is carried out within a range that does not impair the properties of the substance having the original amino acid sequence. In the heavy and light chains of the antibody, if the N-terminal amino acid is glutamic acid, is sometimes cyclized (to form pyroglutamine). In the present invention, glutamine is not distinguishable from normal glutamine in terms of amino acid sequence. In the heavy and light chains of the antibody of the present invention, cysteines are cysteinylated. Such cysteinylated products are also included in the present invention. , and is not distinguishable from normal cysteine ​​in the amino acid sequence.

[0064] The antibodies of the present invention further include human antibodies that bind to HER3. Anti-HER3 human antibodies are human antibodies that contain only the gene sequence of antibodies derived from human chromosomes. The anti-HER3 human antibody is a human chromosome fragment containing the genes for the heavy and light chains of a human antibody. A method using human antibody-producing mice (Tomizuka, K. et al., Na ture Genetics(1997)16,p.133-143;Kuroiwa, Y.et.al.,Nucl.Acids Res.(1998)26,p.3447- 3448;Yoshida,H.et.al.,Animal Cell Techno logy:Basic and Applied Aspects vol.10, p. 69-73(Kitagawa, Y., Matsuda, T. and Iijima, S .eds.), Kluwer Academic Publishers, 1999;T omizuka,K.et.al.,Proc.Natl.Acad.Sci.USA( 2000)97, pp.722-727, etc.)

[0065] 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. Human antibody production is described in detail in WO 2007 / 077028. The contents of WO 2007 / 077028 form part of the disclosure of the present invention.

[0066] 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).

[0067] One aspect of the present invention relates to a protein that binds to HER3. The isolated HER3 binding proteins of the present invention are those selected from the group consisting of (a) SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 6 6, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 11 2, 116, 120, 122, 126, 130, 134, 138, 142, 146, 15 0, 154, 158, 162, 166, 170, 174, 178, 182, 186, 19 0, 194, 198, 202, 206, 210, 214, 218, 222, 226 or 2 (b) CDHR1 contained in the amino acid sequence shown in SEQ ID NOs: 2, 6, 10, and 14; , 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 1 12, 116, 120, 122, 126, 130, 134, 138, 142, 146, 1 50, 154, 158, 162, 166, 170, 174, 178, 182, 186, 1 90, 194, 198, 202, 206, 210, 214, 218, 222, 226 or 230; and (c) a CDRH2 comprising the amino acid sequence set forth in SEQ ID NOs: 2, 6, 10. , 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 10 8, 112, 116, 120, 122, 126, 130, 134, 138, 142, 14 6, 150, 154, 158, 162, 166, 170, 174, 178, 182, 18 6, 190, 194, 198, 202, 206, 210, 214, 218, 222, 22 6 or 230, and a heavy chain amino acid sequence containing CDRH3 included in the amino acid sequence shown in and (d) SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, (e) CDRL1 contained in the amino acid sequence shown in SEQ ID NO: 4, 8 , 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 or 232 (f) a CDRL2 comprised in an amino acid sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 8 2, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 12 8, 132, 136, 140, 144, 148, 152, 156, 160, 164, 16 8, 172, 176, 180, 184, 188, 192, 196, 200, 204, 20 8, 212, 216, 220, 224, 228 or 232 The amino acid sequence of the light chain includes CDRL3.

[0068] The isolated HER3 binding protein of the present invention preferably comprises (a) a sequence selected from the group consisting of SEQ ID NOs: 236, 251, A CDRH comprising an amino acid sequence selected from the group consisting of 252 and 256. (b) one selected from the group consisting of SEQ ID NOs: 258, 278, 280, and 282; (c) a CDRH2 comprising the amino acid sequence shown; and (d) SEQ ID NOs: 283, 285, 309, A CDRH comprising an amino acid sequence selected from the group consisting of 313 and 315. 3, and (d) a heavy chain amino acid sequence comprising SEQ ID NOs: 320, 334, 337, and 340. (e) a CDRL1 comprising an amino acid sequence selected from the group consisting of: an amino acid sequence selected from the group consisting of 343, 356, 351, and 344; and (f) the group consisting of SEQ ID NOs: 360, 381, 385, and 387. A light chain amino acid sequence comprising a CDRL3 containing an amino acid sequence selected from the group consisting of include.

[0069] In another aspect of the invention, the isolated HER3 binding protein of the invention is 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, a heavy chain variable region amino acid sequence selected from the group consisting of 222, 226 and 230, and / or or SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106 , 110, 114, 118, 124, 128, 132, 136, 140, 144, 148 , 152, 156, 160, 164, 168, 172, 176, 180, 184, 188 , 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232.

[0070] In yet another aspect of the invention, the isolated HER3 binding protein of the invention is preferably Preferably, SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, 2 2 and 24, 26 and 28, 30 and 32, 36 and 38, 42 and 44, 46 and 48 , 50 and 52, 54 and 56, 60 and 58, 62 and 64, 66 and 68, 70 and 72, 74 and 76, 78 and 82, 80 and 82, 84 and 86, 88 and 90, 92 and 94, 96 and 98, 100 and 102, 104 and 106, 108 and 110, 1 12 and 114, 116 and 118, 122 and 124, 126 and 128, 130 and 132, 134 and 136, 138 and 140, 142 and 144, 146 and 148, 150 and 152, 154 and 156, 158 and 160, 162 and 164, 166 and and 168, 170 and 172, 174 and 176, 178 and 180, 182 and 184 , 186 and 188, 190 and 192, 194 and 196, 198 and 200, 202 and 204, 206 and 208, 210 and 212, 214 and 216, 218 and 22 0, 222 and 224, 226 and 228, or 230 and 232 The amino acid sequence of the light chain variable region and the amino acid sequence of the light chain variable region, or SEQ ID NOs: 34, 40, 6 0, 62 or 120 and the heavy chain variable region amino acid sequence shown in SEQ ID NO: 58 or The light chain variable region amino acid sequence shown in Table 64 is also included. The isolated HER3 binding protein of the present invention is more preferably selected from the group consisting of SEQ ID NOs: 42, 54, 70, 92 or 96, and the heavy chain variable region amino acid sequence represented by SEQ ID NO: 44, 56, 72, 94 or 98.

[0071] Heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in SEQ ID NOs: 2 and 4 The antibody comprising the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 6 and 8 is designated "U1-39." The antibody comprising the amino acid sequences of the light chain variable region and the light chain variable region is designated "U1-40" and is designated as SEQ ID NOs: 10 and 12. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is referred to as "U1-38 ", the heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 14 and 16. The antibody containing the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 18 and 20 is designated "U1-41." The antibody comprising the amino acid sequence of SEQ ID NO: 22 and the light chain variable region amino acid sequence is designated "U1-42." The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in 24 is referred to as " U1-43”, the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 26 and 28, and the light chain variable region amino acid sequence shown in SEQ ID NOs: 27 and 28. The antibody containing the amino acid sequence of the heavy chain shown in SEQ ID NOs: 30 and 32 is designated "U1-44." The antibody containing the variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-45," and SEQ ID NO: The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in Nos. 36 and 38 are included. The antibody containing the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 42 and 44 was designated "U1-47." The antibody comprising the amino acid sequences of the light chain variable region and the light chain variable region is designated "U1-49" and is shown in SEQ ID NOs: 46 and 48. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is referred to as "U1-50 ", the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 50 and 52. The antibody containing the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 54 and 56 is designated "U1-51." The antibody comprising the amino acid sequence of SEQ ID NO: 60 and the light chain variable region amino acid sequence of SEQ ID NO: 61 is designated "U1-53." 58 is an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in U1-55”, the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 62 and 64, and the light chain variable region amino acid sequence shown in SEQ ID NOs: 63 and 64. The antibody containing the heavy chain amino acid sequence shown in SEQ ID NOs: 66 and 68 is designated "U1-57." The antibody containing the variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-58" and SEQ ID NO: The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in Nos. 70 and 72 are included. The antibody containing the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 74 and 76 is designated "U1-59." The antibody comprising the amino acid sequences of the light chain variable region is designated "U1-52" and is shown in SEQ ID NOs: 78 and 82. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is referred to as "U1-61 ", the heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 80 and 82. The antibody containing the heavy chain variable region shown in SEQ ID NOs: 84 and 86 is designated "U1-61.1." The antibody containing the amino acid sequence and the light chain variable region amino acid sequence is designated "U1-62" and SEQ ID NO: 88 and an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in The heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 92 and 94 are designated "U1-2" and "U1-3", respectively. The antibody containing the variable region amino acid sequence is designated "U1-7" and the heavy chain variable region sequences shown in SEQ ID NOs: 96 and 98 are designated "U1-8". The antibody comprising the variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-9" and SEQ ID NO: The heavy chain variable region amino acid sequences shown in 100 and 102 are The antibody "U1-10" is a heavy chain variable region amino acid sequence shown in SEQ ID NOs: 104 and 106. The antibody comprising the amino acid sequence of the light chain variable region is designated "U1-12", SEQ ID NO: 108 and 1 The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in 10 is referred to as " U1-13”, the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 112 and 114, and the light chain The antibody containing the variable region amino acid sequences is designated "U1-14" and is shown in SEQ ID NOs: 116 and 118. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-15." The heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 122 and 124 are The antibody containing the heavy chain variable region shown in SEQ ID NOs: 126 and 128 is designated "U1-20." The antibody comprising the amino acid sequence of the light chain variable region and the amino acid sequence of the light chain variable region is designated "U1-21" and SEQ ID NO: 1 30 and 132, and the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in The antibody containing the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 134 and 136 is designated "U1-22." The antibody comprising the amino acid sequences of the light chain variable region and the light chain variable region is designated "U1-23" and SEQ ID NOs: 138 and 14 The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in Table 1 is referred to as "U 1-24", the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 142 and 144, and the light chain variable region amino acid sequences shown in SEQ ID NOs: 143 and 144. The antibody containing the variable region amino acid sequence is designated "U1-25" and is shown in SEQ ID NOs: 146 and 148. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-26"; The heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 150 and 152 are The antibody containing the heavy chain variable region shown in SEQ ID NOs: 154 and 156 is designated "U1-27." The antibody containing the amino acid sequence and the light chain variable region amino acid sequence is designated "U1-28" and SEQ ID NO: 15 8 and 160, containing the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in The antibody was designated "U1-31," and the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 162 and 164 were used. and an antibody comprising the light chain variable region amino acid sequences "U1-32" and SEQ ID NOs: 166 and 168 The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in -35", the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 170 and 172, and the light chain variable region amino acid sequences shown in SEQ ID NOs: 171 and 172 The antibody containing the amino acid sequence of the region is designated "U1-36" and the heavy chain ... The antibody comprising the amino acid sequence of the chain variable region and the amino acid sequence of the light chain variable region is designated "U1-37." The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in columns 178 and 180 The antibody comprising the heavy chain variable region sequences shown in SEQ ID NOs: 182 and 184 is designated "U1-34." The antibody comprising the amino acid sequence of SEQ ID NO: 186 and the light chain variable region amino acid sequence is designated "U1-1", SEQ ID NO: 186 and and 188, an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence. The heavy chain variable region amino acid sequences shown in SEQ ID NOs: 190 and 192 are designated "U1-3" and "U1-4", respectively. The antibody containing the amino acid sequences of the chain variable regions is designated "U1-4" and is shown in SEQ ID NOs: 194 and 196. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-5." The heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 198 and 200 are The antibody containing the heavy chain variable region sequences shown in SEQ ID NOs: 202 and 204 is designated "U1-6." The antibody comprising the amino acid sequence of SEQ ID NO: 206 and the light chain variable region amino acid sequence of SEQ ID NO: 207 is designated "U1-8." and 208, an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence. "U1-11", the heavy chain variable region amino acid sequences shown in SEQ ID NOs: 210 and 212, and The antibody containing the light chain variable region amino acid sequence is designated "U1-16" and is shown in SEQ ID NOs: 214 and 216. The antibody containing the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is referred to as "U1-1 7," the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 218 and 220, and the light chain variable region The antibody having the amino acid sequence shown in SEQ ID NOs: 222 and 224 is designated "U1-18." The antibody containing the variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-33" and SEQ ID NO: The heavy chain variable region amino acid sequences and light chain variable region amino acid sequences shown in Nos. 226 and 228 The antibody comprising the heavy chain variable region amino acids shown in SEQ ID NOs: 230 and 232 is designated "U1-29." The antibody comprising the amino acid sequence and the light chain variable region amino acid sequence is designated "U1-30" and is shown in SEQ ID NO: 34. The antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 40 is designated "U1-46." The antibody containing the amino acid sequence of the chain variable region is designated "U1-48" and is shown in SEQ ID NOs: 60 and 58. The antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence is designated "U1-55.1 ", and an antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 120 is referred to as "U1-19 ", the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 62 and 64. The antibody containing the amino acid sequence is called "U1-57.1." These antibodies are described in detail in the Examples. It is written. The isolated HER3 binding protein of the present invention is even more preferably selected from the group consisting of SEQ ID NO: 42 and 44, the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NO: 54 and 56, the heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs. The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in Tables 70 and 72, respectively, The heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in numbers 92 and 94, Alternatively, the heavy chain variable region amino acid sequences and the light chain variable region amino acid sequences shown in SEQ ID NOs: 96 and 98 may be used. and even more preferably, such HER3 binding proteins comprise an anti-HER3 The antibody is U1-49, U1-53, U1-59, U1-7 or U1-9.

[0072] [ka] JPEG0007629500000015.jpg228139JPEG0007629500000016.jpg229147JPEG0007629500000017.jpg228141JPEG00076295000 00018.jpg229140JPEG0007629500000019.jpg228140JPEG0007629500000020.jpg227143JPEG0007629500000021.jpg227144 JPEG0007629500000022.jpg228140JPEG0007629500000023.jpg229142JPEG0007629500000024.jpg228140JPEG00076295000 00025.jpg228146JPEG0007629500000026.jpg227145JPEG0007629500000027.jpg227147JPEG0007629500000028.jpg228149 JPEG0007629500000029.jpg228142JPEG0007629500000030.jpg226145JPEG0007629500000031.jpg228150JPEG00076295000 00032.jpg228143JPEG0007629500000033.jpg227147JPEG0007629500000034.jpg228150JPEG0007629500000035.jpg227148 JPEG0007629500000036.jpg229149JPEG0007629500000037.jpg227146JPEG0007629500000038.jpg227152JPEG00076295000 00039.jpg227146JPEG0007629500000040.jpg228151JPEG0007629500000041.jpg224142JPEG0007629500000042.jpg214162

[0073]

change

[0074] The newly created monoclonal antibodies are U1-49, U1-53, U1-59, and U1 If the antibody binds to a partial peptide or partial conformation to which the U1-7 or U1-9 antibody binds, the antibody The same epitope as U1-49, U1-53, U1-59, U1-7 or U1-9 antibody It can be determined that U1-49, U1-53, U1-59, U1- The antibody competes with the binding of antibody U1-7 or U1-9 to HER3 (i.e., the antibody The binding of U1-49, U1-53, U1-59, U1-7 or U1-9 antibody to HER3 By confirming the presence of a specific epitope, the sequence or structure of the specific epitope can be determined. Even if the antibody is not a U1-49, U1-53, U1-59, U1-7 or U1-9 antibody, It can be determined that the antibody binds to the same epitope as the antibody. is confirmed, the antibody is U1-49, U1-53, U1-59, U1-7 or U1- It is strongly expected that this antibody will have biological activity equivalent to that of antibody No. 9.

[0075] According to the present invention, the HER3 binding protein of the present invention binds to a small portion of the extracellular portion of HER3. The epitope preferably interacts with at least one epitope located in the amino-terminal domain. domain L1 (amino acids 19 to 184), which is the domain Domains S1 (amino acids 185 to 327) and S2 (amino acids 500 to 632) domain L2 (328 to 499) adjacent to two cysteine-rich domains; The epitope is located within a combination of HER3 domains. The epitope is located in part of L1 and S1. Combinations of domains such as (but not limited to) epitopes formed by the above Additionally, the binding proteins of the invention may be characterized in that their binding to HER3 is localized within the HER2 receptor. The present invention is further characterized by reducing signal transduction mediated by 3. Therefore, a decrease in HER3-mediated signaling can result in, for example, the removal of HER3 from the cell surface. downregulation of HER3, resulting in at least partial disappearance of the molecule, or substantially inactivation stabilized forms (i.e., forms that exhibit lower signaling than the unstabilized forms) ) may be caused by stabilization of HER3 on the cell surface. The reduction in signaling mediated by HER3 is due to the increased activity of ligands or other HER family members. binding of GRB2 to HER-2 or binding of GRB2 to SHC by affecting (e.g., reducing or inhibiting) receptor tyrosine phosphorylation, A By inhibiting KT phosphorylation, PYK2 tyrosine phosphorylation, or ERK2 phosphorylation It may also be caused by increasing the HER3 receptor or by reducing tumor invasiveness. The reduction in signaling mediated by HER2 inhibits HER2 signaling with other HER family members. It can also be caused by affecting (e.g., reducing or inhibiting) the formation of 3-containing dimers. One example, among others, is a method for reducing the formation of the HER3-EGFR protein complex. Or it may be inhibiting.

[0076] Furthermore, in the present invention, the amino acid sequence shown in any one of SEQ ID NOs: 1 to 232 is Minor variations in the amino acid sequence are contemplated to be encompassed by the present invention, provided that The variations in the amino acid sequence are at least as large as the sequences shown in any one of SEQ ID NOS: 1 to 232. At least 75%, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% and most preferably 99%. The variations are within the framework regions (i.e., CD They may occur outside the CDRs, within the framework regions, or within the CDRs. Preferred variations in the amino acid sequence shown in 32, i.e., one to several amino acids, Deletions, insertions and / or substitutions occur near boundaries of functional domains. The main focus is to obtain nucleotide and / or amino acid sequence data from public sequence databases or Identification can be achieved by comparison with proprietary sequence databases. and / or sequence motifs occurring in other binding proteins or predicted proteins of function Computerized comparison methods can be used to identify conformational domains Methods for identifying protein sequences that fold into known three-dimensional structures are known. For example, Bowie et al., Science 253, 164 (1991) ;Proteins, Structures and Molecular Princi ples(Creighton, Ed., W.H.Freeman and Compa. ny, New York(1984));Introduction to Protei n Structure(C. Branden and J. Tooze, eds., G Arland Publishing, New York, NY (1991); and Thornton et al., Nature 354:105 (1991). (all of which are incorporated herein by reference). Any person skilled in the art will recognize sequences that can be used to define structural and functional domains in accordance with the present invention. Motifs and structural conformations can be identified. Among the antibodies obtained by combining heavy and light chains that are the same as the original antibody (parent antibody), It is possible to select an antibody superior to the parent antibody. As mentioned above, HER3 antibodies maintain their HER3 binding activity even if there are changes in the amino acid sequence. Hold. In the present invention, "homology" is synonymous with "identity." Homology between two amino acid sequences The accuracy was measured using the Blast algorithm version 2.2.2 (Altschul l, Stephen F.,Thomas L.Madden,Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Gapped BLAST and PSI-BLAST: a new generat ion of protein database search programs” , Nucleic Acids Res.25:3389-3402) This can be determined by using the Blast algorithm. m can be obtained, for example, by visiting www.ncbi.nlm.nih.gov / blast on the Internet. It can also be used.

[0077] 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. The anti-HER3 antibody of the present invention includes MEHD-7945A (or doligotuzumab b), RG-7116, MM-111, MM-121 (or seribantumab) , MM-141, LJM-716, huHER3-8, tri-specific an ti-EGFR / ErbB3 zybody, GSK-2849330, REGN-14 00, KTN-3379, AV-203, monospecific surrobod y (ErbB3), lumretuzumab, MP-EV-20, ZW-9, Dim ercept TM , anti-Erb3 surfactant (SL-175 or SL-1 76), SYM-013, and variants, active fragments, modified forms, etc. thereof are also included.

[0078] 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 Devel 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.

[0079] 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.

[0080] 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 is 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, and eukaryotic microorganisms can be used. In particular, mammalian cells, such as monkey cells, are suitable for use as animal cells. luzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL -1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), Chinese hamster ovary cells (CHO cells, ATCC CCL-61) Folate reductase-deficient strain (Urlaub, G. and Chasin, LA Proc. N atl.Acad.Sci.USA(1980)77,p.4126-4220) Examples include: 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.

[0081] 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. The full-length antibody of the present invention (also referred to simply as "antibody" in the present invention) includes The present invention also includes deletion mutants thereof, and mixtures containing one or more of these deletion mutants. In Akira's "antibody," when the N-terminus is glutamine, it is cyclized to become pyroglutamine. Heavy or light chains containing cysteine ​​residues, and / or cysteinyl residues of which are partly cysteinyl. Also included are those containing heavy or light chains.

[0082] The isotype of the anti-HER3 antibody of the present invention may be, for example, IgA, IgD, IgE, IgG or IgM type, preferably IgG1, IgG2, IgG3, IgG4, IgM1 and IgM2 type, etc., but are not limited thereto. is of the IgG or IgM type, optimally IgG1, IgG2 or IgG4.

[0083] 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 HER3-binding protein is its binding activity to HER3, and preferably its binding activity to HER3. Furthermore, the antibody of the present invention has the cell-internalizing activity. In addition, it may also have ADCC activity, CDC activity and / or ADCP activity.

[0084] In one aspect, for example, antibodies against HER3 are generated as therapeutic candidates. In this regard, the anti-HER3 antibody of the present invention fixes complement and is involved in complement-dependent cytotoxicity (CDC). It may be desirable to administer mouse IgM, mouse IgG2a, mouse Ig G2b, mouse IgG3, human IgM, human IgG1, human IgG3, and human IgA, etc. However, it is not limited to these.) and its involvement in complement fixation and complement-dependent cytotoxicity (CDC). There are many antibody isotypes that can be administered. The antibodies produced will have these isotypes. It is not necessary to have an isotype from the beginning; rather, the antibody produced may have any isotype. and using conventional molecular biology techniques well known in the art, Molecular cloning of constant region genes or cDNAs into suitable expression vectors The cloned V region genes or cDNAs are then added and then transformed using techniques known in the art. It has been discovered that antibodies can be isotype-switched by expressing them in host cells. Isotype-exchanged antibodies have superior CDC compared to naturally occurring variants. It is molecularly processed so that it can be easily detected (Idusogie et al., J Immunol., 16 6, 2571-2575), recombinantly expressed in host cells using techniques known in the art. Such techniques include, inter alia, direct recombinant techniques (e.g., , U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see, e.g., U.S. Pat. No. The use of cells includes the use of soluble cellulose (e.g., cellulose, cellulose acetate, cellulose esters, cellulose esters, cellulose acetate ... In cell fusion techniques, a myeloma or other cell carrying a heavy chain of any desired isotype is used. A cell line (CHO, etc.) is prepared, and then another myeloma or other cell line (CH) carrying the light chain is prepared. Then, these cells are fused to produce cells that express intact antibodies. For example, a strain can be isolated (defining some of the antibody specificity and affinity of the antibody). (which are identical in structure to each other) have the same variable region but are human IgM, human IgG1, or human IgG3 isotypes. To generate the antibody fragment, a human anti-HER3 IgG4 antibody with the desired binding to the HER3 antigen is prepared. Such molecules then fix complement and participate in CDC. It may be possible to provide

[0085] Furthermore, the anti-HER3 antibody of the present invention inhibits the proliferation of monocytes and natural killer (NK) cells. It can bind to Fc receptors on vector cells and mediate antibody-dependent cellular cytotoxicity (ADCC). It may also be desirable to include mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1 and human IgG3, etc. (but are not limited to these), There are many possible antibody isotypes. The antibodies produced will be of these isotypes. It is not necessary for the antibody to have any isotype initially, but rather the antibody produced may have any isotype. and using conventional molecular biology techniques well known in the art. , to a constant region gene or cDNA molecularly cloned into an appropriate expression vector, After adding the cloned V region genes or cDNA, the V region genes are then transfected using techniques known in the art. By expressing the antibody in a host cell using the It is understood that isotype-exchanged antibodies are superior ADCs compared to naturally occurring variants. C (Shields et al. J Biol Chem. , 276, 6591-6604), recombinantly in host cells using techniques known in the art. Such techniques include, inter alia, direct recombinant techniques ( See, e.g., U.S. Pat. No. 4,816,397), cell-cell fusion techniques (e.g., U.S. Pat. No. 4,816,397), See US Patent No. 5,916,771 and US Patent No. 6,207,418. In cell-cell fusion techniques, a cell carrying a heavy chain of any desired isotype can be produced. A myeloma or other cell line (such as CHO) is prepared and cloned into another myeloma or other cell line that possesses a light chain. Cell lines (e.g., CHO) are prepared. These cells are then fused to produce intact antibodies. For example, a cell line expressing the antibody can be isolated. While possessing the same variable region (which defines some of the To generate the antibody fragment, a human anti-HER3 IgG4 antibody with the desired binding to the HER3 antigen is prepared. Such molecules can then be readily isotype-switched to Fc on effector cells. It may be capable of binding to γR and participating in ADCC.

[0086] 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. Examples include: Antibodies can also be purified by using antigen-immobilized carriers and utilizing their binding to antigens. It is possible.

[0087] [Anti-tumor compound] The antitumor compound conjugated to the anti-HER3 antibody-drug conjugate of the present invention is described below. The antitumor compound used in the present invention is a compound having an antitumor effect. There are no particular limitations as long as the compound has a substituent or partial structure that can be bonded to the linker structure. The tumor compound is a compound in which a part or all of the linker is cleaved in tumor cells to form an antitumor compound moiety. When the linker is cleaved at the site where the drug binds, the antitumor effect is achieved. The active compound is released in its unmodified form, exerting its inherent antitumor activity. 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:)

[0088] [ka]

[0089] 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.

[0090] 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 derivatives, other camptothecin or its derivatives (as described in JP-A-6-87746) antitumor agents) and the like.

[0091] 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, a mixture of different numbers of drugs is obtained. The number of drugs bound to one antibody molecule is usually an average value, that is, the average number of drugs bound is In principle, unless otherwise specified, the present invention also Antibodies with specific binding numbers - Antibodies with specific drug binding numbers contained in a drug conjugate mixture Except when drug conjugates are shown, the number of drugs bound represents the average value. The number of exatecan molecules attached to an antibody molecule can be controlled, and the average drug dose per antibody is The average number of exatecan molecules that can be bound is about 1 to 10, but it is preferred The number of the nucleotides is preferably 2 to 8, and more preferably 3 to 8. From the description of the examples, it is possible to design reactions that allow the desired number of drugs to be bound to the antibody, and It is possible to obtain antibody-drug conjugates in which the number of Tecans bound is controlled. The antibody-drug conjugate of the present invention is effective when the number of drugs bound to one antibody molecule is large. However, aggregation, insolubility, fragmentation, etc. are unlikely to occur.

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

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

[0094] [ka]

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

[0096] [ka]

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

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

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

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

[0101] 3.L P L P is a peptide residue consisting of 2 to 7 amino acids. 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)n2 -C(=O)- part It binds to amino groups.

[0102] 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 cerebroside, serine, glutamic acid, and aspartic acid. The number of amino acids ranges from 2 to 7. That's fine.

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

[0104] 4.L a -(CH2)n 2 -C(=O)- 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, L a is a single bond and n 2 It is preferred if is 0.

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

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

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

[0108] The anti-HER3 antibody-drug conjugate of the present invention is a drug that binds to the linker after being transported into tumor cells. The - part is cut off, and NH2-(CH2)n 1 -L a-(CH2)n 2 Drug derivatives with the structure -C(=O)-(NH-DX) It is believed that the antibody-drug conjugate of the present invention releases the As the antitumor derivatives which exhibit antitumor effects when released, the above-mentioned linker -NH-(C H2)n 1 -L a -(CH2)n 2 An antibody having a structural portion in which the terminal of the structure represented by -C(=O)- is an amino group Among the tumorigenic derivatives that may be mentioned are the following, which are particularly preferred: NH2-CH2CH2-C(=O)-(NH-DX), NH2-CH2CH2CH2-C(=O)-(NH-DX), NH2-CH2-O-CH2-C(=O)-(NH-DX), NH2-CH2CH2-O-CH2-C(=O)-(NH-DX). In the case of NH2-CH2-O-CH2-C(=O)-(NH-DX), the aminal structure in the molecule is unstable. Therefore, it further self-decomposes HO-CH2-C(=O)-(NH-DX) These compounds 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. In addition, in the drug-linker structure of the present invention, L P may bind directly to the drug. In this case, L P When the C-terminus of It is either tecan itself or a compound in which glycine is bound to the amino group of exatecan.

[0109] In the antibody-drug conjugate of the present invention in which the drug is exatecan, The drug-linker structural portion of -L 1 -L2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX), or -L 1 -L 2 -L P -(NH-DX) These drug-linker structural moieties are preferably linked to an antibody. The average number of bonds may be 1 to 10, preferably 2 to 8, and more preferably 1 to 10. More preferably, 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)-DGGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX )、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-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-CH2CH2O-CH2CH2-O-CH2CH2-C (=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX)、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Among these, the following are more preferred: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-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), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Furthermore, the following is preferable: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Particularly preferred are the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX).

[0110] In the antibody-drug conjugate of the present application, a linker that connects the anti-HER3 antibody and the drug The structure can be obtained by combining the preferred structures shown in each of the linker parts described above. A preferred linker can be constructed by the following structure. The left end of the structure is the binding site with the antibody, and the right end is the binding site for the drug. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2-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-CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-、 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-。 Among these, the more preferred ones are as follows. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(Succinimid-3-yl-N)-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)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-. Furthermore, the following can be preferably mentioned: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-. Particularly preferred are the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-.

[0111] [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, the compounds are indicated by the numbers shown in each reaction formula. In other words, they are referred to as "compound of formula (1)", "compound (1)", etc. The same applies to mixtures.

[0112] 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.

[0113] [ka]

[0114] [Wherein, AB represents an antibody having a sulfhydryl group, and L 1 'L 1 The linker structure shown in wherein the linker end is a maleimidyl group (following formula):

[0115] [ka]

[0116] (where the nitrogen atom is the binding site). Specifically, -(Succ inimid-3-yl-N)-(CH2)n 2 L represented by -C(=O)- 1 In the structure of the formula (I), the -(Succinimid-3-yl-N The -(NH-DX) represents a linker having a structure in which the -(NH-DX) moiety is a maleimidyl group.

[0117] [ka]

[0118] The structure is represented by the formula: ]

[0119] In the above reaction scheme, in the compound of formula (1), the structure from the drug to the linker terminal is This can be interpreted as a structure in which one structural moiety binds to one antibody. is a convenient description for the purpose of explanation, and in reality, the structural part is present multiple times per antibody molecule. In many cases, several molecules are bonded together. This situation will also be the case in the following description of the manufacturing method. .

[0120] That is, compound (2) which can be obtained by the method described later and a compound having a sulfhydryl group The antibody-drug conjugate (1) is produced by reacting the antibody (3a) with the It is possible. The antibody (3a) having a sulfhydryl group can be obtained by a method well known to those skilled in the art (He rmanson, GT, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press(199 6) For example, Traut's reagent is used to react with the amino groups of antibodies; After reacting S-acetylthioalkanoates with the amino groups of the antibody, the hydroxyl groups Amine reaction: N-succinimidyl 3-(pyridyldithio)propionate After the reaction, a reducing agent is added; dithiothreitol, 2-mercaptoethanol, Applying a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to the antibody and reducing disulfide bonds in the hinge region of the antibody to generate sulfhydryl groups; The methods include, but are not limited to, the following. 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 chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminetetraacetic acid (DTA). These are used at a concentration of 1 mM to 20 mM. Buffer solutions include sodium phosphate, sodium borate, and sodium acetate. Specifically, the antibody is incubated in TCE at 4°C to 37°C for 1 to 4 hours. Antibodies with partially or completely reduced sulfhydryl groups by reacting with P ( 3a) can be obtained. 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 methacrylate (DMF), dimethyl acetamide (DMA), N-methyl-2-pyrrolidone ( Organic solvents such as NMP 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 ​​(N More specifically, NAC is used in an amount of 1 to 2 moles relative to the compound (2) used. The reaction can be terminated by adding an equivalent amount 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. - exchange, purification, antibody concentration, and measurement of the average number of drugs bound per antibody molecule were performed. Identification of the drug conjugate (1) can be performed.

[0121] 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.

[0122] 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. Here, the 280 nm extinction coefficient was calculated from the amino acid sequence of the antibody using a known method (Protein Science, 1995, vol.4, 2411-2423), which differs for each antibody. 80nm absorption coefficient (1.3mLmg -1 cm -1 ~1.8mLmg -1 cm -1 ) was used. U In the case of 1-59, according to the amino acid sequence, 1.768 mL mg -1 cm -1 280nm The extinction coefficient was used as an estimate.

[0123] Common Procedure C: Antibody Buffer Exchange 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 BS6.0 / EDTA. After placing 2.5 mL of antibody solution on the plate, the plate was eluted with 3.5 mL of PBS 6.0 / EDTA. A fraction (3.5 mL) was collected. This fraction was concentrated by common procedure A and purified by common procedure B. After measuring the antibody concentration, the antibody was diluted to 10 mg / mL using PBS6.0 / EDTA. The body concentration was adjusted.

[0124] Common Procedure D: Purification of Antibody-Drug Conjugates Acetate buffer (10 mM, pH 5.5; AB The NAP-25 column was equilibrated with 100 μL of ... Add the antibody-drug conjugate reaction solution (approximately 2.5 mL) and the amount of buffer solution specified by the manufacturer. The antibody fraction was collected by elution with HCl. This collected fraction was then loaded again onto the NAP-25 column. The gel filtration purification procedure, in which the eluate is then eluted with buffer, is repeated two or three times to remove the unbound drug. Linkers and small molecules (tris(2-carboxyethyl)phosphine hydrochloride (TCEP ), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide) The drug conjugate was obtained.

[0125] 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 [ The absorbance of the antibody and the drug differs depending on the conjugation. 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 CA Formula (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Formula (2) where A 280 indicates the absorbance of the antibody-drug conjugate aqueous solution at 280 nm. , A 370 indicates the absorbance of the antibody-drug conjugate aqueous solution at 370 nm, and A A,280 indicates the absorbance of the antibody at 280 nm, and A A,370 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 that A,370 is usually zero. In the case of U1-59, the amino According to the acid sequence, ε A,280 The estimated value was 259400. D,280 and ε D,370 teeth The absorbance of a solution in which the conjugate precursor to be used is dissolved at a certain molar concentration is measured. According to the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length), The A of the antibody-drug conjugate aqueous solution can be obtained by 280 and A 370 Measure By substituting these values ​​into equations (1) and (2) and solving the simultaneous equations, C A and C D Furthermore, C D C A Dividing by this gives the average number of drugs bound per antibody. You can ask for it. In the present invention, the above-described method for determining the average number of drugs bound to one antibody is referred to as "UV It is called "law."

[0126] 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] Drug binding to the L chain (L0) and H chain (H0) of an antibody to which no drug is bound L chains with one drug attached (L chains with one drug attached: L1) and H chains with one drug attached (H chains with one drug attached: L2) H chains with two drugs bound to them: H1, H chains with two drugs bound to them: H2, and H chains with three drugs bound to them: H3 The retention time increases in proportion to the number of substances, resulting in increased hydrophobicity. The peaks are eluted in the order of H2 and H3. By comparing the retention times with L0 and H0, the detected peaks are L0, L1, It can be assigned to either 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.

[0127]

number

[0128]

number

[0129] 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 U1-59, the value estimated from 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.

[0130]

number

[0131] [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 x 0 + L0 peak area ratio x 1 + H0 peak area ratio x 0 + H1 peak area ratio x 1 + H2 peak area ratio x 2 + H3 peak area ratio x 3) / 100 x 2

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

[0133] (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) or (maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -(NH-DX) It is a compound represented by the formula: During the ceremony, 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 group having the following formula: [ka]

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

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

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

[0137] Also, -NH-(CH2)n 1 -L a -(CH2)n 2 - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2 -CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- Preferred as a production intermediate are -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2. Some are more preferable. n 3 As the intermediate, an integer of 2 to 8 is preferred. L 2 is a single bond or -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- where n 4 is an integer between 2 and 4 are preferred as production intermediates.

[0138] Furthermore, n 3 is an integer between 2 and 5, and L 2 is a single bond, and -NH-(CH2)n 1 -L a -(CH2)n 2 - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH 2-O-CH2- or -NH-CH2CH2-O-CH2- are preferred as production intermediates. Among these, -NH-(CH2)n is more preferred. 1 -L a -(CH2)n 2 -, -NH-CH2CH2-, -NH-CH2CH2CH 2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. 3 But integer Preferably, it is 2 or 5 of the above.

[0139] Also, n 3 is an integer between 2 and 5, and L 2 -NH-(CH2-CH2-O)n 4 -CH2-CH2-C(=O)- , n 4 is an integer from 2 to 4, and -NH-(CH2)n 1 -L a -(CH2)n 2 -, -NH-CH2CH2-, -NH-CH2CH2CH 2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH 2- is preferred as a production intermediate. More preferably, n 4 Compounds where the integer is 2 or 4 Furthermore, -NH-(CH2)n 1 -L a - is -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2 CH2-O-CH2- is preferred.

[0140] 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)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2 -C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH- CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、 (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0141] A drug-linker compound selected from the group of intermediate compounds for the production of an anti-Her3 antibody or By reacting with its reactive derivative, disulfide residues present in the hinge region of anti-Her3 antibody can be isolated. A thioether bond is formed at the sulfide bond moiety to form the anti-Her3 antibody-drug complex of the present invention. Conjugates can be prepared using reactive derivatives of anti-Her3 antibodies. It is preferable to use a reactive derivative obtained by reducing an anti-Her3 antibody. It's nice.

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

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

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

[0145] [ka]

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

[0147] 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 the group, NH2-DX [representing exatecan; chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro 2,3-Dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolyl React with dino[1,2-b]quinoline-10,13(9H,15H)-dione](4) or its pharmacologically acceptable salt Compound (6) can be produced by 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 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 the 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 that is easy to use.

[0148] 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; lithium Organometallic bases, such as dialkylaminolithium, such as lithium diisopropylamide; organometallic bases of bissilylamines such as tium bis(trimethylsilyl)amide; or Lysine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine , N-methylmorpholine, diisopropylethylamine, diazabicyclo[5.4.0] Examples include organic bases such as undec-7-ene (DBU).

[0149] 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.

[0150] 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 ) can be produced by selecting the appropriate reagents and conditions for this protecting group. good. Carboxylic acid (11) is converted into an activated ester, a mixed acid anhydride, an acid halide, or the like, The compound (2) can be produced by reacting the obtained compound (10). The reaction conditions, reagents, and The base and inert solvent may be appropriately selected from those described in the synthesis of compound (6). stomach.

[0151] 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 As for the protecting group, if it is a commonly used protecting group, there is no particular restriction. Specifically, the protecting group for the hydroxyl group is an alkoxymethyl group such as a methoxymethyl group. Groups: arylmethyl groups such as benzyl, 4-methoxybenzyl, and triphenylmethyl groups Alkanoyl groups such as acetyl; aroyl groups such as benzoyl; tert-butyldiphenyl Examples of the carboxyl group include methyl groups, ethylene groups, phenylsilyl groups, and the like. alkyl groups such as butyl groups and tert-butyl groups, aryl groups such as allyl groups, and benzyl groups; The amino group can be protected as an ester with tert-butyloxy. alkyloxycarbonyl groups such as hydroxycarbonyl groups, methoxycarbonyl groups, and ethoxycarbonyl groups; allyl group, allyloxycarbonyl group, or 9-fluorenylmethyloxycarbonyl group , benzyloxycarbonyl group, para-methoxybenzyloxycarbonyl group, para (or ortho) nitrobenzyloxycarbonyl group and other arylmethoxycarbonyl groups; alkanoyl groups such as an acetyl group; arylmethyl groups such as a benzyl group and a triphenylmethyl group; aroyl groups such as benzoyl groups; or 2,4-dinitrobenzenesulfonyl groups, arylsulfonyl groups such as trinitrobenzenesulfonyl group; 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 The protecting groups may be selected appropriately from the above-mentioned protecting groups. In this case, the protecting group for the amino group and the protecting group for the carboxy group can be removed by different methods or under different conditions. For example, P 2 is a tert-butyloxycarbonyl group, and P 3 Gaben The combination of protecting groups is a cyclohexyl group. The compound may be selected from the above depending on the properties of the compound protecting the amino group and the carboxy group. When cleaving these protecting groups, 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.

[0152] Compound (2) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (13) 2 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.

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

[0154] [ka]

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

[0156] Compound (11) is converted to an activated ester, a mixed acid anhydride, or the like, and the C-terminus is converted to P in the presence of a base. 4 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).

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

[0158] [ka]

[0159] [In the formula, L P represents the same as above, and L is an acyl group, such as an alkanoyl group, e.g., an acetyl group. X and Y are each an aroyl group such as 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 indicates a protecting group for the carboxy group]

[0160] 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 required. If necessary, the compound can be produced by removing the protecting group or converting the functional group. , an amino acid having a terminal amino group protected, or an acid amino acid of an oligopeptide having a terminal amino group protected The hydroxybenzoates can be obtained by treating the hydroxybenzoates with aldehydes or ketones. 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 are preferred, and paratoluene is particularly preferred. Further, the base may be appropriately selected from the bases already mentioned. In particular, alkali metal alkoxylates such as potassium tert-butoxide are used. Alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, etc. Alkali metal hydrides such as sodium hydride and potassium hydride; Lithium diisopropyl Organometallic bases such as dialkylaminolithium such as pyramide; lithium bis( Preferred are organometallic bases of bissilylamines such as trimethylsilylamide. The solvent used in the reaction is an ether such as tetrahydrofuran or 1,4-dioxane. Solvents: Aromatic hydrocarbon solvents such as benzene and toluene are used. It may also be a mixture of Also, P 5 The amino-protecting group shown in There are no particular limitations as long as the group is a protective group for the amino group described in Production Method 2. In this reaction, P 5 The protecting group of the amino group shown as In this case, it may be reacted with an appropriate amino group-protecting reagent as needed. Then, the protecting group is introduced again. Compound (24) can be prepared by removing the protecting group P of compound (23). 6 It can be produced by removing Here, P6 As the protecting group for the carboxyl group shown in Compounds having the formula (23) are described in the literature, and can be selected appropriately from these. , a protecting group P of the amino group 5 and a protecting group P of the carboxy group 6 Protection that can be removed in different ways or under different conditions It is preferable that the group is a P 5 is a 9-fluorenylmethyloxycarbonyl group Ri, P 6 is a benzyl group. These protecting groups can be selected depending on the properties of the compound protecting the amino and carboxy groups. When removing these protecting groups, reagents and conditions may be selected according to the protecting group. . Carboxylic acid (24) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, 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).

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

[0162] [ka]

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

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

[0165] N-terminus to P 2 The peptide or amino acid (28) protected by the above is reacted with an activated ester, a mixed acid anhydride, etc. and reacting the resulting compound (7) to produce compound (29). The reaction of peptide or amino acid (28) with compound (7) to form an amide bond can be carried out. The conditions, reagents, bases, and solvents are appropriately selected from those described in the synthesis of compound (6). The amino group protecting group P 8 is appropriately selected from those described as the protecting groups of compound (6). The compound may be used in accordance with its properties, etc. As commonly used, the amino acids or peptides that make up a peptide or amino acid (28) The compound (29) can also be produced by repeating the reaction and deprotection of the amide to elongate it. The protecting group P of the amino group of the obtained compound (29) 8 By deprotecting the compound (23 For deprotection, reagents and conditions can be selected according to the protecting group. . N-terminus to P 8 The carboxyl group, hydroxyl group, or amino group on the side chain is P 9 or Peptide (31) is converted to an active ester, mixed acid anhydride, etc., and the resulting compound (30) is By reacting the compound (32), the compound (32) can be produced. The reaction conditions, reagents, bases, and solvents for forming the peptide bond between (31) and compound (30) are as follows: The protecting group P may be appropriately selected from those described in the synthesis of compound (6). 8 and P 9 year For the protecting group of the amino group, carboxy group or hydroxyl group of the compound (6), However, in this case, the protecting group P 9 and protection of side chain functional groups Mamoru P 10 It is necessary to be able to remove P by different methods or conditions. 9 9-Fluorenylmethyl is a methyloxycarbonyl group, and P 10 is a protecting group for the carboxy group, then tert-butyl A protecting group for a hydroxyl group is a methoxymethyl group, and a protecting group for an amino group is a tert Representative examples include a combination of -butyloxycarbonyl group and the like. Protecting group P of the side chain functional group 10 A protecting group that can be removed by subjecting it to acidic conditions is preferred. Preferably, but not limited to, the amino group, carboxyl group or The protecting groups may be selected from those listed above depending on the properties of the hydroxyl group. In this case, reagents and conditions should be selected according to the protecting group. As used, compound (32) is prepared by sequentially reacting and deprotecting the constituent amino acids or peptides. The protection can also be repeated and stretched to produce the film. The protecting group P of the terminal amino group of the obtained compound (32) 9 By deprotecting the compound ( 33) can be produced. This deprotection can be carried out by selecting the appropriate reagents and conditions depending on the protecting group. That's fine. The carboxylic acid derivative (11) is derivatized with an activated ester, a mixed acid anhydride, or an acid halide. and reacting the resulting compound (33) to produce compound (34). Here, the carboxylic acid derivative (11) is L 1 The linker end of ' has a maleimidyl group It is a compound with the following structure. Reaction conditions and reagents for forming a peptide bond between carboxylic acid derivative (11) and compound (33) The base and solvent may be appropriately selected from those described in the synthesis of compound (6). The carboxyl group or hydroxyl group of the amino acid side chain of the peptide portion of the obtained compound (34), Protecting group P for amino group 10 Compound (2) can be produced by deprotecting the The reagents and conditions may be selected depending on the protecting group.

[0166] Compound (29) can also be produced, for example, by the following method. N-terminus to P 8 The peptide or amino acid (28) protected by the above is reacted with an activated ester, a mixed acid anhydride, etc. In the presence of a base, the terminal carboxyl group is converted to P 3 Reaction with the protected amine compound (12) Compound (35) can be produced by the addition of a peptide or amino acid (28). The reaction conditions, reagents, bases, and solvents for forming the peptide bond between (12) and (13) are as follows: The amino group of compound (35) can be appropriately selected from those described in the synthesis of compound (6). Protecting group P 8 The protecting group may be appropriately selected from those described above for the compound (6). Protecting group P of carboxy group 3 As an example, in organic synthetic chemistry, especially in peptide synthesis, A protecting group that is commonly used as a protecting group for the group may be used. Specifically, a protecting group such as a methyl group, an ethylene group, ethyl group, alkyl esters such as tert-butyl, allyl esters, benzyl esters, etc. The protecting group may be appropriately selected from those described above for the compound (6). Protecting group P for amino group 8 and a protecting group P of the carboxy group 3 must be removable in different ways or under different conditions. For example, P 8 is a tert-butyloxycarbonyl group, and P 3 is a benzyl group These protecting groups are used to protect the amino and carboxyl groups. The protecting group may be selected from the above-mentioned compounds depending on the properties of the compound protecting the oxy group. When cleaving the protecting group, the reagents and conditions may be selected according to the protecting group. The protecting group P of the carboxy group of the obtained compound (35) 3 By deprotecting the compound ( 36) can be produced. The deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. good. The obtained compound (36) 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 (29). 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 solvents may be appropriately selected from those described in the synthesis of compound (6). good.

[0167] Compound (32) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (35) 8 Compound (37) is prepared by deprotecting For deprotection, reagents and conditions may be selected according to the protecting group. Amino acid or peptide (31) is converted into an active ester, mixed acid anhydride, acid halide, etc. and reacting it with the resulting compound (37) in the presence of a base to give compound (38). The amide bond between the amino acid or peptide (31) and the compound (37) can be produced. The reaction conditions, reagents, bases and solvents for forming the compound (6) are appropriately selected from those described in the synthesis of the compound (6). Here, the protecting group P of the amino acid or peptide (31) 9 , P 10 transformation The protecting group P of compound (37) 3 Each of these needs to be removed using different methods or conditions. For example, P 9 is a 9-fluorenylmethyloxycarbonyl group, and P 10 tert-butyloxy Carbonyl group, tert-butyl group, or methoxymethyl group, P 3 is a benzyl group, etc. As mentioned above, the functional groups in the side chains The protecting group P 10 The protecting group is preferably a protecting group that can be removed by subjecting it to acidic conditions, but is not limited thereto. The type of the compound to be protected is not limited, and depends on the properties of the amino group, carboxyl group, or hydroxyl group of the compound to be protected. The protecting groups may be selected from those mentioned above depending on the protecting group. The appropriate reagents and conditions can be selected. The protecting group P of the carboxy group of the obtained compound (38) 3 By deprotecting the compound ( 39) can be produced. The deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. good. Compound (39) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with a base. Compound (32) can be produced by reacting compound (4) with benzophenone in the presence of benzophenone. This reaction can be carried out by applying the reaction reagents and conditions normally used in peptide synthesis. The base and solvent may be appropriately selected from those described in the synthesis of compound (6).

[0168] Compound (34) can also be produced, for example, by the following method. The protecting group P of the amino group of compound (38) 9Compound (40) is prepared by deprotecting For 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. and reacting it with the obtained compound (40) in the presence of a base to prepare compound (41). The amide bond between the carboxylic acid derivative (11) and the compound (40) can be formed. The reaction conditions, reagents, bases, and solvents used in the synthesis of compound (6) are appropriately selected from those described in the synthesis of compound (6). Just use it as is. The protecting group P of the carboxy group of the obtained compound (41) 3 By deprotecting the compound ( 42) can be produced. The deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. good. Compound (42) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with a base. Compound (34) can be produced by reacting compound (4) with benzophenone in the presence of benzophenone. This reaction can be carried out by applying the reaction reagents and conditions normally used in peptide synthesis. The base and solvent may be appropriately selected from those described in the synthesis of compound (6).

[0169] Compound (34) can also be produced, for example, by the following method. The carboxylic acid derivative (11) is derivatized with an activated ester, a mixed acid anhydride, or an acid halide. In the presence of a base, the carboxyl group is converted to P 11 The carboxyl group, hydroxyl group or amino group on the side chain is P 10 to prepare compound (44) by reacting with a protected amino acid or peptide (43). The amide bond between the carboxylic acid derivative (11) and the compound (43) can be formed. The reaction conditions, reagents, bases, and solvents used in the synthesis of compound (6) are appropriately selected from those described in the synthesis of compound (6). Here, the protecting group P of compound (44) 10 and P 11 As the compound (6), By appropriately selecting and using the protecting groups for the carboxyl group, hydroxyl group, or amino group, However, in this case, the protecting group P 11 and the protecting group P of the side chain functional group 10 is different It is necessary to be able to remove it by the method or under the conditions. For example, P 11 is a benzyl group, and P 10 Carboxy The protecting group for the hydroxyl group is a methoxymethyl group. For example, a protecting group for an amino group is a tert-butyloxycarbonyl group. Typical examples include the following: a protecting group P for a functional group in a side chain 10 is subjected to acidic conditions A protecting group that can be removed by removing the protecting group is preferred, but is not limited thereto. The compound to be used may be selected from the above-mentioned groups depending on the properties of the amino group, carboxyl group, or hydroxyl group. In cleaving these protecting groups, reagents and conditions may be selected according to the protecting group. good. The protecting group P of the carboxy group of the obtained compound (44) 11 By deprotecting the compound (45) can be produced. This deprotection can be carried out by selecting the reagents and conditions according to the protecting group. Just do that. Compound (45) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with a base. Compound (34) can be produced by reacting compound (30) with This reaction can be carried out by applying the same reaction reagents and conditions as those normally used in peptide synthesis. The agent, base and solvent may be appropriately selected from those described in the synthesis of compound (6). Alternatively, compound (45) may be converted into an active ester, a mixed acid anhydride, an acid halide, or the like. In the presence of a base, the carboxyl group is converted to P 12 and reacting with a protected amino acid or peptide (46) Compound (47) can be produced by this reaction. The reaction conditions, reagents, bases and solvents used in the synthesis of compound (6) can be used accordingly. The protecting group P of compound (47) may be selected appropriately from those described in the synthesis. 10 and P 12 Examples of the protecting groups include those described above for the carboxyl, hydroxyl or amino groups of compound (6). However, in this case, the protecting group P 12 and the protecting group P of the side chain functional group 10 It is necessary to be able to remove P by different methods or conditions. 12 but benzyl group, P 10 is a protecting group for a carboxyl group, such as a tert-butyl group, For example, a protecting group for a methyl group is a methoxymethyl group, and for an amino group is a tert-butyloxy group. A typical example is a combination of a hydroxyl group and a hydroxyl group. Protecting group P of functional group 10 A protecting group that can be removed by subjecting it to acidic conditions is preferred. The present invention is not limited to the above, and the nature of the amino group, carboxyl group or hydroxyl group of the compound to be protected may be varied. The protecting groups may be selected from those mentioned above depending on the above. The reagents and conditions should be selected according to the group. The peptide can also be produced by repeating the reaction and deprotection steps to elongate it. The protecting group P of the carboxy group of the obtained compound (47) 12 By deprotecting the compound (48) can be produced by selecting the appropriate reagents and conditions depending on the protecting group. Compound (48) is converted to an activated ester, a mixed acid anhydride, an acid halide, or the like, and then reacted with a base. Compound (34) can be produced by reacting compound (7) with benzophenone in the presence of benzophenone. This reaction can be carried out by applying the reaction reagents and conditions normally used in peptide synthesis. The base and solvent may be appropriately selected from those described in the synthesis of compound (6). Compound (47) can also be produced, for example, by the following method. Amino acids or peptides (46) can be converted into activated esters, mixed acid anhydrides, or acid halides. In the presence of base, the N-terminus is converted to P 9 The carboxyl group, hydroxyl group or amino group on the side chain is P 10 to prepare peptide (49) by reacting with a protected amino acid or peptide (31) The amino acid or peptide (46) and the amino acid or peptide (31) can be synthesized. The reaction conditions, reagents, bases, and solvents for forming the peptide bond were the same as those described in the synthesis of compound (6). Here, the carboxyl group of the amino acid or peptide (46) can be selected appropriately. Protecting group P of silyl group 12 and a protecting group P of the amino acid or peptide (31). 9 and P 10 As mentioned above, However, they must be removable by different methods or conditions. For example, P 9 9-full is an olefinylmethyloxycarbonyl group, and P 10 If is a protecting group for the carboxy group, ter t-butyl group, etc., for protecting groups of hydroxyl groups, methoxymethyl group, etc., for protecting groups of amino groups, For example, a tert-butyloxycarbonyl group, and P 12 is a benzyl group, etc. The following can be mentioned as a representative example. 10 is subjected to acidic conditions A protecting group that can be removed by, but is not limited to, is preferred. The compound may be selected from the above-mentioned groups depending on the properties of the amino group, carboxyl group, or hydroxyl group of the compound. When cleaving the protecting groups, reagents and conditions may be selected according to the protecting groups. stomach. The N-terminal protecting group P of the obtained peptide (49) 9 The peptide (5 0) can be produced by selecting the reagents and conditions 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 resulting peptide (50) was reacted with the resulting compound (47) in the presence of a base. It can be produced by forming an amide bond between the carboxylic acid derivative (11) and the peptide (50). The reaction conditions, reagents, bases, and solvents used in the synthesis of compound (6) are appropriately selected from those described in the synthesis of compound (6). You can select and use it.

[0170] 6. Manufacturing method 6 In the production intermediate (2), the linker is -L 1 -L 2 -L P -, and the L P teeth, A peptide residue whose N-terminus is a hydrophilic amino acid, For peptide residues in which the amino acid is a hydrophilic amino acid other than glycine, the following method is used: It can also be produced by

[0171] [ka]

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

[0173] L P L p1 and L p2 Since it is formed by combining P The hydrophilic amino acid at the N-terminus of p1 Reason Since it will be p1 The N-terminus of the amino acid may be a hydrophilic amino acid. There may be a plurality of amino acids. p2 If you use a substance that contains hydrophilic amino acids, , depending on its position, L P Multiple hydrophilic amino acids at the N-terminus or at the N-terminus and other positions Contains L P can be manufactured. The N-terminus of the compound described in Production Method 5 is converted to P 8 The peptide or amino acid (28) protected by and reacting it with compound (4) or a salt thereof to obtain compound (5). The peptide (51) can be produced by the reaction of peptide or amino acid (28) with compound (4). The reaction conditions, reagents, bases, and solvents for forming the thiazide bond are the same as those described in the synthesis of compound (6). The protecting group P 8 is as described for the protecting group of compound (6). Any suitable compound may be selected depending on the properties of the compound protecting the amino group. In addition, peptides or amino acids (28) can be constructed as is commonly used in peptide synthesis. The resulting amino acid or peptide is sequentially subjected to reaction and deprotection to elongate the resulting compound (51). can also be manufactured. The protecting group P of the amino group of the obtained compound (51) 8 By deprotecting the compound (52 This deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. good. The N-terminus of the compound according to Production Method 4 is changed to P 9 The carboxyl group, hydroxyl group, or amino group on the side chain is P 1 0 The protected amino acid or peptide (31) is converted to an active ester, a mixed acid anhydride, or the like, The compound (53) can be produced by reacting the obtained compound (52). Reaction conditions for forming a peptide bond between an amino acid or peptide (31) and compound (52) The reagents, bases, and solvents may be appropriately selected from those described in the synthesis of compound (6). Protecting group P 9 and P 10 The procedure is as described in Production Method 5. As is commonly used in synthesis, compound (53) can be prepared by sequentially synthesizing the constituent amino acids or peptides. It can also be produced by repeating the reaction and deprotection and extending the product. The protecting group P of the amino group of the obtained compound (53) 9 By deprotecting the compound (54 This deprotection can be carried out by selecting the appropriate reagents and conditions according to the protecting group. good. The carboxylic acid derivative (11) is derivatized with an activated ester, a mixed acid anhydride, or an acid halide. and reacting the resulting compound (54) to produce compound (55). The reaction conditions for forming a peptide bond between the carboxylic acid derivative (11) and compound (54) are as follows: The conditions, reagents, bases and solvents may be appropriately selected from those described in the synthesis of compound (6). That's fine. The protecting group P of the carboxyl group, hydroxyl group, or amino group of the obtained compound (55) 10 Deprotection Compound (2) can be produced by deprotecting the protecting group. Simply select the appropriate reagents and conditions.

[0174] Compound (53) can also be produced, for example, by the following method. The protecting group P of the carboxy group of peptide (49) described in Production Method 5 12 Deprotecting the Peptide (56) can be prepared by the following procedure. You just need to choose the drug and condition. The resulting peptide (56) was derivatized with an active ester, mixed acid anhydride, or acid halide. and reacting the compound with compound (4) or a salt thereof to produce compound (53). The reaction conditions, reagents, and salts for forming the peptide bond between compound (56) and compound (4) are as follows: The groups and solvents may be appropriately selected from those described in the synthesis of compound (6).

[0175] Compound (55) can also be prepared by the following method, for example. Compound (48) described in Production Method 5 is converted into an active ester, a mixed acid anhydride, or the like, and then reacted with the compound (48) in the presence of a base. or by reacting the compound (4) with the amino acid or peptide according to Production Method 5 in the presence of The thiol (45) is converted to an active ester, a mixed acid anhydride, or the like, and then reacted with the above-mentioned compound (5) in the presence of a base. 2) to produce compound (55). The reaction conditions, reagents, bases, and solvents for forming the thiazide bond are the same as those described in the synthesis of compound (6). It is sufficient to select and use it appropriately from among them.

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

[0177] [ka]

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

[0179] The intermediate compound represented by formula (2) has a linker of -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 - The structure represented by C(=O)- and -L 1 -L 2 -L P There are two variations of the structure indicated by -. The linker uses -L1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 Compound (2) with the structure -C(=O)- can be produced as follows: Compound (57) can be synthesized in the same manner as compound (32) described in Production Method 5. Unlike compound (32), the amino group is protected by a protecting group P 9 and the protecting group P of the side chain functional group 13 Different It is not necessary that the functional groups on the side chains be removable by the method or conditions specified. and the amino group is protected by a protecting group P 9 and a protecting group P of the carboxyl or hydroxyl group in the side chain 13 simultaneously For example, P 9 is a tert-butyloxycarbonyl group, P 13 is a tert-butyl group, a trityl group, or P 3 is a benzyloxycarbonyl group, and P 13 is a benzyl group, etc. The protecting group is selected depending on the properties of the amino group, carboxy group, or hydroxyl group of the compound to be protected. The protecting group may be appropriately selected from those described above for the compound (6), and the cleavage of the protecting group In this case, the reagents and conditions should be selected according to the protecting group. If a protected amino acid or peptide satisfying the properties is used, it can be synthesized in the same manner as in Production Method 5. It is possible. The protecting group P of compound (57) 9 , P 13 and (5) are deprotected sequentially or simultaneously to give compound (5) 1) can be produced by selecting the reagents and conditions according to the protecting group. Compound (58) is L P The functional group of the hydrophilic side chain is not particularly protected, but in the presence of a base, By reacting with compound (11) derived from an activated ester, mixed acid anhydride, etc., the compound The reaction conditions and reagents for forming each peptide bond can be determined. The base and solvent may be appropriately selected from those described in the synthesis of compound (6). The linker uses -L 1 -L 2 -L P Compound (2) having the structure represented by - can be prepared as follows: Cut. Compound (59) can also be synthesized in the same manner as compound (53) described in Production Method 6. Unlike compound (53), the amino group protecting group P 3 and the protecting group P of the side chain functional group 8 different It is not necessary that the functional group on the side chain be removable by a method or conditions. and the amino group protecting group P 9 and a protecting group P of the carboxyl or hydroxyl group in the side chain 13 at the same time It can also be deprotected. For example, P 9 is a tert-butyloxycarbonyl group, and P 1 3 is a tert-butyl group, a trityl group, or P 3 is a benzyloxycarbonyl group, and P 13 is a benzyl group, etc. The protecting group is selected depending on the properties of the amino group, carboxy group, or hydroxyl group of the compound to be protected. The protecting group may be appropriately selected from those described above for the compound (6), and the cleavage of the protecting group In this case, the reagents and conditions should be selected according to the protecting group. If a protected amino acid or peptide satisfying the properties is used, it can be synthesized in the same manner as in Production Method 6. It is possible. Protecting group P of compound (59) 9 , P 13 and (5) are deprotected sequentially or simultaneously to give compound (5) 3) can be produced by selecting the reagents and conditions according to the protecting group. Compound (60) is L P The functional group of the hydrophilic side chain is not particularly protected, but in the presence of a base, By reacting with compound (11) derived from an activated ester, mixed acid anhydride, etc., the compound The reaction conditions and reagents for forming each peptide bond can be determined. The base and solvent may be appropriately selected from those described in the synthesis of compound (6).

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

[0181] [ka]

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

[0183] The amino acid or peptide (31) according to Production Method 5 is converted into an active ester, a mixed acid anhydride, or is derived into an acid halide, etc., and in the presence of a base, glycylglycyl-L-phenylalanyl- N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[a]pyridine Zo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-yl

[0023] glycinamide (a pharmaceutical compound described in WO 1997 / 46260) Compound (62) can be prepared by reacting compound (61) or a salt thereof with Reaction conditions for forming a peptide bond between amino acid or peptide (31) and compound (61) The reagents, bases and solvents may be appropriately selected from those described in the synthesis of compound (6). N-terminal protecting group P 3 , a protecting group P of the functional group of the side chain 10 As for the manufacturing method 5, In addition, the protecting group P of the functional group of the side chain 10 It is not necessary to have an amino acid sequence protected only at the N-terminus. The reaction can be carried out using a monoacid or peptide (31) to give compound (62).

[0184] 9. Manufacturing method 9 Among the compounds represented by formula (2), the linker is -L 1 -L 2 -L P - is the structure shown in The L P is an oligopeptide whose C-terminus consists of two or more glycines and which binds to a drug. When the N-terminus of the peptide residue is a hydrophilic amino acid, the peptide is glycine. When the amino acid is a tido residue, it can be prepared, for example, by the following method.

[0185] [ka]

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

[0187] L P L p1 and L p2 Since it is formed by combining P The C-terminus of Therefore, the number of glycines is L P The number of C-terminal glycines contained in the The design should take into account the number of iterations to be performed. Peptide (63) is an oligopeptide whose C-terminus consists of two or more glycines. When the N-terminus of the peptide residue is a hydrophilic amino acid, the peptide is glycine. a tide residue, and the N-terminus is P 14 Peptide (63) is a peptide synthesis As is commonly used in the synthesis of hydroxybenzoates, the constituent amino acids or peptides are sequentially condensed and deprotected. Protection can be synthesized by repeating the process. Peptide (63) is converted into an active ester, a mixed acid anhydride, or the like, to give compound (4) or a salt thereof. Compound (64) can be produced by reacting peptide (63) with The reaction conditions, reagents, bases and solvents for forming the peptide bond of compound (4) are the same as those for compound (6). The protecting group P may be appropriately selected from those described in the synthesis of 14 is the compound (6) The composition may be appropriately selected from those described above. Compound (64) also has an N-terminus converted to P 14 The protected amino acid or peptide (65) was activated. and reacting the resulting compound with the compound (52) described in Production Method 6. It can also be produced by reacting an amino acid or peptide (65) with compound (52). The reaction conditions, reagents, bases, and solvents for forming the peptide bond are as described in the synthesis of compound (6). The protecting group P may be appropriately selected from the above. 14 The same as described in the synthesis of compound (6) It is sufficient to select and use it appropriately from the above. The protecting group P of the amino group of the obtained compound (64) 14 By deprotecting the compound (6 6) can be produced by selecting the reagents and conditions according to the protecting group. The carboxylic acid derivative (11) is derivatized with an activated ester, a mixed acid anhydride, or an acid halide. The compound (2) can be produced by reacting the resulting compound (66). The reaction conditions for forming an amide bond between the carboxylic acid derivative (11) and compound (66) can be The reagents, bases and solvents may be appropriately selected from those described in the synthesis of compound (6). stomach. Compound (2) can also be produced by the following method. Compound (67) is L p1 The N-terminal glycine of 2 and the compound ( The amino acid or peptide (4) described in Production Method 5 can be synthesized in the same manner as in Production Method 45. 6) is converted to an activated ester, a mixed acid anhydride, or an acid halide, etc., to give compound (67). By reacting the amine compound (68), the compound (68) can be produced. The amino acid or peptide (46) is a glycine or its C-terminus consists of two or more glycines. It is an oligopeptide having a C-terminus of P 12 Protected by: Amino acid or peptide The reaction conditions, reagents, bases, and solvents for forming the amide bond between (46) and compound (67) are as follows: The compound may be appropriately selected from those described in the synthesis of compound (6). Compound (68) can be prepared by converting compound (11) into an active ester, mixed acid anhydride, or the like, and then cleaving the C-terminal P 12 It can also be prepared by reacting the protected peptide (69) with Here, peptide (69) is an oligopeptide whose C-terminus consists of two or more glycines. When the N-terminus is a hydrophilic amino acid, the peptide residue is glycine. Peptide (69) is prepared by dissolving its constituent amino acids in the same manner as in conventional peptide synthesis. Amino acids or peptides can be synthesized by repeating the condensation reaction and deprotection. Peptide (69) The reaction conditions, reagents, bases and solvents for forming the peptide bond between compound (11) and compound (12) are as follows: The protecting group P may be appropriately selected from those described in the synthesis of 6). 12 is deprotected under acidic conditions. Protective groups that can be used are preferred, but are not limited thereto, and are the same as those described in the synthesis of compound (6). It is sufficient to select an appropriate one from the above and use it. The protecting group P of the carboxy group of the obtained compound (68) 12 By deprotecting the compound (70) can be produced. This deprotection can be carried out by selecting the reagents and conditions according to the protecting group. Just do that. Compound (70) is converted into an active ester, a mixed acid anhydride, or the like, to compound (4) or a salt thereof. By reacting compound (70) with compound (2), compound (2) can be produced. The reaction conditions, reagents, bases, and solvents for forming the peptide bond of (4) are determined by the synthesis of compound (6). It is sufficient to select and use an appropriate one from those described above. Alternatively, compound (2) can be produced by the following method. Compound (52) described in Production Method 6 is converted into an active ester, a mixed acid anhydride, or the like, and then reacted with the compound (52) in the presence of a base. Compound (2) can be produced by reacting compound (67) under the above conditions. The reaction conditions, reagents, bases, and solvents for forming the peptide bond between compound (67) and compound (52) are as follows: The solvent may be appropriately selected from those described in the synthesis of compound (6).

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

[0189] The antibody-drug conjugate of the present invention can be stored in the atmosphere or subjected to purification procedures such as recrystallization. When this is done, the material may absorb water or absorbed water may adhere to it, resulting in the formation of a hydrate. Such compounds or 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 contain unnatural atomic isotopes. The atomic isotopes may also include, for example, deuterium ( 2 H), tritium ( 3 H), Yo Uranium-125( 125 I) or carbon-14( 14 C) and the like. In addition, the compound of the present invention is For example, tritium ( 3 H), iodine-125( 125 I), carbon-14( 14 C), copper-64( 64 Cu), Ruthenium-89( 89 Zr), Iodine-124( 124 I), Fluorine-18( 18 F), Indium-11 1( 111 I), carbon-11( 11 C) or Iodine-131( 131 Radiolabeled with radioisotopes such as I) Radiolabeled compounds can be used as therapeutic or prophylactic agents, research reagents, e.g., assay reagents. The antibody-drug complexes of the present invention are useful as drugs and diagnostic agents, for example, as in vivo diagnostic imaging agents. All isotopic variations of conjugates, whether radioactive or not, are within the scope of the present invention. Included.

[0190] [Pharmaceuticals] The anti-HER3 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. Cut. That is, the anti-HER3 antibody-drug conjugate of the present invention is a major therapeutic method for cancer treatment. and can be used as a drug of choice for chemotherapy, resulting in the It can slow the growth, inhibit proliferation, and even destroy cancer cells. Cancer patients can be relieved from symptoms caused by cancer and their quality of life can be improved, saving their lives. Even if the destruction of cancer cells is not achieved, the proliferation of cancer cells is suppressed. By suppressing and controlling the immune system, cancer patients can achieve a higher quality of life and live longer. This allows the achievement of survival. In addition to the use of drugs alone in such drug therapy, other drugs may be used in adjuvant therapy. It can also be used as a drug in combination with other therapies, such as surgery, radiation therapy, and hormone therapy. Furthermore, it can be used as a drug in neoadjuvant therapy. It can also be used as a In addition to the above therapeutic uses, it is also effective in suppressing the growth of minute metastatic cancer cells and even destroying them. In particular, the expression of HER3 has been confirmed in primary cancer cells. In such cases, administration of the anti-HER3 antibody-drug conjugate of the present invention can suppress cancer metastasis and For example, it can suppress and destroy cancer cells in body fluids during the metastasis process. It has the effect of destroying cancer cells, as well as the effect of suppressing and destroying minute cancer cells immediately after implantation in any tissue. Therefore, it is expected to have an inhibitory or preventive effect on cancer metastasis, especially after surgical removal of cancer. Results can be expected. The anti-HER3 antibody-drug conjugate of the present invention is administered to patients as a systemic therapy. In addition, it can be administered locally to cancer tissues and is expected to have a therapeutic effect. The antibody-drug conjugate (1) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, it showed anti-breast cancer, anti-lung cancer, and anti-melanoma activity. The antibody-drug conjugate (2) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, it showed anti-breast cancer, anti-lung cancer, anti-colon cancer, and anti-melanoma effects, and in vivo, it showed anti- It showed stronger anti-breast cancer and anti-melanoma effects than 1-59. The antibody-drug conjugate (3) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, anti-breast cancer, anti-lung cancer, anti-ovarian cancer, anti-colon cancer, and anti-melanoma effects were observed. Ivo has stronger anti-breast cancer, anti-lung cancer, anti-colon cancer, anti-gastric cancer effects than U1-59. It showed anti-melanoma activity. The antibody-drug conjugate (4) has excellent antitumor activity, safety, and physical properties, and has been reported in v It showed anti-breast cancer activity in vitro. The antibody-drug conjugate (5) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, it showed anti-breast cancer, anti-lung cancer, and anti-melanoma effects. The antibody-drug conjugate (6) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, it has anti-breast cancer, anti-lung cancer, and anti-melanoma effects, and in vivo it has stronger effects than U1-59 It showed anti-breast cancer activity. The antibody-drug conjugate (7) has excellent antitumor activity, safety, and physical properties. In vitro, it showed anti-breast cancer, anti-lung cancer, and anti-melanoma effects. The antibody-drug conjugate (8) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, it has anti-breast cancer, anti-lung cancer, and anti-melanoma effects, and in vivo it has stronger effects than U1-59 It showed anti-breast cancer activity. The antibody-drug conjugate (9) has excellent antitumor activity, safety, and physical properties, and has been reported in v In vitro, it has been shown to have anti-breast cancer, anti-lung cancer, anti-ovarian cancer, anti-colon cancer, and anti-melanoma effects. Ta. The antibody-drug conjugate (10) has excellent antitumor activity, safety, and physical properties. It has anti-breast cancer, anti-lung cancer, anti-colon cancer and anti-melanoma effects in vitro and anti-melanoma effects in vivo. It has stronger anti-breast cancer, anti-lung cancer, anti-colon cancer, anti-gastric cancer, and anti-melanocorticoid effects than U1-59. It showed anti-inflammatory effects. The antibody-drug conjugate (11) has excellent antitumor activity, safety, and physical properties. It showed anti-breast cancer activity in vitro. The antibody-drug conjugate (12) has excellent antitumor activity, safety, and physical properties. It has been shown to have anti-breast cancer, anti-lung cancer, anti-ovarian cancer, anti-colon cancer and anti-melanoma effects in vitro. did. The antibody-drug conjugate (13) has excellent antitumor activity, safety, and physical properties. It has anti-breast cancer, anti-lung cancer, anti-colon cancer and anti-melanoma effects in vitro and anti-melanoma effects in vivo. Stronger anti-breast cancer (including triple-negative breast cancer) effects than U1-59, anti-lung cancer effects, and anti-colonial effects It showed anti-cancer activity, anti-gastric cancer activity, anti-pancreatic cancer activity, and anti-melanoma activity. The antibody-drug conjugate (14) has excellent antitumor activity, safety, and physical properties. It showed anti-breast cancer activity in vitro. The antibody-drug conjugate (16a) has excellent antitumor activity, safety, and physical properties. In vivo anti-breast cancer (including luminal and triple-negative breast cancer) activity, anti-melanoma anti-ovarian cancer, anti-bladder cancer, anti-lung cancer, anti-head and neck cancer and anti-gastric cancer effects. alone or in combination with trastuzumab, gefitinib, cetuximab, panitumumab or pertuzumab This was shown in combination with izumab.

[0191] Examples of cancer types to which the anti-HER3 antibody-drug conjugate of the present invention can be applied include: , lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, Metastatic breast cancer, luminal breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical Cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, glioblastoma multiforme, liver cancer, hepatocellular carcinoma, Colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer Examples include adenocarcinoma, liver carcinoma, anal carcinoma, and penile carcinoma. In particular, triple carcinoma of breast cancer. Negative breast cancer (HER2, estrogen receptor, progesterone receptor) The only treatment currently available for non-expression of this disease is chemotherapy, and it is said to have a poor prognosis. There are few reports on HER3 expression in breast cancer. If observed in patients with triple-negative breast cancer, the anti-HER3 antibody-drug conjugates of the present invention Gates can be used as a therapeutic and / or prophylactic agent. Cancer cells express a protein that can be recognized by the antibody in the antibody-drug conjugate There is no limitation to these as long as the cells are cancer cells. The anti-HER3 antibody-drug conjugate of the present invention exerts an anti-HER3 activity in cancer cells to be treated. Cancer cells expressing the HER3 protein, which can be recognized by the antibody in the antibody-drug conjugate, are treated. As used herein, "cancer expressing HER3 protein" refers to cancers that express HER3 protein on the cell surface. Cancers that contain cells that have the HER3 protein or that secrete the HER3 protein into the blood. HER3 protein is overexpressed in various human tumors and is detected in tumor (primary and metastatic) specimens. Immunohistochemistry (IHC) and HER3 staining to assess overexpression of HER3 protein in the body Fluorescence in situ hybridization (FISH) to assess gene amplification, and blood Enzyme-linked immunosorbent assay (ELISA) to assess the overexpression of HER3 protein in specimens, The evaluation can be carried out using methods commonly practiced in the field. Furthermore, the anti-HER3 antibody-drug conjugate of the present invention is capable of inhibiting the growth of cancer cells by inhibiting the growth of cancer cells. Antitumor effects are achieved by recognizing and internalizing the HER3 protein expressed on the surface. Therefore, the therapeutic target of the anti-HER3 antibody-drug conjugate of the present invention is "HER3 The present invention is not limited to "cancers expressing R3 protein," but may also include leukemia, malignant lymphoma, and Plasmocytoma, myeloma, or sarcoma may also be treated.

[0192] The anti-HER3 antibody-drug conjugate of the present invention can be suitably administered to mammals. The animal may be any animal, but is more preferably a human.

[0193] The anti-HER3 antibody-drug conjugate of the present invention is used in a pharmaceutical composition comprising the conjugate. The substances to be administered are those that are comparable in dosage and concentration to those commonly used in this field. It is possible to appropriately select and apply from among others.

[0194] The anti-HER3 antibody-drug conjugates of the present invention contain one or more pharmaceutically compatible components. For example, the pharmaceutical composition may be administered as a pharmaceutical composition containing one or more of the following: The above pharmaceutical carriers (e.g., sterile liquids) are included. Here, liquids include, for example, water and Oils include, for example, lacquer oils (oils of petroleum, animal, vegetable or synthetic origin). The oil may be seitan, soybean oil, mineral oil, sesame oil, etc. Water is used when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and Aqueous glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients can be appropriately selected from those known in the art. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of suitable pharmaceutical carriers are found in "Remington The formulation is described in "The Journal of Pharmaceutical Sciences." Corresponding to the mode of administration.

[0195] Various delivery systems are known, and can be used to administer the anti-HER3 antibody-drug conjugates of the present invention. Methods of administration include intradermal, intramuscular, intraperitoneal, intravenous, and transdermal. The following routes may be mentioned, but are not limited to: In certain preferred embodiments, the above-mentioned ligands may be administered intravenously or by bolus injection. Administration of the end-drug conjugate is by injection. Parenteral administration is the preferred route of administration. be.

[0196] 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 individually or in unit dosage form or mixed together in a syringe or syringe. In this case, it is dispensed, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the above medicine is administered by injection, an ampoule of sterile water for injection or saline solution may be used. For example, the components may be provided in a pre-mixed form so that they can be mixed prior to administration.

[0197] The pharmaceutical composition of the present invention contains the anti-HER3 antibody-drug conjugate of the present invention as an active ingredient. or may comprise an anti-HER3 antibody-drug conjugate and at least one other The anti-HER3 antibody-drug conjugates of the present invention may contain other drugs (e.g., cancer therapeutic agents). It can also be administered with other cancer treatments, thereby enhancing the anti-cancer effect. Other drugs such as anticancer drugs used for such purposes may, for example, and administering a pharmaceutical composition containing the anti-HER3 antibody-drug conjugate of the present invention as an active ingredient to or administering a pharmaceutical composition containing an anti-HER3 antibody-drug conjugate as an active ingredient. or administered simultaneously with the antibody-drug conjugate, separately (individually), or sequentially. The antibodies of the present invention may be administered to an individual at different intervals. In the present invention, the HER3 antibody-drug conjugate is referred to as an antibody-drug conjugate and other When the drugs are administered simultaneously as a single formulation, and when the drugs are administered simultaneously as separate formulations, Whether the antibody-drug conjugate is administered continuously or at staggered intervals, As such a cancer therapeutic agent, 5-F U, trastuzumab, trastuzumab emtansine (T-DM 1), cetuximab, gefitinib, panitumumab, pertu zumab, abraxane, erlotinib, carboplatin, cis platin, gemcitabine, capecitabine, irinotec an(CPT-11), paclitaxel, docetaxel, pemetrex ed, sorafenib, vinblastin, vinorelbine, vemu rafenib or a drug described in WO 2003 / 038043, In addition, LH-RH analogs (leuprorelin, goserelin, etc.), estramustine folate Sulfate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase enzyme inhibitors (anastrozole, letrozole, exemestane, etc.) However, there is no limitation as long as the drug has antitumor activity. Based on the target, cetuximab, gefitinib, panitumumab, etc. anti-FGER agents, anti-H agents such as trastuzumab, T-DM1, and pertuzumab ER2 agents, anti-HER3 agents such as patritumab, MM-121, and MM-111, They can be classified into anti-VEGF agents such as fliximab and adalimumab. Furthermore, anti-EGFR antibodies such as cetuximab and panitumumab, Anti-HER2 antibodies such as uzumab and pertuzumab, patritumab, MM -121, MM-111, and other anti-HER3 antibodies, infliximab, adalimum The anti-HER3 antibody-drug combination of the present invention can be classified into anti-VEGF antibodies such as anti-HER3 antibody-drug combinations. Jugate is an anti-HER2 agent used in the treatment of gastric cancer, breast cancer, triple-negative breast cancer, etc. or when administered in combination with an anti-HER2 antibody, or ii) lung cancer, head and neck cancer, gastric cancer, breast cancer In combination with anti-EGFR agents or anti-EGFR antibodies in the treatment of breast cancer, triple-negative breast cancer, etc. When administered in combination, it produces excellent therapeutic effects. The drug may be an anticancer drug or a drug for reducing side effects caused by a concomitant drug.

[0198] In the present invention, a "pharmaceutical composition comprising an anti-HER3 antibody-drug conjugate and another drug" is "Anti-HER3 antibody-drug conjugates" are "medicinal products administered in combination with other drugs." In the present invention, the anti-HER3 antibody-drug conjugate and other "Administered in combination" means that drugs are administered in the body of a recipient for a certain period of time. This means that the anti-HER3 antibody-drug conjugate and other pharmaceutical agents are incorporated. Even if a formulation containing the ER3 antibody-drug conjugate and other drugs in a single formulation is administered, Alternatively, each may be formulated separately and administered separately. In this case, the timing of administration is not particularly limited, and they may be administered simultaneously or at different times. The anti-HER3 antibody-drug conjugate and the anti-HER3 antibody-drug conjugate may be administered at the same time or on different days. When other drugs are administered at different times or on different days, the order of administration is not particularly limited. Usually, each drug product is administered according to its own administration method. The doses may be administered at the same frequency or at different frequencies. When the preparations are formulated separately, the administration method (administration route) of each preparation may be the same or different. The anti-HER3 antibody-drug conjugate may be administered by any of the following administration methods (administration routes). The other drug does not need to be present in the body at the same time, and should be administered for a certain period of time (e.g., one month, preferably It is preferably taken into the body within a period of at least one week, more preferably several days, and even more preferably one day. It is sufficient that one active ingredient is contained in the other, and even if the other active ingredient is eliminated from the body when one is administered, good. "Pharmaceutical composition administered in combination with an anti-HER3 antibody-drug conjugate and other drugs" Examples of dosage forms of the "drug" include: 1) an anti-HER3 antibody-drug conjugate and another drug; 2) administering the anti-HER3 antibody-drug conjugate and the other agent separately; 3) simultaneous administration of two types of formulations obtained by formulating them into The same administration route for two formulations obtained by separately formulating a drug conjugate and another drug 4) Anti-HER3 antibody-drug conjugates and other drugs are prepared separately. 5) Simultaneous administration of two formulations obtained by formulation using different administration routes, and 6) anti-HER3 antibody-drug The conjugate and other drugs are formulated separately, resulting in two different formulations, each administered via a different route. Examples include administration of anti-HER3 antibody-drug conjugates at different times. dosage, administration interval, administration form, etc. of "pharmaceutical composition administered in combination with other drugs" The formulations and the like are those of pharmaceutical compositions containing the anti-HER3 antibody-drug conjugate of the present invention. It is similar to, but not limited to, the following. When such a pharmaceutical composition is formulated into two different formulations, a kit containing them may be prepared. may be. In the present invention, the "combination" of an anti-HER3 antibody-drug conjugate and another drug is "administered in combination" with an anti-HER3 antibody-drug conjugate and another drug. means.

[0199] 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

[0200] The composition and concentration of the pharmaceutical composition vary depending on the administration method. The anti-HER3 antibody-drug conjugates used in this study are directed against the antigen of the antibody-drug conjugate. The affinity of the antibody to the antigen, i.e., the dissociation constant (Kd value), is high (K The lower the d value, the more effective the drug can be even at a smaller dose. When determining the dosage of the antibody-drug conjugate, the antibody-drug conjugate and the antibody The dosage can also be determined based on the affinity of the antibody to the antigen. When the adjugate is administered to humans, for example, about 0.001 to 100 mg / kg is used. It may be administered once or multiple times at intervals of once every 1 to 180 days. [Example]

[0201] 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

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

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

[0204] Step 1: tert-Butyl (4-{[(1S,9S)-9-ethyl-5-fluoro-9 -Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-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 Rubodimide hydrochloride (0.216 g, 1.13 mmol) was added and stirred for 1 hour. The reaction solution was diluted with exatecan mesylate (0.500 g, 0.94 mmol) and triethyl ether. N,N-dimethylformamide (0.157 mL, 1.13 mmol) was added. The mixture was added dropwise to a solution of 10 mL of acetic acid and stirred at room temperature for one day. The product was purified by silica gel column chromatography [chloroform to chloroform:methanol = 8:2 (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) + .

[0205] Step 2: 4-amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy] 4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-1-yl]butanamide trifluoroacetate The compound obtained in step 1 above (0.388 g, 0.61 mmol) was dissolved in dichloromethane (9 ml 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)0.87(3H,t,J=7.2Hz), 1.79-1.92(4H,m),2.10-2.17(2H,m),2.27(2H, t,J=7.0Hz),2.40(3H,s),2.80-2.86(2H,m),3. 15-3.20(2H,m),5.15(1H,d,J=18.8Hz),5.26(1 H,d,J=18.8Hz),5.42(2H,s),5.54-5.61(1H,m) ,6.55(1H,s),7.32(1H,s),7.72(3H,brs),7.82 (1H,d,J=11.0Hz),8.54(1H,d,J=8.6Hz). MS(APCI)m / z:521(M+H) + .

[0206] Step 3: N-(tert-butoxycarbonyl)glycylglycyl-L-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 the above step 2 (0.080 g) and stirred for 3.5 hours. , 0.15mmol) was added dropwise to N,N-dimethylformamide solution (1.5mL) The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. Chromatography [chloroform to chloroform:methanol = 8:2 (v / v)] Purification 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) + .

[0207] Step 4: Glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9 -ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3 ,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-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=7.4Hz ),1.71-1.73(2H,m),1.82-1.90(2H,m),2.12-2 .20(4H,m),2.40(3H,s),2.75(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(1H ,d,J=5.9Hz),3.72(1H,d,J=5.5Hz),3.87(1H,d d,J=16.8,5.9Hz),4.50-4.56(1H,m),5.16(1H, d,J=19.2Hz),5.25(1H,d,J=18.8Hz),5.42(2H, s),5.55-5.60(1H,m),7.17-7.27(5H,m),7.32( 1H,s),7.78-7.81(2H,m),7.95-7.97(3H,m),8. 33-8.35(2H,m),8.48-8.51(2H,m). MS(APCI)m / z:839(M+H) + .

[0208] Step 5: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )hexanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9 S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4 (oxobutyl)glycinamide N,N-dimethylformamide (337 mg, 0.353 mmol) of the compound obtained in step 4 above To a solution of methyl amide (1.2 mL), triethylamine (44.3 mL, 0.318 mmol) ), N-succinimidyl 6-maleimidohexanoate (119.7 mg, 0.388 mm The solvent was evaporated under reduced pressure, and the resulting residue was Column chromatography [chloroform to chloroform:methanol = 5:1 (v / v )] to obtain the title compound (278.0 mg, 76%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.3Hz ),1.12-1.22(2H,m),1.40-1.51(4H,m),1.66-1 .76(2H,m),1.80-1.91(2H,m),2.05-2.21(6H,m ),2.39(3H,s),2.79(1H,dd,J=14.0,9.8Hz),2. 98-3.21(5H,m),3.55-3.77(8H,m),4.41-4.48( 1H,m),5.15(1H,d,J=18.9Hz),5.24(1H,d,J=18 .9Hz),5.40(1H,d,J=17.1Hz),5.44(1H,d,J=17 .1Hz),5.54-5.60(1H,m),6.53(1H,s),6.99(2H ,s),7.20-7.27(5H,m),7.30(1H,s),7.70(1H,t ,J=5.5Hz),7.80(1H,d,J=11.0Hz),8.03(1H,t, J=5.8Hz),8.08(1H,t,J=5.5Hz),8.14(1H,d,J= 7.9Hz), 8.25(1H,t,J=6.1Hz),8.46(1H,d,J=8. 5Hz). MS(APCI)m / z:1032(M+H) + .

[0209] Step 6: Antibody-drug conjugate (1) Reduction of antibody: U1-59 prepared in Reference Example 1 was subjected to the common procedure B and Using the PBS6.0 / EDTA buffer, the medium was replaced with PBS6.0 / EDTA and the antibody concentration was adjusted to 10 mg / mL. This solution (1.00 mL) was placed in a 2.0 mL polypropylene tube. 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0307 mL; per antibody molecule) 4.6 equivalents relative to the amount of phosphate) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesqu e, Inc.; 0.050 mL) was added. The pH of this solution was kept within 7.4 ± 0.1. After confirming this, incubate at 37°C for 1 hour to remove disulfides from the hinge region of the antibody. The amide bond was reduced. Conjugation of antibody and drug linker: Incubate the above solution in a 22°C water bath for 10 minutes. After cooling, dimethyl sulfoxide (Sigma-Aldrich Co. LLC; 0 0.0586 mL) and a dimethyl sulfoxide solution ( 0.0615 mL (9.2 equivalents per antibody molecule) was added and incubated in a water bath at 22°C for 40 minutes. The antibody was incubated to allow the drug linker to bind to the antibody. Add an aqueous solution (0.0123 mL) of ma-Aldrich Co. LLC and further add the mixture at room temperature. Stir for 20 minutes using a tube rotator (MTR-103, AS ONE Corporation) at 4°C. The drug linker reaction was stopped. Purification: The above solution was purified by the common procedure D described in Production Method 1 (using ABS as the buffer solution). Purification using this method was carried out to obtain 6 mL of a solution containing the title antibody-drug conjugate. Characterization: Common procedure E (as the molar extinction coefficient of the drug linker) described in Preparation Method 1 , ε D,280 =7280ε D,370 =23400) to obtain the following characteristic values. Antibody concentration: 1.29 mg / mL, antibody yield: 7.74 mg (77%), using common procedure E The average number of drugs bound per antibody molecule (n) measured was 4.9.

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

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

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

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

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

[0215] Step 1: Antibody-drug conjugate (4) Reduction of antibody: U1-59 prepared in Reference Example 1 was subjected to the common procedure B and Using the common procedure C, the medium was replaced with PBS 6.0 / EDTA and 10 mg / mL of antibody was added. This solution (1.00 mL) was placed in a 1.5 mL polypropylene tube. , here 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0187 mL; antibody 2.8 equivalents per molecule) and 1M dipotassium hydrogen phosphate aqueous solution (Nacalai T esque, Inc.; 0.0170 mL) was added. The pH of this solution was kept within 7.0 ± 0.1. After confirming that the antibody is soluble in the hinge region, the antibody is incubated at 37°C for 1 hour. The disulfide bonds of the ATP were reduced. Conjugation of antibody and drug linker: Add the compound obtained in step 5 to the above solution. Dimethyl sulfoxide solution containing 0 mM ATP (0.0314 mL; 4.7 equivalents per antibody molecule) (amount) was added at room temperature and incubated at 15°C for 1 hour to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0 Add 0.0123 mL (18.4 equivalents per antibody molecule) and incubate at room temperature for another 20 minutes. The reaction of the drug linker was stopped by incubation. Purification: The above solution was purified by the common procedure D described in Production Method 1 (using ABS as the buffer solution). Purification was carried out using 100 mL of 100 mL of 100 mL of a solution containing the antibody-drug conjugate. The solution was concentrated using procedure A. Characterization: Common procedure E (as the molar extinction coefficient of the drug linker) described in Preparation Method 1 , ε D,280 =5000, ε D,370 = 19000) to obtain the following characteristic values. Antibody concentration: 1.02 mg / mL, antibody yield: 6.1 mg (61%), measured using common procedure E Average number of drugs bound per antibody molecule (n): 2.9; common procedure F (drug linker) Carr's molar extinction coefficient, ε D,280 per antibody molecule measured using a 5000 mAb Average number of drug bonds (n): 3.2.

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

[0217] Step 1: tert-Butyl (5S,14S)-5-benzyl-1-{[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3 ',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-14- {[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1,4,7,10 ,13-Pentaoxo-3,6,9,12-tetraazahexadecan-16-oate Under ice cooling, glycylglycyl-L-phenylalanyl-N-[(1S,9S)-9-ethyl 5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4': 6,7]indolizino[1,2-b]quinolin-1-yl]glycinamide (International Publication No. Free form of the pharmaceutical compound described in No. 1997 / 46260; 0.250g, 0.332 N-hydroxysuccinimide (57.2 mg, 0.497 mmol), and and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-aspartic acid 4 -tert-butyl (0.205 g, 0.497 mmol) N,N-dimethylformamide A solution of N,N'-dicyclohexylcarbodiimide (0.123 mL) in 10 mL of methyl 2-(2-methyl-1,2-dihydro-2-methyl-2-propanol) was added to the solution. g, 0.497 mmol) was added and stirred at room temperature for 2 days. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform to chloroform:methanol). ール=9:1(v / v)]にて Refined, labeled compound (0.278g, 73%), light yellow solid The body is the same as the body. 1 H-NMR(400MHz,DMSO-d6)δ:0.86(3H,t,J=7.1Hz ),1.35(9H,s),1.79-1.90(2H,m),2.03-2.25(2 H,m),2.40(3H,s),2.40-2.51(2H,m),2.64-2.8 2(2H,m),2.98(1H,dd,J=13.7,4.6Hz),3.16(2H ,brs),3.55(1H,dd,J=16.7,5.7Hz),3.63-3.80 (4H,m),4.16-4.34(3H,m),4.36-4.50(2H,m),5 .23(2H,s),5.37(1H,d,J=16.5Hz),5.43(1H,d, J=16.5Hz),5.51-5.62(1H,m),6.52(1H,s),7.1 0-7.25(5H,m),7.26-7.33(3H,m),7.39(2H,t,J =7.3Hz),7.65-7.72(3H,m),7.80(1H,d,J=11.0 Hz),7.86(2H,d,J=7.3Hz),7.98(1H,t,J=5.5Hz ),8.07(1H,d,J=7.8Hz),8.15(1H,t,J=5.5Hz), 8.31(1H,t,J=5.5Hz),8.41(1H,d,J=8.7Hz). MS (ESI) m / z: 1147 (M+H) + .

[0218] Project 2: tert-ブチル(5S,14S)-14-アミノ-5-ベンジル-1-{[ (1S,9S)-9-エチル-5-フルオロ-9-ヒドロキシ-4-メチル-10,13 -Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d e]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]a amino}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazahexa Decane-16-oate N,N-dimethylformamide (0.279 g, 0.242 mmol) of the compound obtained in step 1 above A solution of fluoromethane (2.00 mL) in piperidine (0.240 mL, 2.42 mmol) The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. Chromatography [chloroform to chloroform:methanol = 2:1 (v / v)] The resulting mixture was purified by the procedure described above to give the title compound (0.265 g, quantitative) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.2Hz ),1.39(9H,s),1.81-1.94(1H,m),2.07-2.28(2 H,m),2.37(1H,dd,J=15.8,8.0Hz),2.43(3H,s) ,2.60(1H,dd,J=15.8,4.9Hz),2.75-2.82(1H,m ),3.00(1H,dd,J=13.9,4.5Hz),3.16-3.25(2H, m),3.50-3.61(2H,m),3.65-3.81(5H,m),4.40- 4.51(1H,m),5.27(2H,dd,J=24.1,19.0Hz),5.4 3(2H,dd,J=21.3,16.2Hz),5.56-5.65(1H,m),6 .55(1H,s),7.15-7.28(5H,m),7.33(1H,s),7.8 3(1H,d,J=11.0Hz),8.04(1H,t,J=5.7Hz),8.09 (1H,d,J=8.2Hz),8.26-8.39(2H,m),8.44(1H,d ,J=8.2Hz).

[0219] Step 3: tert-Butyl (5S,14S)-5-benzyl-14-{[6-(2,5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}- 1-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1 0,13-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzyl Nzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1- yl]amino}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazoline Hexadecane-16-oate The compound obtained in step 2 above (0.100 g, 0.108 mmol) was added to N,N-dimethylformamide. N-succinimidyl 6-maleimidohexanoate ( The mixture was stirred at room temperature for 2 days. The solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform :methanol = 9:1 (v / v)] to obtain the title compound (80.0 mg, 66%). Obtained as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.2Hz ),1.13-1.23(2H,m),1.37(9H,s),1.42-1.54(4 H,m),1.80-1.96(2H,m),2.08-2.25(4H,m),2.3 5-3.76(15H,m),2.43(3H,s),4.39-4.49(1H,m) ,4.55-4.67(1H,m),5.21-5.34(2H,m),5.43(2H ,dd,J=21.1,16.4Hz),5.56-5.64(1H,m),6.55( 1H,s),7.01(2H,d,J=0.8Hz),7.16-7.26(5H,m) ,7.33(1H,s),7.83(1H,d,J=11.3Hz),8.04-8.1 8(3H,m),8.30-8.37(1H,m),8.43(1H,d,J=8.6H z). MS(ESI)m / z:1118(M+H) + .

[0220] Step 4: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )Hexanoyl]-L-α-aspartylglycylglycyl-L-phenylalanyl-N -[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl ]Glycinamide Under ice cooling, the compound obtained in step 3 above (70.0 mg, 62.6 μmol) was added to trifluoro Acetic acid (4.00 mL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to give the title compound. (55.0 mg, 83%) was obtained as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.4Hz ),1.14-1.24(2H,m),1.41-1.53(4H,m),1.79-1 .95(2H,m),2.08-2.28(4H,m),2.37-2.60(2H,m ),2.42(3H,s),2.63-2.82(2H,m),2.99(1H,dd, J=14.1,5.1Hz),3.12-3.25(2H,m),3.29-3.44( 1H,m),3.52-3.80(6H,m),4.38-4.48(1H,m),4. 56(1H,dd,J=13.7,7.4Hz),5.27(2H,dd,J=24.3 ,18.8Hz),5.43(2H,dd,J=21.5,16.4Hz),5.57- 5.62(1H,m),6.55(1H,s),7.01(2H,s),7.15-7. 26(5H,m),7.33(1H,s),7.82(1H,d,J=11.0Hz), 7.98(1H,brs),8.08(1H,d,J=6.7Hz),8.15(1H, d,J=7.8Hz),8.34(1H,brs),8.44(1H,d,J=8.6H z), 12.26(1H,brs). MS(ESI)m / z:1062(M+H) + .

[0221] Step 5: Antibody-drug conjugate (5) Using U1-59 prepared in Reference Example 1 and the compound obtained in the above step 4, The title antibody-drug conjugate was obtained by the same method as in 6. Antibody concentration: 1.36 mg / mL, antibody yield: 8.16 mg (82%), common procedure E ( The molar extinction coefficient of the lag linker is ε D,280 =7620, ε D,370 Use =23700 ) The average number of drugs bound per antibody molecule (n): 5.0.

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

[0223] Step 1: Antibody-drug conjugate (6) Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 4 of Example 5, The title antibody-drug conjugate was obtained by the same method as in step 6. Antibody concentration: 11.5 mg / mL, antibody yield: 224.2 mg (90%), common procedure E( The molar extinction coefficient of the drug linker is ε D,280 =7620, ε D,370 Use =23700 The average number of drugs bound per antibody molecule (n) measured by ELISA (for ELISA): 4.6.

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

[0225] Step 1: tert-Butyl (3S,12S)-12-benzyl-21-{[(1S,9 S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-3 -{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4,7,10, 13,16,21-Hexaoxo-5,8,11,14,17-pentaazahenicosane 1-Noate (2S)-4-tert-butoxy-2-{[(9H-fluoren-9-ylmethoxy )carbonyl]amino}-4-oxobutanoic acid (0.625 g, 1.52 mmol) Dissolve N-hydroxysuccinimide (0.175 g) in dichloromethane (10.0 mL). , 1.52 mol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Imidazole hydrochloride (0.291 g, 1.52 mmol) was added and stirred for 1 hour. The compound obtained in Step 4 of Example 1 (1.00 g, 1.01 mmol) was added to N,N-dimethylformamide. The mixture was added dropwise to a dimethylformamide solution (10.0 mL) and stirred at room temperature for 20 hours. The residue was purified by silica gel column chromatography [chloroform, The product was purified with a 200 ml / min mixture of chloroform and methanol (8:2 (v / v)) to give the title compound (0.873 g, 7 0%) as a pale yellow solid. 1 H-NMR(400MHz,DMSO-d6)δ:0.88(3H,t,J=7.4Hz ),1.37(9H,s),1.68-1.78(2H,m),1.81-1.93(2 H,m),2.10-2.23(4H,m),2.41(3H,s),2.68-2.8 5(3H,m),2.99-3.22(5H,m),3.58-3.81(6H,m), 4.19-4.36(3H,m),4.38-4.52(2H,m),5.17(1H, d,J=19.2Hz),5.25(1H,d,J=19.2Hz),5.43(2H, s),5.54-5.62(1H,m),6.55(1H,s),7.15-7.34( 8H,m),7.41(2H,t,J=7.2Hz),7.66-7.75(4H,m) ,7.81(1H,d,J=11.0Hz),7.88(2H,d,J=7.4Hz), 8.01-8.06(1H,m),8.14(1H,d,J=8.2Hz),8.17- 8.22(1H,m),8.25-8.30(1H,m),8.47(1H,d,J=8 .6Hz). MS(APCI)m / z:1232(M+H) + .

[0226] Step 2: tert-Butyl (3S,12S)-12-benzyl-3-{[6-(2,5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}- 21-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H- Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 -yl]amino}-4,7,10,13,16,21-hexaoxo-5,8,11,1 4,17-Pentaazahenicosan-1-noate The compound obtained in step 1 above (0.800 g, 0.649 mmol) was dissolved in N,N-dimethylformamide. Dissolved in fluoromethane (3.00 mL) and piperidine (0.643 mL, 6.49 mmol) The solvent was evaporated to dryness under reduced pressure, and the resulting residue was dissolved in N,N-dimethylform. The resulting solution was dissolved in 10 mL of N-succinimidyl 6-maleimidohexanoate (0. The mixture was stirred for 20 hours. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform to chloroform:methanol). The title compound (0.224 g, 29%) was obtained as a pale yellow solid. Got it as a body. 1 H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.6Hz ),1.15-1.22(2H,m),1.35(9H,s),1.44-1.47(4 H,m),1.71-1.73(2H,m),1.80-1.91(2H,m),2.0 8(2H,t,J=7.6Hz),2.13-2.20(4H,m),2.40(3H, s),2.67(1H,dt,J=11.1,4.8Hz),2.78(1H,dd,J =13.6,9.4Hz),2.99-3.17(6H,m),3.31-3.36(2 H,m),3.57-3.76(6H,m),4.45-4.47(1H,m),4.5 7-4.60(1H,m),5.16(1H,d,J=18.7Hz),5.25(1H ,d,J=18.7Hz),5.42(2H,s),5.55-5.60(1H,m), 6.53(1H,s),6.99(2H,s),7.15-7.27(5H,m),7. 31(1H,s),7.70(1H,t,J=5.4Hz),7.80(1H,d,J= 10.9Hz),7.99(1H,t,J=5.7Hz),8.09-8.12(3H, m),8.25(1H,t,J=6.0Hz),8.45(1H,d,J=9.1Hz) . MS(APCI)m / z:1203(M+H) + .

[0227] Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )Hexanoyl]-L-α-aspartylglycylglycyl-L-phenylalanyl-N -(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H -benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- 1-yl]amino}-4-oxobutyl)glycinamide The compound obtained in Step 2 above (0.224 g, 0.186 mmol) was treated with Step 2 of Example 1. The same reaction was carried out to obtain the title compound (21.2 mg, 10%) as a pale yellow solid. 1H-NMR(400MHz,DMSO-d6)δ:0.87(3H,t,J=7.2Hz ),1.13-1.21(2H,m),1.42-1.45(6H,m),1.70-1 .72(2H,m),1.85-1.88(2H,m),2.06-2.20(6H,m ),2.39(3H,s),2.63-2.67(1H,m),2.78-2.81(1 H,m),3.04-3.12(6H,m),3.63-3.70(6H,m),4.4 6-4.52(2H,m),5.16(1H,d,J=19.2Hz),5.25(1H ,d,J=18.8Hz),5.42(2H,s),5.55-5.58(1H,m), 6.53(1H,s),6.99(2H,s),7.18-7.23(6H,m),7. 30(1H,s),7.71(1H,t,J=5.5Hz),7.79(1H,d,J= 10.9Hz),7.99-8.02(1H,m),8.10-8.11(3H,m), 8.27-8.30(1H,m),8.47-8.50(1H,m). MS(APCI)m / z:1147(M+H) + .

[0228] Step 4: Antibody-drug conjugate (7) Using U1-59 prepared in Reference Example 1 and the compound obtained in the above step 3, The title antibody-drug conjugate was obtained by the same method as in 6. Antibody concentration: 1.39 mg / mL, antibody yield: 8.34 mg (83%), common procedure E ( The molar extinction coefficient of the lag linker is ε D,280 =7670, ε D,370 Use =24800 ) The average number of drugs bound per antibody molecule (n): 4.7.

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

[0230] Step 1: Antibody-drug conjugate (8) Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 3 of Example 7, The title antibody-drug conjugate was obtained by the same method as in step 6. Antibody concentration: 11.2 mg / mL, antibody yield: 228.5 mg (91%), common procedure E( The molar extinction coefficient of the drug linker is ε D,280 =7670, ε D,370 Use =24800 The average number of drugs bound per antibody molecule (n) measured by ELISA (for ELISA): 4.7.

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

[0232] [ka]

[0233] Step 1: N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl} 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 of Example 1 (100 mg, 0.119 mmol) was added to 6-maleimide 3-(2-(2-(3-maleimidopropyl))-N-succinimidyl dodecanoate N-succinimidyl propanamide ethoxyethoxypropanoate (50.7 mg, 0.119 mmol) was reacted in the same manner as in Step 5 of Example 1 to obtain the title compound (66 0.5 mg, 48%) 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) + .

[0234] Step 2: Antibody-drug conjugate (9) Using U1-59 prepared in Reference Example 1 and the compound obtained in the above Step 1, The title antibody-drug conjugate was obtained by the same method as in 6. Antibody concentration: 2.08 mg / mL, antibody yield: 18.7 mg (94%), common procedure E ( The molar extinction coefficient of the lag linker is ε D,280 =4964, ε D,370 Use =18982 ) The average number of drugs bound per antibody molecule (n): 5.6.

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

[0236] Step 1: Antibody-drug conjugate (10) Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 1 of Example 9, The title antibody-drug conjugate was obtained by the same method as in step 6. Antibody concentration: 19.7 mg / mL, antibody yield: 236.4 mg (95%), common procedure E( The molar extinction coefficient of the drug linker is ε D,280 =4964, ε D,370 Use =18982 Average number of drugs bound per antibody molecule (n): 6.2; The molar extinction coefficient of the lag linker is ε D,280 Antibodies measured using 4964 Average number of drugs bound per molecule (n): 6.4.

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

[0238] [ka]

[0239] Step 1: Antibody-drug conjugate (11) Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 1 of Example 9, The title antibody-drug conjugate was obtained by the same method as in step 1. Antibody concentration: 0.88 mg / mL, antibody yield: 5.28 mg (53%), common procedure E ( The molar extinction coefficient of the lag linker is ε D,280 =4964, ε D,370 Use =18982 Average number of drugs bound per antibody molecule (n): 3.0; The molar extinction coefficient of the linker is ε D,280 Antibody measured using 4964 Average number of drug conjugates per molecule (n): 3.3.

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

[0241] Step 1: ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}a Methylaminoacetate N-9-Fluorenylmethoxycarbonylglycylglycine (4.33g, 12.2m mol), tetrahydrofuran (120 mL), and toluene (40.0 mL). The mixture was treated with pyridine (1.16 mL, 14.7 mmol) and lead tetraacetate (6.84 g, 1 4.7 mmol) was added and the mixture was heated under reflux for 5 hours. After the reaction mixture was cooled to room temperature, the insoluble matter was removed. The solvent was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The mixture was washed with water and saturated saline, and the organic layer was dried over anhydrous magnesium sulfate. After distilling off the solvent under reduced pressure, the resulting residue was purified by silica gel column chromatography [Hexa The product was purified using a mixture of ethanol and ethyl acetate (9:1 (v / v) to ethyl acetate) to give the title compound (3.00 g , 67%) was obtained as a colorless solid. 1 H-NMR(400MHz,CDCl3)δ:2.07(3H,s),3.90(2H, d,J=5.1Hz),4.23(1H,t,J=7.0Hz),4.46(2H,d, J=6.6Hz),5.26(2H,d,J=7.0Hz),5.32(1H,brs) ,6.96(1H,brs),7.32(2H,t,J=7.3Hz),7.41(2H ,t,J=7.3Hz),7.59(2H,d,J=7.3Hz),7.77(2H,d ,J=7.3Hz).

[0242] Step 2: Benzyl [({N-[(9H-fluoren-9-ylmethoxy)carbonyl] Glycyl(amino)methoxy]acetate 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).

[0243] 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 obtain 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).

[0244] Step 4: 9H-fluoren-9-ylmethyl (2-{[(2-{[(1S,9S)-9- Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3, 9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4 ':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-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 The solvent was evaporated under reduced pressure, and the resulting residue was concentrated in a silica gel column. 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 a pale brown solution. Obtained as a 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) + .

[0245] Step 5: N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy -4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-Benzo[de]pyrano[3',4':6,7]indolizino[1,2-b ]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide 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.

[0246] Step 6: N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl -L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro -9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]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 The compound described in Patent Publication No. 351 (0.440 g, 0.878 mmol) of N,N-dimethylformamide N,N'-dicyclohexylcarbodiimide in ethylformamide (50.0 mL) (0.181 g, 0.878 mmol) was added and stirred at room temperature for 4 days. The solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography [chloroform to chloroform The resulting mixture was purified with a hexanes / methanol ratio of 9:1 (v / v) to give the title compound (0.269 g, 58%). was obtained as a pale orange solid. MS(ESI)m / z:1063(M+H) + .

[0247] Step 7: Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3' ,4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-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.

[0248] Step 8: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S, 9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]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) + .

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

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

[0251] Step 1: Antibody-drug conjugate (13) Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 8 of Example 12, The title antibody-drug conjugate was obtained by the same method as in step 6 of 1. Antibody concentration: 22.2 mg / mL, antibody yield: 244.2 mg (98%), common procedure E( The molar extinction coefficient of the drug linker is ε D,280 =5178, ε D,370 =20217 Average number of drugs bound per antibody molecule (n): 6.2; The molar extinction coefficient of the lag linker is ε D,280 Antibodies measured using 5178 Average...

Claims

1. The anti-HER3 antibody was treated under reducing conditions and then reacted with the following compound: (maleimid-N-yl)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-DGGFG-(NH-DX), The following drug-linker structure: -(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-DGGFG-(NH-DX) A method for producing an antibody-drug conjugate in which the above is bound 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, -DGGFG- represents the pentapeptide residue -Asp-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 according to any one of claims 1 to 3, wherein the anti-HER3 antibody comprises CDRH1 to H3 and CDRL1 to L3 of U1-49, U1-53, U1-59, U1-7 or U1-9 in its heavy and light chains, respectively.

5. The method according to any one of claims 1 to 3, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region of U1-49, U1-53, U1-59, U1-7 or U1-9 in its heavy chain and light chain, respectively.

6. The method according to any one of claims 1 to 3, wherein the anti-HER3 antibody comprises the amino acid sequences shown in SEQ ID NOs: 42 and 44, 54 and 56, 70 and 72, 92 and 94, or 96 and 98 in its heavy chain and light chain, respectively.

7. The method according to any one of claims 1 to 3, wherein the anti-HER3 antibody comprises the amino acid sequences shown in SEQ ID NOs: 583 and 584 in its heavy and light chains, respectively.

8. The method according to claim 7, wherein the lysine residue at the carboxyl terminus of the heavy chain of the anti-HER3 antibody is deleted.

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