Novel method for producing antibody-drug conjugate

Abstract

Provided are a novel method for producing exatecan, which is a component of an antibody-drug conjugate, and a novel method for producing an antibody-drug conjugate using exatecan.SOLUTION: A method for producing a compound represented by the formula (C), which comprises a step of converting a compound represented by the formula (B) (wherein R1 is an amino group protected by a protective group) to a compound represented by the formula (C) (wherein R1 is as defined above) by intramolecular cyclization.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel method for producing exatecan, a component of an antibody-drug conjugate; and a novel method for producing an antibody-drug conjugate using the same. [Background technology]

[0002] Antibodies that bind to antigens that are expressed on the surface of cancer cells and can be internalized into the cells are used to produce cytotoxic antibodies. Antibody-drug conjugates (Antibody-Drug Conjugates) ADCs (anti-cancer drug delivery systems) are effective in treating cancer by selectively delivering drugs to cancer cells. It is expected that drugs will accumulate inside cells and kill cancer cells (Non-patent documents 1-5). .

[0003] One of the antibody-drug conjugates is a combination of an antibody and an exon, a topoisomerase I inhibitor. Antibody-drug conjugates containing satecan as a component are known (Patent Documents 1 to 8, Non-patent documents 6, 7) These antibody-drug conjugates have excellent antitumor effects and safety. Clinical trials are currently underway.

[0004] Known methods for producing exatecan include those described in Patent Documents 9 to 11. . [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 057687 [Patent Document 2] International Publication No. 2014 / 061277 [Patent Document 3] International Publication No. 2015 / 098099 [License 4] International Publication No. 2015 / 115091 [Patent Document 5] International Publication No. 2015 / 146132 [License 6] International Publication No. 2015 / 155976 [License 7] International Publication No. 2015 / 155998 [License 8] International Publication No. 2018 / 135501 [License 9] Special Announcement No. 5-59061 [License 10] Special Announcement No. 8-337584 [License 11] International Publication No. 96 / 26181 [Non-licensed literature]

[0006] [Non-licensed Document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-licensed Document 2] Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [Non-licensed Document 3] Damle NK Expert Opin. Biol. Ther. (2004) 4, 1445-1452. [Non-licensed Document 4] Senter PD, et al., Nature Biotechnology (2012) 30, 631-637. [Non-licensed Document 5] Howard A. et al., J Clin Oncol 29: 398-405. [Non-licensed Document 6] Ogitani Y. et al., Clinical Cancer Research (2016) 22(20), 5097-5108. [Non-Patent Document 7] Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046. Summary of the Invention [Problem to be solved by the invention]

[0007] Exatecan is a compound of formula (2)

[0008] [ka]

[0009] and is a compound represented by the formula (I): It is a mixture.

[0010] Known methods for producing exatecan include those described in Patent Documents 9 to 11. This manufacturing method can be expressed as follows: That is, the compound represented by formula (19) The compound is reacted with succinic anhydride to convert it to a compound represented by formula (20), which is then reduced to give a compound represented by formula (2 1), which is then converted to a compound represented by formula (22) by intramolecular cyclization. The compound represented by formula (23) is converted into an oxime by Beckmann rearrangement. 4), which is then converted by ring-opening reaction into a compound represented by formula (25), The amino group is protected to convert it to a compound represented by formula (26), which is then hydrolyzed to give a compound represented by formula (27). This is converted to a compound represented by formula (28) by intramolecular cyclization, and then reduced to to the compound represented by formula (29), which is then converted by oxidation to the compound represented by formula (9). Then, a nitrogen atom is introduced to convert it into a compound represented by formula (10), and selective deprotection is performed to obtain and converting the compound represented by formula (11) into a compound represented by formula (1), The compound is converted to the compound represented by formula (12) by the der reaction, and finally, the acetyl group is deprotected. This is a method for producing a compound represented by formula (2), i.e., exatecan, by However, this manufacturing method requires repeated ring-opening and ring-closing reactions and oxidation and reduction reactions. Therefore, the number of steps is long and complicated operations are required. There is a need to develop a method for this.

[0011] [ka]

[0012] One object of the present invention is to provide a novel method for producing exatecan which requires fewer steps and is industrially superior. Furthermore, we will develop a new method for producing antibody-drug conjugates using this method. Another object of the present invention is to achieve this. [Means for solving the problem]

[0013] As a result of extensive research into a method for producing exatecan, the present inventors have discovered a method that requires fewer steps and can be produced industrially. We have discovered a novel and excellent method for producing exatecan. A new method for producing antibody-drug conjugates using xatecan was developed. That is, the present invention provides: [1] Formula (B)

[0014] [ka]

[0015] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group) is By doing so, Formula (C)

[0016] [ka]

[0017] A compound represented by the formula (wherein R 1 has the same meaning as defined above), C) A method for producing a compound represented by the formula: [2] R 1 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. an amino group protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group; ] The manufacturing method described in. [3] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group, The manufacturing method described above. [4] R 1 is an amino group protected with an acetyl group. [5] Intramolecular cyclization involves reacting a compound of formula (B) with trifluoroacetic anhydride. The method according to any one of [1] to [4], wherein the method comprises the steps of: [6] The method according to [5], wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid. [7] The process wherein the intramolecular cyclization comprises reacting a compound of formula (B) with thionyl chloride. The method according to any one of [1] to [4], wherein the manufacturing method is carried out by [8] The method according to [7], wherein the intramolecular cyclization is carried out in the presence of aluminum chloride. [9] Formula (J)

[0018] [ka]

[0019] (wherein Y represents a leaving group, and R 1 represents an amino group protected by a protecting group. (shown in Fig. 1) is subjected to intramolecular cyclization to give Formula (C)

[0020] [ka]

[0021] A compound represented by the formula (wherein R 1 has the same meaning as defined above), C) A method for producing a compound represented by the formula:

[10] R 1 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. an amino group protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group; ] The manufacturing method described in.

[11] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group, The manufacturing method described above.

[12] R 1 is an amino group protected with an acetyl group.

[13] The method according to any one of [9] to

[12] , wherein Y is a chloro group.

[14]

[13] The process according to any one of [9] to

[12] , wherein Y is a trifluoroacetoxy group. Construction method.

[15] The method according to

[13] , wherein the intramolecular cyclization is carried out in the presence of aluminum chloride.

[16] The method according to

[14] , wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid. .

[17] Formula (D)

[0022] [ka]

[0023] (wherein X represents a leaving group, and R 1 indicates an amino group protected by a protecting group. ) and 3-butenoic acid are coupled, Formula (E)

[0024] [ka]

[0025] A compound represented by the formula (wherein R 1 has the same meaning as above), and then a step of converting the compound of formula ( E) by reducing a compound represented by the formula Formula (B)

[0026] [ka]

[0027] A compound represented by the formula (wherein R 1 has the same meaning as above), and then a step of converting the compound of formula ( B) is subjected to intramolecular cyclization of a compound represented by the formula (I), Formula (C)

[0028] [ka]

[0029] A compound represented by the formula (wherein R1 has the same meaning as defined above), A method for producing a compound represented by formula (C), comprising:

[18] X is a bromo group, an iodo group, a trifluoromethanesulfonyloxy group, or an arylsulfonyl group. The method according to

[17] , wherein the aryloxy group is a phenyloxy group.

[19] The method according to

[17] , wherein X is a bromo group.

[20] The method according to

[17] , wherein X is an iodo group. [twenty one] R 1 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method for producing the compound according to any one of

[0017] to

[20] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group. [twenty two] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group, [7]

[10] The method for producing the present invention described in any one of

[10] . [twenty three] R 1 Any one of

[17] to

[20] , wherein The manufacturing method described in [twenty four] The compound represented by formula (D) is coupled with 3-butenoic acid to obtain the compound represented by formula (E). The process of converting the compound into a compound prepared from palladium(II) acetate and tri(o-tolyl)phosphine

[17] to

[23] , which is carried out in the presence of a palladium complex The manufacturing method described above. [twenty five] The compound represented by formula (E) is dissolved in a basic aqueous solution and washed with a first organic solvent. Then, an acid is added to the basic aqueous solution, and the compound represented by formula (E) is reacted with a second organic solvent.

[24] The method according to any one of

[17] to

[24] , further comprising a step of subjecting the mixture to liquid separation and extraction. Law.

[26] The method according to

[25] , wherein the first organic solvent is 2-methyltetrahydrofuran. .

[27]

[25] or

[26] , wherein the second organic solvent is 2-methyltetrahydrofuran. The manufacturing method described above.

[28] Any one of

[25] to

[27] , wherein the basic aqueous solution is an aqueous solution of sodium hydroxide. The manufacturing method described in paragraph .

[29] The step of reducing the compound represented by formula (E) and converting it into the compound represented by formula (B) comprises the step of: A method of reacting a compound represented by (E) with hydrogen in a solvent in the presence of a palladium carbon catalyst The method according to any one of

[17] to

[28] , wherein the manufacturing method is carried out by

[30] A step of intramolecularly cyclizing the compound represented by formula (B) to convert it into a compound represented by formula (C). by a process comprising reacting a compound of formula (B) with trifluoroacetic anhydride. The manufacturing method according to any one of

[17] to

[29] , wherein the manufacturing method is carried out by

[31] The method according to

[30] , wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid. .

[32] A step of intramolecularly cyclizing the compound represented by formula (B) to convert it into a compound represented by formula (C). is carried out by a process comprising reacting a compound of formula (B) with thionyl chloride. The method according to any one of

[17] to

[29] ,

[33] The method according to

[32] , wherein the intramolecular cyclization is carried out in the presence of aluminum chloride.

[34] Produced by the method described in any one of [1] to

[33] Formula (C)

[0030] [ka]

[0031] The method is characterized in that a compound represented by the formula (I) is used as a starting material, The compound represented by formula (C) Formula (F)

[0032] [ka]

[0033] A compound represented by the formula (wherein R 1 is R according to any one of claims 1 to 33 1 Agree with Showing righteousness, R 2 represents an amino group protected by a protecting group), and then ) a compound represented by Formula (G)

[0034] [ka]

[0035] A compound represented by the formula (wherein R 2 has the same meaning as above), and then a step of converting the compound of formula ( G) a compound represented by the formula Formula (1)

[0036] [ka]

[0037] Condensing a compound represented by the formula: Formula (H)

[0038] [ka]

[0039] A compound represented by the formula (wherein R 2 has the same meaning as above), and then a step of converting the compound of formula ( H) a compound represented by Formula (2)

[0040] [ka]

[0041] A method for producing a compound represented by formula (2), comprising a step of converting a compound represented by formula (2) into a compound represented by formula (2).

[35] R 2 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method for producing the compound according to the above item

[0034] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[36] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[34] The manufacturing method described in

[37] R 2 is an amino group protected with an acetyl group.

[38] The step of converting the compound represented by formula (C) into the compound represented by formula (F) comprises the steps of: (i) forming a salt a step of reacting the compound with a nitrite ester in the presence of a group to introduce a nitroso group, and then (ii) ) introducing a protecting group onto the nitrogen atom derived from the nitroso group, and (iii) using a platinum-carbon catalyst.

[34] to

[37] , comprising a step of reducing the compound with hydrogen in the presence of The manufacturing method described above.

[39] The step of converting the compound represented by formula (F) into the compound represented by formula (G) is carried out using a hydrochloric acid / ethanol The method according to any one of

[34] to

[38] , wherein the method is carried out in a solvent containing ethanol. Law.

[40] A compound represented by formula (G) and a compound represented by formula (1) are condensed to obtain a compound represented by formula (H). The method according to any one of

[34] to

[0039] , wherein the step of converting the compound into a compound having the formula (I) is carried out in a solvent containing o-cresol.

[41] The step of converting the compound represented by formula (H) into the compound represented by formula (2) is The method according to any one of

[34] to

[40] , which is carried out in a solvent containing sulfonic acid. Law.

[42] Any of

[34] to

[41] , wherein the compound represented by formula (2) is a methanesulfonate salt. 1. The manufacturing method according to any one of claims 1 to 9.

[43] The compound represented by formula (2) is methanesulfonate·m hydrate (where m is 0 to 3). The method according to any one of

[34] to

[41] , wherein the range is

[44] The method according to any one of

[34] to

[0041] , wherein the compound represented by formula (2) is methanesulfonate anhydride.

[45] The method according to any one of

[34] to

[0041] , wherein the compound represented by formula (2) is methanesulfonate monohydrate.

[46] The method according to any one of

[34] to

[0041] , wherein the compound represented by formula (2) is methanesulfonate dihydrate.

[47] The method according to any one of

[34] to

[0041] , wherein the compound represented by formula (2) is methanesulfonate trihydrate.

[48] Formula (3)

[0042] [ka]

[0043] A compound represented by Formula (4)

[0044] [ka]

[0045] and then converting the compound represented by formula (4) into a compound represented by formula (5): Formula (5)

[0046] [ka]

[0047] and then converting the compound represented by formula (5) into a compound represented by formula (6): Formula (6)

[0048] [ka]

[0049] and then reacting the compound represented by formula (6) with 3-butenoic acid. Coupling Formula (7)

[0050] [ka]

[0051] and then converting the compound represented by formula (7) into a compound represented by formula (8). Formula (8)

[0052] [ka]

[0053] and then intramolecularly cyclizing the compound represented by formula (8), Formula (9)

[0054] [ka]

[0055] and then converting the compound represented by formula (9) into a compound represented by formula (9): Formula (10)

[0056] [ka]

[0057] and then converting the compound represented by formula (10) into a compound represented by formula (11): Formula (11)

[0058] [ka]

[0059] and then a compound represented by formula (11), Formula (1)

[0060] [ka]

[0061] Condensing a compound represented by the formula: Formula (12)

[0062] [ka]

[0063] and then converting the compound represented by formula (12) into a compound represented by formula (13): Formula (2)

[0064] [ka]

[0065] A method for producing a compound represented by formula (2), comprising a step of converting a compound represented by formula (2) into a compound represented by formula (2).

[49] The compound represented by formula (6) is coupled with 3-butenoic acid to give the compound represented by formula (7). The process of converting the compound into a compound prepared from palladium(II) acetate and tri(o-tolyl)phosphine The production method according to

[48] , wherein the reaction is carried out in the presence of a palladium complex.

[50] The compound represented by formula (7) is dissolved in a basic aqueous solution and washed with a first organic solvent. Then, an acid is added to the basic aqueous solution, and the compound represented by formula (7) is reacted with a second organic solvent.

[48] ​​or

[49] , comprising a step of subjecting the mixture to liquid separation and extraction.

[51] The method according to

[50] , wherein the first organic solvent is 2-methyltetrahydrofuran. .

[52]

[50] or

[51] , wherein the second organic solvent is 2-methyltetrahydrofuran. The manufacturing method described above.

[53] Any one of

[50] to

[52] , wherein the basic aqueous solution is an aqueous solution of sodium hydroxide. The manufacturing method described in paragraph .

[54] A step of intramolecularly cyclizing the compound represented by formula (8) to convert it into a compound represented by formula (9). However, by a method comprising reacting a compound represented by formula (8) with trifluoroacetic anhydride The manufacturing method according to any one of

[50] to

[53] , wherein the manufacturing method is carried out by

[55] The method according to

[54] , wherein the intramolecular cyclization is carried out in a solvent containing trifluoroacetic acid. .

[56] The step of converting a compound represented by formula (9) into a compound represented by formula (10) comprises the steps of: (i) a step of reacting with a nitrite in the presence of a base to introduce a nitroso group, and then (i i) introducing a protecting group onto the nitrogen atom derived from the nitroso group, and (iii) introducing a protecting group onto the nitrogen atom derived from the nitroso group,

[48] ​​to

[55] , comprising a step of reducing the compound with hydrogen in the presence of a catalyst. The manufacturing method described above.

[57] The step of converting the compound represented by formula (10) into the compound represented by formula (11) comprises the step of: / The process according to any one of

[48] to

[56] , which is carried out in a solvent containing ethanol. Construction method.

[58] A compound represented by formula (11) and a compound represented by formula (1) are condensed to obtain a compound represented by formula (12). The step of converting the compound to the compound to be obtained is carried out in a solvent containing o-cresol,

[48]

[57] The method according to any one of the preceding items.

[59] The step of converting the compound represented by formula (12) into the compound represented by formula (2) is carried out by using methane

[58] The process according to any one of

[48] to

[58] , which is carried out in a solvent containing sulfonic acid. method.

[60] Any of

[48] to

[59] , wherein the compound represented by formula (2) is a methanesulfonate salt. 1. The manufacturing method according to any one of claims 1 to 9.

[61] The compound represented by formula (2) is methanesulfonate·m hydrate (where m is 0 to 3). The method according to any one of

[48] to

[59] , wherein the range is

[62] The method according to any one of

[48] to

[0059] , wherein the compound represented by formula (2) is methanesulfonate anhydride.

[63] The method according to any one of

[48] to

[0059] , wherein the compound represented by formula (2) is methanesulfonate monohydrate.

[64] The method according to any one of

[48] to

[0059] , wherein the compound represented by formula (2) is methanesulfonate dihydrate.

[65] The method according to any one of

[48] to

[0059] , wherein the compound represented by formula (2) is methanesulfonate trihydrate.

[66] Formula (D)

[0066] [ka]

[0067] (wherein X represents a leaving group, and R 1 indicates an amino group protected by a protecting group. ) and 3-butenoic acid are coupled, Formula (E)

[0068] [ka]

[0069] A compound represented by the formula (wherein R 1 is as defined above), ) A method for producing a compound represented by the formula:

[67] X is a bromo group, an iodo group, a trifluoromethanesulfonyloxy group, or an arylsulfonyl group. The method according to

[66] , wherein the aryloxy group is a phenyloxy group.

[68] The method according to

[66] , wherein X is a bromo group.

[69] The method according to

[66] , wherein X is an iodo group.

[70] R 1are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method according to any one of

[0066] to

[69] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[71] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[66]

[69] A manufacturing method according to any one of

[69] to

[69] .

[72] R 1 Any one of

[66] to

[69] , wherein The manufacturing method described in

[73] Palladium complexes prepared from palladium(II) acetate and tri(o-tolyl)phosphine. The method according to any one of

[66] to

[72] , wherein the method is carried out in the presence of a living body.

[74] The compound represented by formula (E) is dissolved in a basic aqueous solution and washed with a first organic solvent. Then, an acid is added to the basic aqueous solution, and the compound represented by formula (E) is reacted with a second organic solvent.

[73] , further comprising a step of subjecting the mixture to liquid separation and extraction. Law.

[75] The method according to

[74] , wherein the first organic solvent is 2-methyltetrahydrofuran. .

[76]

[74] or

[75] , wherein the second organic solvent is 2-methyltetrahydrofuran. The manufacturing method described above.

[77] Any one of

[74] to

[76] , wherein the basic aqueous solution is an aqueous solution of sodium hydroxide. The manufacturing method described in paragraph .

[78] Formula (E)

[0070] [ka]

[0071] A compound represented by the formula (wherein R 1 represents an amino group protected by a protecting group), Formula (B)

[0072] [ka]

[0073] A compound represented by the formula (wherein R 1 is as defined above), ) A method for producing a compound represented by the formula:

[79] R 1 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method for producing the compound according to the above

[0078] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[80] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[78] The manufacturing method described in

[81] R 1 is an amino group protected with an acetyl group.

[82] The compound represented by formula (E) is reacted with hydrogen in a solvent in the presence of a palladium carbon catalyst. The method according to any one of

[78] to

[81] , wherein the method is carried out by the method.

[83] Formula (C)

[0074] [ka]

[0075] A compound represented by the formula (wherein R 1represents an amino group protected by a protecting group), (i) reacting with a nitrite in the presence of a base to introduce a nitroso group; (ii) introducing a protecting group onto the nitrogen atom derived from the nitroso group; and (iii) platinum By including a step of reducing with hydrogen in the presence of a carbon catalyst, Formula (F)

[0076] [ka]

[0077] A compound represented by the formula (wherein R 1 has the same meaning as above, and R 2 is an amino group protected by a protecting group. A method for producing a compound represented by formula (F), comprising a step of converting a hydroxyl group into a hydroxyl group (representing a hydroxyl group).

[84] R 1 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method for producing the compound according to

[0083] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[85] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[83] The manufacturing method described in

[86] R 1 is an amino group protected with an acetyl group.

[87] R 2 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method according to any one of

[0083] to

[86] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[88] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[83]

[86] A method for producing a compound according to any one of the preceding claims.

[89] R 2 Any one of

[83] to

[86] , wherein The manufacturing method described in

[90] Formula (F)

[0078] [ka]

[0079] A compound represented by the formula (wherein R 1 and R 2 indicates an amino group protected by a protecting group) In a solvent containing acid / ethanol, Formula (G)

[0080] [ka]

[0081] A compound represented by the formula (wherein R 2 has the same meaning as above), ) A method for producing a compound represented by the formula:

[91] R 1 are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method for producing the compound according to

[0090] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[92] R 1 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[90] The manufacturing method described in

[93] R 1 is an amino group protected with an acetyl group.

[94] R 2are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method according to any one of

[0090] to

[93] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[95] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[90]

[93] A method for producing a compound according to any one of

[93] to

[93] .

[96] R 2 Any one of

[90] to

[93] , wherein The manufacturing method described in

[97] Formula (G)

[0082] [ka]

[0083] A compound represented by the formula (wherein R 2 represents an amino group protected by a protecting group), and Formula (1)

[0084] [ka]

[0085] in a solvent containing o-cresol, Formula (H)

[0086] [ka]

[0087] A compound represented by the formula (wherein R 2 is as defined above), ) A method for producing a compound represented by the formula:

[98] R 2are acetyl, methoxyacetyl, trifluoroacetyl, and trichloroacetyl groups. The method for producing the compound according to

[0097] , wherein the amino group is protected by a methyl group, a pivaloyl group, a formyl group, or a benzoyl group.

[99] R 2 is an amino group protected with an acetyl group or a trifluoroacetyl group.

[97] The manufacturing method described in

[0100] R 2 is an amino group protected with an acetyl group.

[0101] Any of [1] to

[0100] , characterized in that no chromatography is used. 1. The method of manufacturing according to claim 1.

[0102] Formula (6)

[0088] [ka]

[0089] A compound represented by the formula:

[0103] Formula (34)

[0090] [ka]

[0091] A compound represented by the formula:

[0104] Formula (7)

[0092] [ka]

[0093] A compound represented by the formula:

[0105] Formula (8)

[0094] [ka]

[0095] A compound represented by the formula:

[0106] Produced by the method described in any one of

[34] to

[65] Formula (2)

[0096] [ka]

[0097] The method is characterized in that a compound represented by the formula (I) is used as a starting material, A compound represented by formula (2), Formula (13)

[0098] [ka]

[0099] By condensing a compound represented by Formula (14)

[0100] [ka]

[0101] A method for producing a compound represented by formula (14), comprising:

[0107]

[0106] Formula (14)

[0102] [ka]

[0103] The method is characterized in that a compound represented by the formula: (i) reducing the antibody, and then (ii) The compound represented by formula (14) prepared by the above method is reacted with a reduced antibody. a process of Including, Formula (15)

[0104] [ka]

[0105] (wherein A represents the binding site to the antibody) and an antibody-drug conjugate in which the antibody is bound to a drug linker represented by the formula: Method for producing Jugate.

[0108] The antibody is anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, or is an anti-GPR20 antibody, a manufacturing method described in

[0107] .

[0109] A manufacturing method described in

[0108] , wherein the antibody is an anti-HER2 antibody.

[0110] The anti-HER2 antibody comprises an amino acid sequence set forth in amino acid numbers 1 to 449 in SEQ ID NO: 1. a heavy chain consisting of a sequence of amino acids 1 to 214 in SEQ ID NO: 2; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1; and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2. The manufacturing method described above.

[0111] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 7 to A manufacturing method described in

[0109] or

[0110] , in which the number of the compounds is in the range of 8.

[0112] A manufacturing method described in

[0108] , wherein the antibody is an anti-HER3 antibody.

[0113] The anti-HER3 antibody comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a An antibody comprising a light chain consisting of the amino acid sequence set forth above, or the carboxyl terminal of the heavy chain of said antibody. The method for producing an antibody described in

[0112] , wherein the antibody has a deleted lysine residue.

[0114] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 7 to A manufacturing method described in

[0112] or

[0113] , in which the number of the compounds is in the range of 8.

[0115] A manufacturing method described in

[0108] , in which the antibody is an anti-TROP2 antibody.

[0116] The anti-TROP2 antibody comprises the amino acid sequence set forth in amino acid numbers 20 to 470 of SEQ ID NO:5. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 6; an antibody comprising a light chain consisting of a lysine residue at the carboxyl terminus of a heavy chain of said antibody; A method for producing an antibody having a missing group, as described in

[0115] .

[0117] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 3 to A manufacturing method described in

[0115] or

[0116] , in which the number of the compounds is in the range of 5.

[0118] A manufacturing method described in

[0108] , wherein the antibody is an anti-B7-H3 antibody.

[0119] The anti-B7-H3 antibody is selected from the group consisting of amino acids 20 to 471 in SEQ ID NO:7. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 8; an antibody comprising a light chain consisting of a lysine residue at the carboxyl terminus of a heavy chain of said antibody; A method for producing an antibody having a missing group, as described in

[0118] .

[0120] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 3 to A manufacturing method described in

[0118] or

[0119] , in which the number of the compounds is in the range of 5.

[0121] A manufacturing method described in

[0108] , wherein the antibody is an anti-GPR20 antibody.

[0122] The anti-GPR20 antibody is a polypeptide comprising the amino acids 20 to 472 of SEQ ID NO: 9. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 10; an antibody comprising a light chain consisting of the carboxyl-terminal lysine of said antibody heavy chain, A method for producing an antibody having deleted residues, as described in

[0121] .

[0123] The average number of drug linkers per antibody in antibody-drug conjugates ranges from 7 to A manufacturing method described in

[0121] or

[0122] , in which the number of the compounds is in the range of 8. Regarding. [Effects of the Invention]

[0106] The present invention provides a novel method for producing exatecan which has a short number of steps and is industrially excellent. Furthermore, a novel method for producing an antibody-drug conjugate using this is provided. It is possible. [Brief explanation of the drawings]

[0107] [Figure 1] 1 shows the amino acid sequence of the anti-HER2 antibody heavy chain (SEQ ID NO: 1). [Figure 2] 1 shows the amino acid sequence of the anti-HER2 antibody light chain (SEQ ID NO: 2). [Figure 3] 1 shows the amino acid sequence of the anti-HER3 antibody heavy chain (SEQ ID NO: 3). [Figure 4] 1 shows the amino acid sequence of the anti-HER3 antibody light chain (SEQ ID NO: 4). [Figure 5] The amino acid sequence of the anti-TROP2 antibody heavy chain (SEQ ID NO: 5) is shown. [Figure 6] The amino acid sequence of the anti-TROP2 antibody light chain (SEQ ID NO: 6) is shown. [Figure 7] The amino acid sequence of the anti-B7-H3 antibody heavy chain (SEQ ID NO: 7) is shown. [Figure 8] The amino acid sequence of the anti-B7-H3 antibody light chain (SEQ ID NO: 8) is shown. [Figure 9] The amino acid sequence of the anti-GPR20 antibody heavy chain (SEQ ID NO: 9) is shown. [Figure 10] The amino acid sequence of the anti-GPR20 antibody light chain (SEQ ID NO: 10) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0108] A preferred embodiment of the present invention will be described below. The embodiment shows an example of a typical embodiment of the present invention, and the present invention The scope will not be interpreted narrowly.

[0109] [Antibody-drug conjugates]

[0110] The antibody-drug conjugate produced by the present invention is preferably Formula (15)

[0111] [ka]

[0112] (wherein A represents the binding site to the antibody) and an antibody-drug conjugate in which the antibody is bound to a drug linker represented by the formula: It's Jugate.

[0113] In the present invention, the portion of the antibody-drug conjugate consisting of a linker and a drug The structure is called a "drug linker." This drug linker binds to the disulfide bond between the antibody chains. Thiol groups (in other words) generated at positions (two heavy chain-heavy chain interchains and two heavy chain-light chain interchains) In other words, it is bound to the sulfur atom of a cysteine ​​residue.

[0114] The drug linker of the present invention comprises exatecan, a topoisomerase I inhibitor, as a component. Exatecan is a compound of formula (2)

[0115] [ka]

[0116] and is a camptothecin derivative having antitumor effects.

[0117] The antibody-drug conjugate used in the present invention is Formula (16)

[0118] [ka]

[0119] It can also be expressed as

[0120] Here, the drug linker is bound to the antibody via a thioether bond. The so-called average number of drug-antibody bindings (DAR; Drug-to-Antibody Ratio) It is synonymous with and indicates the average number of drug linkers bound per antibody. The antibody-drug conjugate used in the present invention, after being transported into cancer cells, Formula (18)

[0121] [ka]

[0122] The compound represented by the formula (I) is released, thereby exerting an antitumor effect.

[0123] The compound represented by formula (18) is an antibody-drug conjugate produced by the present invention. It is believed to be the main cause of the antitumor activity of α-tocopherol, and has been confirmed to have topoisomerase I inhibitory activity. (Ogitani Y. et al., Clinical Cancer Research, 2016, Oct 15;22(20):509 7-5108, Epub 2016 Mar 29).

[0124] The compound represented by formula (18) is an antibody-drug conjugate produced by the present invention. This is thought to be caused by cleavage of the linker portion of Formula (17)

[0125] [ka]

[0126] It is believed that this occurs due to the decomposition of the aminal structure of the compound represented by the formula: The antibody-drug conjugates produced by the present invention have a bystander effect It is also known that cancer cells are involved in the regulation of inflammatory bowel disease (Ogitani Y. et al., Cancer Science (2016) 107, 1039-1046). This bystander effect is due to the fact that the antibody-drug conjugates produced according to the present invention After being internalized into the target-expressing cancer cells, the released compound represented by formula (18) expresses the target. This is achieved by exerting an antitumor effect on nearby cancer cells that have not yet expressed the tumor.

[0127] [Exatecan manufacturing] The exatecan of the present invention can be produced according to the following method.

[0128] [ka]

[0129] [In the formula, X represents a leaving group, and is preferably a bromo group, an iodo group, or a trifluoromethanesulfonyl group.] and more preferably a bromo group or an iodine group. R represents a halogen group, and even more preferably represents a bromo group; 1 represents an amino group protected by a protecting group. It represents a group, and preferably represents an acetyl group, a methoxyacetyl group, a trifluoroacetyl group, a tri Amino groups protected with chloroacetyl, pivaloyl, formyl, or benzoyl groups and more preferably an amino group protected with an acetyl group or a trifluoroacetyl group. represents an amino group protected with an acetyl group; R 2 is protected represents an amino group, and is preferably an acetyl group, a methoxyacetyl group, or a trifluoroacetyl group , trichloroacetyl group, pivaloyl group, formyl group, or benzoyl group protected It represents an amino group, more preferably an amino group protected with an acetyl group or a trifluoroacetyl group. and more preferably an amino group protected with an acetyl group.

[0130] Step 1: In this step, the compound represented by formula (D) is coupled with 3-butenoic acid to give The compound represented by formula (D) can be obtained by the known method. The amount of 3-butenoic acid used in this step can be determined by the reaction There is no limitation as long as the reaction proceeds, but preferably, the reaction is carried out in a ratio of 1 to 1. 5 equivalents.

[0131] The coupling reaction can be carried out in the presence of a transition metal catalyst, preferably palladium. The palladium catalyst used in this step is a catalyst that allows the reaction to proceed. There is no particular limitation as long as it is a compound that can be used. For example, palladium (II) acetate, trifluoroacetic acid, Palladium(II) chloride, palladium(II) bromide, palladium(II) iodide divalent palladium(II), bis(triphenylphosphine)palladium(II) chloride, etc. Palladium salts and their complexes, palladium black, palladium carbon, tetrakis Triphenylphosphinepalladium(0), bis(dibenzylideneacetone)palladium( Palladium metal with a valence of 0, such as palladium acetate, and its complexes can be used. Radium (II) can be used. The amount of palladium catalyst used in this step is There are no limitations as long as the reaction proceeds, but it is preferable to use 0. 0.03 to 0.03 equivalents.

[0132] Furthermore, in this step, in addition to the above-mentioned palladium catalyst, a palladium complex is preferably used in the reaction system. The ligands that can be used in this step include For example, triphenylphosphine, tri(o-tolyl)phosphine, tri(3-methoxy)phosphine, tri(4-chlorophenyl)phosphine, tri(2-furyl) Phosphine, tri(2-thienyl)phosphine, 1,2-bis(diphenylphosphino) Ethane, and the Buchwald ligand (2-dicyclohexylphosphino-2',6' -Dimethoxybiphenyl (SPhos), 2-dicyclohexylphosphino-2',4 ',6'-triisopropylbiphenyl (XPhos), etc.) can be used, and For this purpose, tri(o-tolyl)phosphine can be used. The amount of is not limited as long as the reaction proceeds, but is preferably The amount is 0.006 to 0.06 equivalents.

[0133] This step can be preferably carried out in the presence of a base. The solvent is not particularly limited as long as the reaction proceeds, but examples thereof include triethylamine, tributylamine, and the like. amine, diisopropylethylamine, N-methylmorpholine, N-methylpyrrolidine, N -Methylpiperidine, pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 4- Dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, 1,8-di Azabicyclo[5.4.0]undec-7-ene and 1,5-diazabicyclo[4.3 .0]undec-7-ene and other organic bases, potassium carbonate, potassium hydroxide, potassium bicarbonate, etc. Sodium, sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, vinegar Potassium tert-oxide, sodium methoxide, sodium ethoxide, and potassium tert- Examples of suitable inorganic bases include triethylamine, tributyl butoxide, etc. Amine, diisopropylethylamine, potassium carbonate, potassium hydroxide, potassium bicarbonate Sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, and vinegar Potassium carbonate is preferred, and diisopropylethylamine is more preferred. The amount of base used in this step is not limited as long as the reaction proceeds. The amount is 2 to 3 equivalents relative to the compound represented by formula (D).

[0134] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Methyl-2-pyrrolidone, dimethyl sulfoxide, and water, or a mixture thereof, were used. A preferred example is tetrahydrofuran.

[0135] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 45 to 85°C. The reaction temperature in this step is preferably a temperature at which tetrahydrofuran is heated to reflux. The reaction time is not limited as long as the reaction proceeds, but is preferably 2.5 to 10 hours.

[0136] The compound represented by formula (E) can be prepared by dissolving the compound represented by formula (E) in a basic aqueous solution. a step of separating and washing with a first organic solvent, followed by adding an acid to the basic aqueous solution and washing with a second organic solvent; and extracting the compound represented by formula (E) with a solvent. The first organic solvent is preferably 2-methyltetrahydrofuran. The second organic solvent is preferably 2-methyltetrahydrofuran. , preferably an aqueous sodium hydroxide solution.

[0137] The compound represented by formula (E) has two geometric isomers, E and Z. All of these are included in the compounds represented by formula (E) and are within the scope of the present invention. The compound represented by formula (E) may be a mixture of E and Z isomers, and the mixture may be used in the next step. It can be used to a certain extent.

[0138] In this step, 3-butenoic acid ester can also be used instead of 3-butenoic acid. In this case, the compound represented by formula (D) is coupled with a 3-butenoic acid ester to form a compound represented by formula (D). The resulting product is converted into a compound represented by formula (E) by hydrolysis. can be done.

[0139] Step 2: In this step, the compound represented by formula (E) is reduced to give the compound represented by formula (B). This is the process of converting the compound into a compound.

[0140] The reduction method in this step is not limited as long as the reaction proceeds, but is preferably carried out under a hydrogen atmosphere. (Preferably under a hydrogen stream of 0.05 to 0.6 MPa) The reaction can be carried out using a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst, and more preferably , can be carried out using a palladium catalyst, and even more preferably using palladium on carbon This can be carried out using, even more preferably, 5% palladium on carbon. The amount of 5% palladium carbon used in the process is not limited as long as the reaction proceeds. The amount is 5 to 80% by weight based on the compound represented by formula (D) used in step 1.

[0141] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example is 2-methyltetrahydrofuran.

[0142] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 20 to 60°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 It's time.

[0143] Step 3: In this step, a compound represented by formula (B) is subjected to intramolecular cyclization to give a compound represented by formula (C). The intramolecular cyclization is preferably carried out by intramolecular Friedel-Cr This can be achieved by the intramolecular Friedel reaction. The method for the Crafts acylation reaction is not limited as long as the reaction proceeds. For this purpose, a method using trifluoroacetic anhydride or a method using thionyl chloride, sulfuryl chloride, Include methods using oxalyl, phosphorus oxychloride, phosphorus trichloride, or phosphorus pentachloride. More preferably, a method using trifluoroacetic anhydride or thionyl chloride is used. A more preferred method is to use trifluoroacetic anhydride. The amount of trifluoroacetic anhydride used in this step is not limited as long as the reaction proceeds. However, it is preferably 1 to 3 equivalents relative to the compound represented by formula (B). The amount of thionyl chloride used is not limited as long as the reaction proceeds, but is preferably a thionyl chloride represented by formula (B): The amount is 1 to 3 equivalents relative to the compound to be treated.

[0144] In the case of the method using trifluoroacetic anhydride, this step is preferably carried out in the presence of an acid. More preferably, the reaction is carried out in the presence of trifluoroacetic acid. oxalyl chloride, phosphorus oxychloride, phosphorus trichloride or phosphorus pentachloride In this case, this step is preferably carried out in the presence of aluminum chloride. The amount of aluminum chloride to be used is not limited as long as the reaction proceeds, but it is preferably a compound represented by the formula (B): The amount is 1 to 5 equivalents relative to the compound represented by the formula:

[0145] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, dichloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane Tantan, hexane, pentane, heptane, cyclohexane, ethylcyclohexane, benzene Examples of suitable solvents include benzene, toluene, and chlorobenzene, as well as mixtures thereof. Examples of the solvent include methylene chloride. When trifluoroacetic acid is used, Suitably, trifluoroacetic acid may be included as a solvent.

[0146] The reaction temperature in this step is not limited as long as the reaction proceeds. In the case of the method using thionyl chloride, the temperature is preferably -10°C to 20°C. The reaction temperature is preferably 10° C. to 40° C. The reaction time in this step is not limited as long as the reaction proceeds. In the case of a method using trifluoroacetic anhydride, the reaction time is preferably, but not limited to, 2 to 8 hours. In the case of the method using thionyl chloride, the reaction time is preferably 1 to 4 hours.

[0147] This process can also be carried out in the following two stages.

[0148] [ka]

[0149] [In the formula, Y represents a leaving group, and is preferably a chloro group, a bromo group, an iodo group, a fluoro group, or represents a trifluoroacetoxy group, and more preferably a chloro group or a trifluoroacetoxy group. R indicates an oxy group; 1 represents an amino group protected with a protecting group, and is preferably an acetyl group, trifluoroacetyl, trichloroacetyl, pivaloyl, hydroxyacetyl, It is preferably an amino group protected by a methyl group or a benzoyl group, more preferably an acetyl group or represents an amino group protected with a trifluoroacetyl group, and more preferably represents an acetyl group represents an amino group protected by

[0150] Step 3A is a step of converting a compound represented by formula (B) into a compound represented by formula (J). is.

[0151] When Y is a chloro group, preferred are thionyl chloride, sulfuryl chloride, oxalyl chloride, thionyl chloride, thionyl chloride, sulfuryl chloride, thion ... This step can be carried out by a method using phosphorus dichloride, phosphorus trichloride or phosphorus pentachloride. More preferably, this step can be carried out by a method using thionyl chloride. The amount of thionyl chloride used in is not limited as long as the reaction proceeds, but is preferably a thionyl chloride represented by the formula ( B) is 1 to 3 equivalents relative to the compound represented by B). In this case, this step can be preferably carried out by a method using trifluoroacetic anhydride. The amount of trifluoroacetic anhydride used in the step is not limited as long as the reaction proceeds. The solvent used in this step is preferably 1 to 3 equivalents relative to the compound represented by formula (B). The solvent is not particularly limited as long as it does not inhibit the reaction. For example, dichloromethane ethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, pentachloroethane Heptane, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene benzene, trifluoroacetic acid, and mixed solvents thereof; thiochloride, In the case of the method using an anhydride, methylene chloride is preferable. In the case of the method using trifluoroacetic acid, trifluoroacetic acid is preferable.

[0152] Step 3B is a step of converting a compound represented by formula (J) into a compound represented by formula (C). In the case of the method using thionyl chloride, the reaction is preferably carried out in the presence of aluminum chloride. The amount of aluminum chloride used in this step is determined based on the amount of aluminum chloride that is used as the reaction proceeds. Although not limited thereto, it is preferably 1 to 5 equivalents relative to the compound represented by formula (B). In the case of a method using trifluoroacetic anhydride, this step is preferably carried out in the presence of an acid. More preferably, this step can be carried out in the presence of trifluoroacetic acid.

[0153] The reaction temperature in Step 3A and Step 3B is not limited as long as the reaction proceeds. In the case of the method using trifluoroacetic anhydride, the temperature is preferably 10°C to 40°C. In the case of the method using the above-mentioned method, the reaction temperature is preferably from -10°C to 20°C. The reaction time is not limited as long as the reaction proceeds. In the case of the method using thionyl chloride, the reaction time is preferably The reaction time is 1 to 4 hours, and in the case of a method using trifluoroacetic anhydride, it is preferably 2 hours. The time is approximately 8 hours.

[0154] Step 4: This step is a step of converting the compound represented by formula (C) into the compound represented by formula (F). This step is preferably carried out by (i) nitrosating (or oximating) the α-position of the carbonyl group. (ii) a step of introducing a protecting group onto the nitrogen atom derived from the nitroso group (or oxime group); (ii) and (iii) a reducing step. The order of these steps may be reversed, or they may be performed simultaneously.

[0155] The nitrosating agent (or oximating agent) used in step (i) is represented by formula (C): Any compound capable of nitrosating (or oximating) the α-position of the carbonyl group of the compound to be treated is particularly suitable. Although not limited thereto, nitrite esters can be preferably used, and nitrous acid esters are more preferably used. amyl, n-butyl nitrite, or tert-butyl nitrite can be used, and even more preferably Preferably, amyl nitrite can be used. The amount of the mil is not limited as long as the reaction proceeds, but it is preferable to use the compound represented by formula (C) The amount is 1 to 1.6 equivalents.

[0156] Step (i) preferably uses a base. is a nitrosation (or oximation) of the α-position of the carbonyl group of the compound represented by formula (C). There is no particular limitation as long as it is applicable, but potassium tert-butoxide is preferably used. The amount of potassium tert-butoxide used in step (i) is not limited as long as the reaction proceeds, but is preferably , 1 to 1.5 equivalents.

[0157] The solvent used in step (i) is not particularly limited as long as it does not inhibit the reaction. Although not used, for example, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran Ran, 2-methyltetrahydrofuran, 1,4-dioxane, hexane, pentane, heptane toluene, cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, and dimethyl sulfoxide, and mixed solvents thereof, and preferably Mention may be made of tetrahydrofuran.

[0158] The reaction temperature in step (i) is not limited as long as the reaction proceeds, but is preferably −1 The reaction time in this step is not limited as long as the reaction proceeds. The average time is 1.5 to 30 hours. Step (ii) is R 2 The reaction conditions are adjusted appropriately depending on the type of protecting group for the amino group in It can be set. 2 When is an amino group protected with an acetyl group, (ii ) step can preferably use acetic anhydride in acetic acid.

[0159] Step (iii) is carried out under a hydrogen atmosphere (preferably, hydrogen gas at 0.15 to 1.2 MPa). For example, platinum-carbon catalysts can be used, or zinc powder can be used. However, it is preferably carried out using a platinum-carbon catalyst, more preferably a 2% The amount of 2% or 5% platinum on carbon catalyst is As long as the reaction proceeds, there is no limitation, but it is preferable to use a compound represented by formula (C) in a ratio of 5 to The content of the solvent in step (iii) is 60% by weight. It is possible. The reaction temperature of steps (ii) and (iii) is not limited as long as the reaction proceeds. However, the temperature is preferably 0 to 40°C. The total reaction time is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours. .

[0160] Step 5: In this step, the protecting group of the aromatic amino group of the compound represented by formula (F) is selectively deprotected, This step converts the compound represented by formula (G) into a compound represented by formula (G). 1 and R 2 In Ami The reaction conditions can be set appropriately depending on the type of protecting group for the R 1 and R 2 I'm flustered When the amino group is protected with a methyl group, this step is preferably carried out using hydrochloric acid. More preferably, the reaction can be carried out using 2N hydrochloric acid / ethanol. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 40 to 60°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 2 to 14 hours. It is between.

[0161] Step 6: In this step, a compound represented by formula (G) is condensed with a compound represented by formula (1) to obtain a compound represented by formula (H The compound represented by formula (1) is disclosed in U.S. Pat. It can be manufactured by referring to the description in No. 778891, etc., or a commercially available product can be used. The amount of the compound represented by formula (1) used in this step is not limited as long as the reaction proceeds. Although it is not limited to this, it is preferably 0.8 to 1.2 equivalents relative to the compound represented by formula (G).

[0162] This step is carried out in the presence of an acid catalyst. The acid catalyst used in this step is preferably Examples of the acid catalyst used in this step include lysinium p-toluenesulfonate. As long as the reaction proceeds, there is no limitation, but preferably, the compound represented by formula (G) 0.03 to 0.3 equivalents.

[0163] This step is preferably carried out in a solvent containing cresol or phenol, and more preferably The reaction is carried out in toluene containing o-cresol. The presence of the compound represented by formula (H) improves the precipitation behavior of the compound, improving the yield and reducing the reaction time. This is because it has the effect of shortening the time required.

[0164] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 90 to 130°C. and more preferably, a temperature at which toluene is heated to reflux. There are no limitations as long as the reaction proceeds, but it is preferably 16 to 64 hours.

[0165] In this step, the compound represented by formula (K) and the compound represented by formula (L) are used as reaction intermediates. It is thought that this occurs via a compound.

[0166] [ka]

[0167] Step 7: This step is a step of converting a compound represented by formula (H) into a compound represented by formula (2). The compound represented by formula (2) may be a salt or a hydrate. All of these are included in the scope of the "compound represented by formula (2)" in the present invention. This step can be preferably carried out in the presence of an acid, more preferably methanesulfonic acid and water. The process can be carried out in the presence of

[0168] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. Preferably, a solvent containing 2-methoxyethanol and ethylcyclohexane is used. When the acid is further contained as a solvent, more preferably, methanesulfonic acid, water, 2 A mixed solvent of 2-methoxyethanol and ethylcyclohexane can be used.

[0169] This step is not limited as long as the reaction proceeds, but is preferably carried out at 80 to 160°C. More preferably, methanesulfonic acid, water, 2-methoxyethanol, and ethyl acetate are used. The reaction can be carried out at a temperature where the mixed solvent of cyclohexane and hexane is heated to reflux. The reaction time is not limited as long as the reaction proceeds, but is preferably 4 to 16 hours.

[0170] The compound represented by formula (2) can be preferably obtained as a methanesulfonate salt. , more preferably as methanesulfonate·m hydrate (where m is 0-3) and even more preferably, methanesulfonate anhydride, methanesulfonate as monohydrate, methanesulfonate dihydrate, or methanesulfonate trihydrate It can be obtained, and even more preferably, as the methanesulfonate dihydrate. Any of these can be used in the production method of the present invention. This can be controlled by adjusting the humidity during crystal collection and drying. The compound represented by formula (2) can be more preferably produced by the following method: do.

[0171] [ka]

[0172] Step 8: In this step, the compound represented by formula (3) is brominated to convert it into the compound represented by formula (4). The compound represented by formula (3) can be prepared by a known method or commercially available. can be used.

[0173] The brominating agent used in this step is not limited as long as the reaction proceeds. For example, Examples of suitable bromine include bromine and N-bromosuccinimide, and preferably N-bromosuccinimide. The amount of N-bromosuccinimide used in this step is determined by the reaction is not limited as long as the reaction proceeds, but preferably, 1 to 1 This step is preferably carried out in a mixture of sulfuric acid and other solvents. do.

[0174] The other solvents are not particularly limited as long as they do not inhibit the reaction. For example, chloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, Pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, Examples of the solvent include chlorobenzene and a mixture thereof, and preferably, heptane. It is possible.

[0175] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 50 to 70°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 It's time.

[0176] Step 9: In this step, the nitro group of the compound represented by formula (4) is reduced to an amino group to give the compound represented by formula (5). The reducing agent used in this step is Any compound capable of selectively reducing only the nitro group is sufficient, and preferably, in the presence of hydrogen (preferably For this purpose, a platinum carbon catalyst can be used under a hydrogen flow of 0.05 to 0.2 MPa. More preferably, a 1% platinum-carbon catalyst can be used. The amount of is not limited as long as the reaction proceeds, but is preferably the amount of The solvent used in this step is 5 to 40% by weight based on the compound to be reacted. There is no particular limitation as long as it does not inhibit the reaction. For example, methanol, ethanol, 1,2- Dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dihydrofuran Examples of the solvent include hexane, ethyl acetate, and water, and mixtures thereof, and preferably acetic acid. The reaction temperature in this step is not limited as long as the reaction proceeds. However, the reaction temperature is preferably 50 to 70° C. The reaction time in this step is not limited as long as the reaction proceeds. However, it is preferably 2 to 8 hours.

[0177] Step 10: In this step, the amino group of the compound represented by formula (5) is acetylated to give a compound represented by formula (6). The acetylating agent used in this step is, for example, acetic anhydride or Acetyl chloride is an example, and acetic anhydride is preferable. The amount of acetic anhydride used is not limited as long as the reaction proceeds, but it is preferably the same as that used in step 8. The amount of the base is preferably 0.5 to 1 equivalent relative to the compound represented by formula (3) used in this step. The base is not limited as long as the reaction proceeds, but is preferably The amount of the base is not limited as long as the reaction proceeds, but it is preferably is 0.75 to 1.5 equivalents relative to the compound represented by formula (3) used in step 8. The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. For example, acetonitrile, dichloromethane, chloroform, methanol, ethanol, dichloromethane, ethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran furan, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane hexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone, 2- Butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl Examples of solvents include 2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixtures thereof. The reaction temperature in this step is adjusted to the temperature at which the reaction proceeds. The reaction temperature is not limited as long as the reaction is carried out at a temperature of 10 to 40°C. There is no limitation as long as the reaction proceeds, but it is preferably 3 to 12 hours.

[0178] Step 11: This step involves coupling the compound represented by formula (6) with 3-butenoic acid to give This step is a step of converting the compound represented by formula (7) into a compound represented by formula (7). This can be done in the same way. The compound represented by formula (7) has two geometric isomers, E and Z. All of these are included in the compound represented by formula (7) and are within the scope of the present invention. The compound represented by formula (7) may be a mixture of E and Z isomers, and the mixture may be used in the next step. It can be used to a certain extent.

[0179] Step 12: In this step, the compound represented by formula (7) is reduced to give the compound represented by formula (8). This is the process of converting the compound into a compound.

[0180] The reduction method in this step is not limited as long as the reaction proceeds, but is preferably carried out under a hydrogen atmosphere. (Preferably under a hydrogen stream of 0.05 to 0.2 MPa) The reaction can be carried out using a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst, and more preferably , can be carried out using a palladium catalyst, and even more preferably using palladium on carbon More preferably, 5% palladium on carbon can be used. The amount of 5% palladium carbon used in the step (1) is not limited as long as the reaction proceeds. , 5 to 40% by weight based on the compound represented by formula (7) used in step 11.

[0181] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example is 2-methyltetrahydrofuran.

[0182] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 20 to 60°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 0.5 to 2 It's time.

[0183] Step 13: In this step, a compound represented by formula (8) is subjected to intramolecular cyclization to give a compound represented by formula (9). This step can be carried out in the same manner as in step 3. Cut.

[0184] Step 14: This step is a step of converting the compound represented by formula (9) into the compound represented by formula (10). This step can be carried out in the same manner as in step 4.

[0185] Step 15: This step is carried out by selectively deprotecting the protecting group of the aromatic amino group of the compound represented by formula (10). This step is a step of converting the compound represented by formula (11) into a compound represented by formula (11). It can be done similarly.

[0186] Step 16: In this step, a compound represented by formula (11) is condensed with a compound represented by formula (1) to obtain a compound represented by formula ( 12) This step can be carried out in the same manner as in step 6. It can be done. In this step, the compound represented by formula (30) and / or the compound represented by formula (31) are used as reaction intermediates. ) is thought to be the route via the compound represented by the formula:

[0187] [ka]

[0188] Step 17: This step is a step of converting a compound represented by formula (12) into a compound represented by formula (2). This step can be carried out in the same manner as in step 7. The compound represented by formula (2) can also be produced according to the following method.

[0189] [ka]

[0190] Step 18: In this step, the compound represented by formula (3) is iodized and converted into the compound represented by formula (32). The compound represented by formula (3) can be prepared by a known method or commercially available. Commercially available products can be used. The iodinating agent used in this step is not limited as long as the reaction proceeds. For example, Examples of the iodine include iodine and N-iodosuccinimide, and preferably N-iodosuccinimide. The amount of N-iodosuccinimide used in this step is As long as the reaction proceeds, there is no limitation, but it is preferable to use a compound represented by formula (3) in a ratio of 1 to This step can be preferably carried out in a mixed solvent of sulfuric acid and other solvents. .

[0191] The other solvents are not particularly limited as long as they do not inhibit the reaction. For example, chloromethane, chloroform, diethyl ether, 1,2-dimethoxyethane, hexane, Pentane, heptane, cyclohexane, ethylcyclohexane, benzene, toluene, Examples of the solvent include chlorobenzene and a mixture thereof, and preferably, heptane. It is possible. The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably −10 to 10° C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 1 to 4 hours. It is between.

[0192] Step 19: In this step, the nitro group of the compound represented by formula (32) is reduced to an amino group, and the compound represented by formula (33) The reducing agent used in this step is Any compound capable of selectively reducing only the nitro group without reducing the nitro group is sufficient, and preferably, a compound capable of selectively reducing only the nitro group in the presence of hydrogen ( Preferably, a platinum carbon catalyst can be used under a hydrogen stream of 0.05 to 0.2 MPa. The amount of the platinum-carbon catalyst used in this step is not limited as long as the reaction proceeds. In this case, the amount is 5 to 40% by weight based on the compound represented by formula (3) used in step 18. The solvent used in the process is not particularly limited as long as it does not inhibit the reaction. For example, methanol, ethanol, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyl ethyltetrahydrofuran, 1,4-dioxane, ethyl acetate, and water, and mixtures thereof A suitable solvent is ethyl acetate. The reaction temperature is not limited as long as the reaction proceeds, but is preferably 50 to 70°C. The reaction time is not limited as long as the reaction proceeds, but is preferably 2 to 8 hours.

[0193] Step 20: In this step, the amino group of the compound represented by formula (33) is acetylated to give a compound represented by formula (34). The acetylating agent used in this step is, for example, acetic anhydride. Examples of the acid include acetic acid and acetyl chloride, and preferably acetic anhydride. The amount of acetic anhydride used in step 1 is not limited as long as the reaction proceeds. The amount of the compound represented by formula (3) used in step 8 is 0.5 to 1 equivalent. The base is not limited as long as the reaction proceeds, but is preferably The amount of the base is not limited as long as the reaction proceeds, but is preferably triethylamine. Preferably, 0.75 to 1.5 equivalents relative to the compound represented by formula (3) used in step 18 The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran tetrahydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetonitrile N,N-dimethylformamide, N,N-dimethylacetamide, 1 -methyl-2-pyrrolidone, dimethyl sulfoxide, and water, and mixed solvents thereof The reaction temperature in this step is The reaction temperature is not limited as long as the reaction proceeds, but is preferably 10 to 40°C. The reaction time is not limited as long as the reaction proceeds, but is preferably 3 to 12 hours.

[0194] Step 21: This step involves coupling the compound represented by formula (34) with 3-butenoic acid. This step is a step of converting the compound represented by formula (7) into a compound represented by formula (7). It can be done similarly.

[0195] Step 22: In this step, the compound represented by formula (7) is reduced to give the compound represented by formula (8). This is the process of converting the compound into a compound.

[0196] This step can be carried out in the same manner as in step 12, but the remaining iodide ions can be used. Since the catalytic activity may decrease due to the addition of more catalyst and hydrogen, the amount of catalyst and hydrogen pressure are higher than in step 12. It is preferable.

[0197] The reduction method in this step is not limited as long as the reaction proceeds, but is preferably carried out under a hydrogen atmosphere. (Preferably under a hydrogen stream of 0.15 to 0.6 MPa) The reaction can be carried out using a nickel catalyst, a ruthenium catalyst, or a rhodium catalyst, and more preferably , can be carried out using a palladium catalyst, and even more preferably using palladium on carbon More preferably, 5% palladium on carbon can be used. The amount of 5% palladium carbon used in the step (1) is not limited as long as the reaction proceeds. , 20 to 160% by weight based on the compound represented by formula (34) used in step 21.

[0198] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, for example, acetonitrile, dichloromethane, chloroform, methanol, ethanol , diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Hydrofuran, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, Cyclohexane, ethylcyclohexane, benzene, toluene, chlorobenzene, acetone , 2-butanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1- Examples of solvents include methyl-2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents of these. A preferred example is 2-methyltetrahydrofuran.

[0199] The reaction temperature in this step is not limited as long as the reaction proceeds, but is preferably 20 to 60°C. The reaction time of this step is not limited as long as the reaction proceeds, but is preferably 4 to 16 hours. It is between.

[0200] Step 23: In this step, a compound represented by formula (8) is subjected to intramolecular cyclization to give a compound represented by formula (9). This step can be carried out in the same manner as in step 3. Cut.

[0201] Step 24: This step is a step of converting the compound represented by formula (9) into the compound represented by formula (10). This step can be carried out in the same manner as in step 4.

[0202] Step 25: This step is carried out by selectively deprotecting the protecting group of the aromatic amino group of the compound represented by formula (10). This step is a step of converting the compound represented by formula (11) into a compound represented by formula (11). It can be done similarly.

[0203] Step 26: In this step, a compound represented by formula (11) is condensed with a compound represented by formula (1) to obtain a compound represented by formula ( 12) to a compound represented by the formula (12). This step is the same as the method described in step 16. can be done.

[0204] Step 27: This step is a step of converting a compound represented by formula (12) into a compound represented by formula (2). This step can be carried out in the same manner as in step 7. In the reactions of each of the above steps, after the reaction is completed, the target compound of each step is The target compound can be isolated from the reaction mixture according to well-known methods. Insoluble matters such as catalysts are removed by filtration depending on the reaction mixture, and (ii) a water-immiscible solvent (e.g., For example, methylene chloride, diethyl ether, ethyl acetate, or 2-methyltetrahydrofuran (iii) the organic layer is washed with water and then extracted with magnesium sulfate anhydride. (iv) the solvent is distilled off to obtain the obtained product. The target compound can be recovered, if necessary, by methods well known in the field of organic chemistry (e.g., recrystallization, reprecipitation, Further, by silica gel column chromatography or high performance liquid chromatography, Although the compound can be further purified by chromatography, the production method of the present invention is preferably This can be done without the use of

[0205] The compound represented by formula (2) obtained by the production method of the present invention is preferably a compound represented by formula (15): and an antibody-drug conjugate in which the antibody is bound to a drug linker represented by the formula: Other chemical structures can be used for the preparation of, but not limited to, adjuvants. It can also be used for the production of antibody-drug conjugates having the above structure and other applications. .

[0206] [Preparation of Drug Linker Intermediates]

[0207] A drug linker intermediate that is preferably used in the production of the antibody-drug conjugate of the present invention is , a compound represented by formula (14).

[0208] [ka]

[0209] The compound represented by formula (14) can be prepared as follows.

[0210] [ka]

[0211] The compound represented by formula (2) can be produced by the production method of the present invention. The compound represented by formula (13) can be prepared according to the method described in WO 2014 / 057687, WO 2014 / 057688, WO 2014 / 057689 ... International Publication No. 2015 / 098099, International Publication No. 2015 / 115091, International Publication No. The present invention can be produced by referring to the description in No. 15 / 155998.

[0212] The conversion to the compound of formula (14) can be achieved by reacting the compound of formula (13) with an active ester. and then reacting the compound represented by formula (2) in the presence of a base. This can be done by reacting with a compound represented by the formula:

[0213] The activated ester can be prepared by, for example, converting the compound represented by formula (13) into N,N'-dicyclohexyl Dicarboxylic acid carbodiimide (DCC), or 1-ethyl-3-(3-dimethylaminopropyl) carboxylate Using a condensation agent such as sucrose dihydrochloride (WSCD HCl), 1-hydroxybenzoxazole Triazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt) By reacting with additives such as methyl methyl ester, N-hydroxysuccinimide, or p-nitrophenol, The activated ester can be produced by reacting the compound represented by formula (13) , O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl Uronium hexafluorophosphate pentafluorophenyl trifluoroacetate (H ATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl Uronium hexafluorophosphate (HBTU), diethyl cyanophosphonate, or 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium It can also be produced by reacting with a condensing agent such as dimethyl methyl methacrylate (DMTMM).

[0214] The mixed acid anhydride can be prepared, for example, by reacting a compound represented by formula (13) with a base in the presence of a base, if necessary. It can be produced by reacting it with isobutyl chlorocarbonate.

[0215] The acid halide can be reacted with thionyl chloride or oxalyl chloride, if necessary in the presence of a base. The compound can be prepared by treating the compound with an acid halide such as methyl methyl ether.

[0216] The base used in this step is not particularly limited as long as the reaction proceeds. Triethylamine, tributylamine, diisopropylethylamine, N-methylmorpholine N-methylpyrrolidine, N-methylpiperidine, pyridine, 2-methylpyridine, 2 ,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2. 2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5 -diazabicyclo[4.3.0]undec-7-ene, and other organic bases, potassium carbonate, water Potassium oxide, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate um, sodium acetate, potassium acetate, sodium methoxide, sodium ethoxide, Examples of inorganic bases include potassium tert-butoxide, and preferably triethylene Aminomethylamine, tributylamine, diisopropylethylamine, potassium carbonate, potassium hydroxide Um, potassium bicarbonate, sodium carbonate, sodium hydroxide, sodium bicarbonate, vinegar Examples of suitable amines include sodium acetate and potassium acetate, and more preferably, triethylamine, diethylamine, Mention may be made of isopropylethylamine or N-methylmorpholine.

[0217] The solvent used in this step is not particularly limited as long as it does not inhibit the reaction. However, acetonitrile, dichloromethane, chloroform, methanol, ethanol, diethyl ether, 1,2-dimethoxyethane, tetrahydrofuran, 2-methyltetrahydrofuran Furan, 1,4-dioxane, ethyl acetate, hexane, pentane, heptane, cyclohexane Benzene, ethylcyclohexane, toluene, chlorobenzene, acetone, 2-butylene Tanone, N,N-dimethylformamide, N,N-dimethylacetamide, 1-methyl- 2-pyrrolidone, dimethyl sulfoxide, and water, as well as mixed solvents thereof and preferably, acetonitrile, dichloromethane, methanol, tetrahydrofuran , 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide , 1-methyl-2-pyrrolidone, dimethyl sulfoxide, and water, and their mixed solvents Examples of suitable solvents include:

[0218] [Antibody production]

[0219] The antibodies used to produce the antibody-drug conjugates of the present invention may be derived from any species. The antibodies may be derived from any of various origins, but are preferably derived from humans, rats, mice, and rabbits. If derived from a species other than human, it is preferred to chimerize or humanize it 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.

[0220] The antibody used to produce the antibody-drug conjugate of the present invention preferably targets cancer cells. It has the property of being able to recognize cancer cells and bind to them. characteristics, ability to be taken up and internalized by cancer cells, and / or cytocidal activity against cancer cells It is preferable that the material has properties such as

[0221] The binding of antibodies to cancer 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. This can be confirmed using the P assay (BioTechniques 28:162-165, January 2000). The toxin is a recombinant complex of the catalytic domain of diphtheria toxin and protein G. Proteins can also be used.

[0222] Anti-tumor activity of antibodies can be assessed in vitro by measuring their ability to inhibit cell proliferation. For example, a cancer cell line that overexpresses the antibody's target protein can be cultured and Antibodies were added at various concentrations to the cells, and the effects on focus formation, colony formation, and spheroid growth were evaluated. In vivo, for example, the inhibitory activity of a target protein can be measured. The antibody is administered to nude mice transplanted with the cancer cell line expressing the antibody, and changes in the cancer cells are measured. By this, antitumor activity can be confirmed.

[0223] It is preferable that the antibody itself has an anti-tumor effect, but the antibody-drug conjugate may also have an anti-tumor effect. Since the antibody is bound to a compound that exerts an antitumor effect, the antibody itself does not necessarily have an antitumor effect. For the purpose of specifically and selectively exerting the cytotoxicity of antitumor compounds on cancer cells, It is important and preferred that the antibody has the property of being internalized and transported into cancer cells.

[0224] The antibody used to produce the antibody-drug conjugate of the present invention can be obtained by known means. For example, antigenic polynucleotides can be prepared by a method commonly used in the art. It is obtained by immunizing an animal with the peptide and collecting and purifying the antibodies produced in the body. The origin of the antigen is not limited to humans, but can be derived from animals other than humans, such as mice and rats. In this case, the antigen that binds to the obtained heterologous antigen can be used to immunize animals. Antibodies applicable to human diseases are selected by testing their cross-reactivity with human antigens. can.

[0225] Also, known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Ke nnet, 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.

[0226] 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.

[0227] The antibody used to produce the antibody-drug conjugate of the present invention is a heterologous antigen for humans. Recombinant antibodies that have been artificially modified to reduce their sensitivity, for example, Preferably, the antibody is a chimeric antibody or a humanized antibody. or an antibody having only the gene sequence of a human-derived antibody, i.e., a human antibody. These antibodies can be produced using known methods.

[0228] 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)).

[0229] Humanized antibodies are characterized by the complementarity determining regions (CDRs) of heterologous antibodies. The antibody was developed by incorporating only the ity determining region (IL-1) into a human-derived antibody. (Nature (1986) 321, p.522-525), by CDR grafting method In addition to the CDR sequences of the heterologous antibody, amino acid residues of some of the frameworks of the heterologous antibody are also included. Humanized antibodies (WO 90 / 07861), gene conversion mutagenesis (gene conversion mutagenesis) strategy Examples include immobilized antibodies (US Pat. No. 5,821,337).

[0230] Human antibodies include those derived from humans having human chromosome fragments containing the heavy and light chain genes of human antibodies. Antibodies generated using antibody-producing mice (Tomizuka, K. et al., Nature Genetics (1997) 1 6, p.133-143;Kuroiwa, Y. et. al., Nucl. Acids Res.(1998) 26, p.3447-3448;Yoshida , H. et. al., Animal Cell Technology:Basic and Applied Aspects vol.10, p.69-73(K itagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; (See Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.) Alternatively, a phage display selected from a human antibody library can be used. The antibody obtained by the method (Wormstone, IM et al., Investigative Ophthalmology & Vis (2002)43 (7), p.2301-2308;Carmen, S. et. al., Briefings in Function al Genomics and Proteomics(2002), 1(2), p.189-203;Siriwardena, D. et. al., Ophth See, for example, Almology (2002) 109(3), pp. 427-431.

[0231] The antibodies used to produce the antibody-drug conjugates of the present invention also include modified antibodies. The modified antibody is an antibody according to the present invention that has been chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, N-bonds or O-bonds. - Chemical modifications include those with chemical moieties attached to the carbohydrate chain. The body contains post-translational modifications (e.g., addition of N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, etc. By expressing this in a prokaryotic host cell, a methionine residue is added to the N-terminus. In addition, the present invention also includes those that enable the detection or isolation of the antibody or antigen of the present invention. Such modifications include those labeled with an enzyme label, a fluorescent label, and an affinity label. Such modified antibodies according to the present invention are included in the meaning of the term "antibody." The modified antibodies according to the present invention are intended to improve the stability and circulation of the antibodies. These are useful for improving the affinity, reducing the antigenicity, detecting or isolating antibodies or antigens, etc.

[0232] Furthermore, it is possible to regulate sugar chain modification (glycosylation, decarboxylation) of the antibody of the present invention. Antibody-dependent cellular cytotoxicity can be enhanced by glycosylation of antibodies. Techniques for controlling chain modification include those described in WO 99 / 54342 and WO 00 / 6173 No. 9, International Publication No. 02 / 31140, etc. are known, but are not limited to these. The antibodies according to the present invention also include antibodies in which the glycosylation has been regulated.

[0233] In addition, antibodies produced in mammalian cultured cells have a lysine residue at the carboxyl terminal of the heavy chain. It is known that the hydroxyl group is deleted (Journal of Chromatography A, 705: 129-134 (1995) ), and also, two amino acid residues, glycine and lysine, are deleted at the carboxyl terminal of the heavy chain. It is known that the proline residue at the carboxyl terminus is newly amidated ( Analytical Biochemistry, 360: 75-83(2007)). However, the deletion and Modifications affect the antigen-binding ability and effector functions of antibodies (complement activation and antibody-dependent cellular cytotoxicity). Therefore, the antibody of the present invention does not have any effect on the antibody's function (use, etc.). Also included are antibodies and functional fragments of such antibodies, which have one or two amino acids at the carboxyl terminus of the heavy chain. Deletions in which the acid is deleted and amidated deletions (e.g., carboxyl-terminal However, the antigen-binding ability and the effector - As long as the function is maintained, the carboxyl-terminal deletion of the heavy chain of the antibody according to the present invention can be carried out in the manner described above. The two heavy chains constituting the antibody of the present invention are not limited to the type. The heavy chain may be any one of heavy chains selected from the group consisting of α- and β-blockers, or a combination of any two of heavy chains. The ratio of the amounts of each deletion fragment is determined based on the mammalian culture medium producing the antibody of the present invention. Although the type of cultured cells and culture conditions may affect the antibody of the present invention, it is preferable to use two antibodies. List those in which one amino acid residue is deleted at the carboxyl terminus of both heavy chains. can be done.

[0234] The isotype of the antibody of the present invention may be, for example, IgG (IgG1, IgG2, Ig Examples of the antibody include IgG1 and IgG2. It is possible.

[0235] The antibodies that can be used to produce the antibody-drug conjugates of the present invention are not particularly limited, but examples include: For example, anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD 3 antibodies, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD9 8 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FG Examples of antibodies include anti-FR4 antibodies, anti-FOLR1 antibodies, anti-VEGF antibodies, and anti-GPR20 antibodies. and preferably an anti-HER2 antibody, an anti-HER3 antibody, an anti-TROP2 antibody, an anti-B7-H3 antibody, Examples of antibodies include anti-GPR20 antibodies.

[0236] In the present invention, the term "anti-HER2 antibody" refers to an antibody against HER2 (Human Epidermal Growth Factor-2) l Growth Factor Receptor Type 2; ErbB-2) and preferably binds to HER2, thereby being internalized in HER2-expressing cells. The figures show antibodies with localizing activity.

[0237] Examples of anti-HER2 antibodies include trastuzumab (US Patent No. 5821337) and pertuzumab (International Publication No. No. 01 / 00245), and preferably trastuzumab. do.

[0238] In the present invention, "trastuzumab" refers to the amino acid sequence of SEQ ID NO: 1 (FIG. 1). a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 2) to 449; A humanized anti-HER2 monoclonal antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NOs. 1 to 214. It is a clonal antibody.

[0239] In the present invention, the term "anti-HER3 antibody" refers to an antibody against HER3 (Human Epidermal Growth Factor-3) l Growth Factor Receptor Type 3; ErbB-3) and preferably binds to HER3 on the surface of HER3-expressing cells to bind to the HER3. Antibodies with the activity of being internalized into expressing cells are shown.

[0240] Examples of anti-HER3 antibodies include patritumab (U3-1 287), U1-59 (International Publication No. 2007 / 077028), MM-121 (ser ibantumab), the anti-ERBB3 antibody described in WO 2008 / 100624, R G-7116 (lumretuzumab) and LJM-716 (elgemtuma b) and preferably include patritumab and U1-59. do.

[0241] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor r-associated calcium signal transducer 2 ; EGP-1) and preferably binds to TROP2, thereby 1 shows antibodies that have the activity of being internalized into ROP2-expressing cells.

[0242] Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. 2015 / 003366). No. 098099).

[0243] In the present invention, the term "anti-B7-H3 antibody" refers to a B7-H3 (B cell antibody PD-L3; CD276) and specifically binds to Preferably, the antibody has the activity of being internalized into B7-H3-expressing cells by binding to B7-H3. The antibodies shown are:

[0244] Examples of anti-B7-H3 antibodies include M30-H1-L4 (International Publication No. 2014 / 05 No. 7687).

[0245] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that binds to GPR20 (G Protein-coated Specifically binds to GPR20, preferably GPR20. The figures show antibodies that have the activity of being internalized into GPR20-expressing cells when combined with GPR20. Examples of anti-GPR20 antibodies include h046-H4e / L7 (International Publication No. 2018 / 003366). No. 135501).

[0246] [Conjugation of antibodies and drug-linker intermediates]

[0247] The antibody-drug conjugate of the present invention comprises a drug linker intermediate (preferably represented by the formula (14) ) and an antibody having a thiol group (also called a sulfhydryl group). It can be produced by reacting

[0248] Antibodies having sulfhydryl groups can be obtained by methods well known to those skilled in the art (Hermanson, G. T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) 0.3 to 3 molar equivalents per intra-chain disulfide of antibody are used, and ethylenediamine By reacting with the antibody in a buffer containing a chelating agent such as EDTA, Antibodies with partially or completely reduced interchain disulfides and sulfhydryl groups can be obtained.

[0249] Additionally, 2 to 20 molar equivalents of a drug linker per antibody having a sulfhydryl group are used. - Using an intermediate (preferably a compound represented by formula (14)), two to three compounds per antibody Antibody-drug conjugates with eight drugs attached can be produced.

[0250] The average number of drugs bound per antibody molecule of the produced antibody-drug conjugate can be calculated using the following formula: For example, antibody-drug conjugates and their conjugates at two wavelengths of 280 nm and 370 nm. The UV method is used to calculate the denaturation rate by measuring the UV absorbance of the denaturation precursor. The compound-drug conjugate was treated with a reducing agent, and each of the resulting fragments was analyzed by HPLC. This can be done by a method of quantitative determination and calculation (HPLC method).

[0251] Conjugation of antibodies with drug linker intermediates and antibody-drug conjugates The average number of drugs bound per molecule was calculated according to the method described in WO 2014 / 057687 and WO 2014 / 057687. Publication No. 2015 / 098099, International Publication No. 2015 / 115091, International Publication No. 015 / 155998 and WO 2018 / 135501, etc. This can be done.

[0252] In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate. Antibody-drug conjugates in which the antibody in the gate is an anti-HER2 antibody are shown.

[0253] The anti-HER2 antibody preferably comprises the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 1. a heavy chain consisting of the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence of SEQ ID NO: 1 to 214; an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 1; and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:2.

[0254] Drug per antibody in the anti-HER2 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 8, and More preferably, it is 7 to 8, even more preferably, it is 7.5 to 8, and even more preferably, it is It is about 8.

[0255] The anti-HER2 antibody-drug conjugate is a drug-phosphoric acid conjugate produced by the production method of the present invention. The Kerr intermediate (preferably a compound represented by formula (14)) is used to produce a compound according to International Publication No. 2015 / 1 The manufacturing method can be carried out by referring to the description in No. 15091.

[0256] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate. Antibody-drug conjugates in which the antibody in the gate is an anti-HER3 antibody are shown.

[0257] The anti-HER3 antibody preferably comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 and a An antibody comprising a light chain consisting of the amino acid sequence set forth in No. 4, or a heavy chain carbohydrate of said antibody. It is an antibody lacking the lysine residue at the xyl terminus.

[0258] Drug per antibody in the anti-HER3 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 8, and More preferably, it is 7 to 8, even more preferably, it is 7.5 to 8, and even more preferably, it is It is about 8.

[0259] The anti-HER3 antibody-drug conjugate is a drug-phosphoric acid conjugate produced by the production method of the present invention. The Kerr intermediate (preferably a compound represented by formula (14)) is used to produce a compound according to International Publication No. 2015 / 1 The present invention can be produced by referring to the description in No. 55998.

[0260] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to an antibody-drug conjugate. 1 shows an antibody-drug conjugate in which the antibody in the conjugate is an anti-TROP2 antibody.

[0261] The anti-TROP2 antibody preferably has a sequence similar to that shown in amino acids 20 to 470 of SEQ ID NO:5. and a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 6 at amino acid numbers 21 to 234. An antibody comprising a light chain consisting of the amino acid sequence set forth above, or the carboxyl terminal of the heavy chain of said antibody. It is an antibody in which the lysine residue is deleted.

[0262] The amount of drug per antibody in the anti-TROP2 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 5, and More preferably, it is 3.5 to 4.5, and even more preferably, it is about 4.

[0263] The anti-TROP2 antibody-drug conjugate is a drug linker produced by the production method of the present invention. The present invention relates to a method for producing a compound represented by formula (14) using an anchor intermediate (preferably a compound represented by formula (14)). The present invention can be produced by referring to the description in No. 098099.

[0264] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to an antibody-drug conjugate. 1 shows an antibody-drug conjugate in which the antibody in the conjugate is an anti-B7-H3 antibody.

[0265] The anti-B7-H3 antibody preferably comprises the amino acid sequence set forth in SEQ ID NO:7 from amino acid number 20 to 471. and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 8, which is represented by amino acid numbers 21 to 233. An antibody comprising a light chain consisting of the amino acid sequence set forth above, or the carboxyl terminal of the heavy chain of said antibody. It is an antibody in which the lysine residue is deleted.

[0266] The drug content per antibody of the anti-B7-H3 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 5, and More preferably, it is 3.5 to 4.5, and even more preferably, it is about 4.

[0267] The anti-B7-H3 antibody-drug conjugate is a drug linker produced by the production method of the present invention. The present invention relates to a method for producing a compound represented by formula (14) using an intermediate copolymer of formula (14) and formula (14) of formula (14). The compound can be produced by referring to the description in No. 057687.

[0268] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to an antibody-drug conjugate. 1 shows an antibody-drug conjugate in which the antibody in the conjugate is an anti-GPR20 antibody.

[0269] The anti-GPR20 antibody preferably comprises a sequence corresponding to amino acid numbers 20 to 472 in SEQ ID NO:9. a heavy chain consisting of the amino acid sequence set forth above and a heavy chain consisting of amino acid sequences 21 to 234 in SEQ ID NO: 10; An antibody comprising a light chain consisting of the amino acid sequence described above, or the heavy chain carboxyl terminal of said antibody. This is an antibody in which the terminal lysine residues are deleted.

[0270] The drug content per antibody of the anti-GPR20 antibody-drug conjugate produced by the present invention The average number of bonds in the linker is preferably 2 to 8, more preferably 3 to 8, and More preferably, it is 7 to 8, even more preferably, it is 7.5 to 8, and even more preferably, it is is about 8.

[0271] The anti-GPR20 antibody-drug conjugate is a drug linker produced by the production method of the present invention. The present invention relates to a method for producing a compound represented by formula (14) using an anchor intermediate (preferably a compound represented by formula (14)). The compound can be produced by referring to the description in No. 135501.

[0272] [Pharmaceutical composition] The antibody-drug conjugates produced according to the present invention may be prepared by administering to the patient one or more pharmaceutically compatible The pharmaceutically compatible component can be administered in the form of an antibody-drug mixture prepared according to the present invention. Depending on the dosage and administration concentration of the compound conjugate, a formulation commonly used in this field may be used. Additives and other additives can be appropriately selected and applied. For example, The antibody-drug conjugate may be prepared by adding a buffer such as a histidine buffer, sucrose or trehalose. and surfactants such as polysorbate 80 or polysorbate 20. The compound may be administered as a pharmaceutical composition containing the compound.

[0273] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be administered to a patient. In addition to being applied as a systemic therapy, it can also be applied locally to cancer tissues to expect therapeutic effects. can.

[0274] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be administered to a mammal. It can be preferably used for animals, but more preferably for humans. .

[0275] The pharmaceutical composition comprising the antibody-drug conjugate produced by the present invention is preferably It can be used as an injection, more preferably as an aqueous injection or a freeze-dried injection. It can be used as a lyophilized injection, and more preferably, it can be used as a lyophilized injection.

[0276] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention is an aqueous injection. In some cases, it may be preferably administered intravenously after dilution with an appropriate diluent. Examples of diluents include glucose solution (preferably 5% glucose solution) and physiological saline solution. It is possible.

[0277] The pharmaceutical composition containing the antibody-drug conjugate produced by the present invention is freeze-dried and can be used for injection. In the case of a pharmaceutical preparation, it is preferably dissolved in water for injection, and then diluted with an appropriate diluent to the required amount. After that, it can be administered intravenously by drip infusion. As a diluent, a glucose solution (preferably 5% Examples of the solution include glucose solution and physiological saline.

[0278] For administering a pharmaceutical composition containing an antibody-drug conjugate produced according to the present invention. Routes of introduction that can be used include, for example, intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal. The route may be mentioned, preferably the intravenous route.

[0279] The antibody-drug conjugates produced according to the present invention can be administered to humans for 1 to 180 days. The dose may be administered once every week, two weeks, three weeks, or four weeks. It can be administered at intervals, and even more preferably, at intervals of once every three weeks. Furthermore, the antibody-drug conjugates produced according to the present invention can be administered in a single dose of approximately 0. It can be administered at a dose of 0.001 to 100 mg / kg, preferably 0.8 mg / kg per dose. The antibody produced by the present invention can be administered at a dose of up to 12.4 mg / kg. When the drug conjugate is an anti-HER2 antibody-drug conjugate, preferably It can be administered at a dose of 5.4, 6.4, or 7.4 mg / kg per dose, and even more preferably More preferably, it is administered at a dose of 5.4 mg / kg or 6.4 mg / kg per dose. can be done.

[0280] A pharmaceutical composition comprising an antibody-drug conjugate produced by the present invention can be used in the treatment of cancer. It can be used for the treatment of breast cancer, gastric cancer (sometimes called gastric adenocarcinoma), and the like. , colon cancer (sometimes called colorectal cancer, including colon cancer and rectal cancer), lung cancer ( including small cell lung cancer and non-small cell lung cancer), esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma , bile duct cancer, Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial cancer, prostate Adenocarcinoma, bladder cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal Liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, endometrial cancer, uterine cancer, kidney cancer, Vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neurological Epithelial tissue tumors, nerve sheath tumors, head and neck cancer, skin cancer, pharyngeal cancer, gallbladder cancer, bile duct cancer It is used for the treatment of at least one cancer selected from the group consisting of carcinoma, mesothelioma, and sarcoma. For example, the antibody-drug conjugate produced by the present invention can be used as an anti-H In the case of an ER2 antibody-drug conjugate, it is more preferably an ER2 antibody-drug conjugate for use in treating breast cancer, gastric cancer, or colon cancer. , non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, bile duct cancer, page at least one selected from the group consisting of pancreatic cancer, ovarian cancer, and uterine carcinosarcoma and more preferably, it can be used for the treatment of breast cancer, stomach cancer, colon cancer, Non-small cell lung cancer, esophageal cancer, salivary gland cancer, gastroesophageal junction adenocarcinoma, bile duct cancer, and pegylated The present invention can be used to treat at least one cancer selected from the group consisting of: and even more preferably for the treatment of breast cancer, gastric cancer, colon cancer, or non-small cell lung cancer. can be used for.

[0281] The pharmaceutical composition comprising the antibody-drug conjugate produced by the present invention is useful for the treatment of cancer. It can be used as a drug of choice for drug therapy, which is the main treatment, and as a result, As a result, it can slow the growth of cancer cells, inhibit their proliferation, and even destroy them. These effects can help cancer patients relieve symptoms caused by cancer and improve their quality of life. This can achieve improvement, preserving the life of cancer patients and achieving therapeutic effects. Even if the drug is not effective, it may have beneficial effects on cancer patients by suppressing or controlling the growth of cancer cells. It is possible to achieve a higher quality of life and longer survival.

[0282] In addition to the use of the drug alone in such drug therapy, the antibody produced by the present invention -Pharmaceutical compositions containing drug conjugates may be used in adjuvant therapy in combination with other therapies. It can also be used in combination with surgery, radiation therapy, hormone therapy, etc. Furthermore, it can be used as a drug therapy in neoadjuvant therapy. do.

[0283] In addition to the above-mentioned therapeutic uses, the antibody-drug conjugates produced by the present invention can be used in various therapeutic applications. The pharmaceutical composition containing the compound has a preventive effect of suppressing the growth of minute metastatic cancer cells and even destroying them. For example, it can suppress and destroy cancer cells in body fluids during the metastasis process. and the effects of suppressing or destroying minute cancer cells immediately after implantation in any tissue. Therefore, it is expected to be effective in suppressing and preventing cancer metastasis, especially after surgical removal of cancer. The effect is expected.

[0284] Pharmaceutical compositions containing antibody-drug conjugates produced according to the present invention may be used in other cancer treatments. It can also be administered in combination with other therapeutic agents, which can enhance the antitumor effect. Other cancer treatments used for this purpose include 5-fluorouracil (5-FU). , Pertuzumab, Trastuzumab , Paclitaxel, Carboplatin ), Cisplatin, Gemcitabine, Capecitabine, Irinotecan ( CPT-11, docetaxel, pemetrexed exed), sorafenib, vinblastine in), vinorelbine, everolimus ), Tanespimycin, Bevacizumab mab), oxaliplatin, lapatinib ib), trastuzumab emtansine (T-DM1) and the drugs described in WO 2003 / 038043, and further LH-RH Analogues (Leuprorelin, Goserelin) in), etc.), Estramustine Phos phate), estrogen antagonists (tamoxifen, raloxifene) Raloxifene, etc.), aromatase inhibitors (anastrozole, astrozole, letrozole, and exemestane However, as long as the drug has antitumor activity, There is no limitation. [Example]

[0285] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. It is not something that can be done. In the example, 1 H-NMR" and " 13 "C-NMR" stands for "nuclear magnetic resonance spectrum" The CDCl3 in parentheses represents the measurement solvent, deuterated chloroform, and DMSO-d6 represents D2O means deuterated water, which is the measurement solvent. TMS (tetramethylsilane) was used as the internal standard substance. 1 H-NMR Multiplicities are s=singlet, d=doublet, t=triplet, q=qua rtet, quint=quintet, m=multiplet, and brs=bro ad means singlet.

[0286] Example 1 N-(3-bromo-5-fluoro-4-methylphenyl)acetamide

[0287] [ka]

[0288] 2-Fluoro-1-methyl-4-nitrobenzene (10.0 g, 64.5 mmol) After heating a solution of concentrated sulfuric acid (90% or more, 50 mL) and heptane (50 mL) to approximately 60°C, N-bromosuccinimide (13.8 g, 77.4 mmol) was added in six portions. After stirring at about 60°C for 1 hour, the reaction mixture was cooled to room temperature. Toluene (50 mL) was added, and the mixture was separated, after which the aqueous layer was removed. Water (50 mL), 6.5 wt% aqueous sodium bicarbonate solution (50 mL), 5 wt% sulfurous acid The resulting aqueous layer was washed with water (50 mL), activated carbon (1 After stirring at room temperature for 1 hour, the insoluble matter was filtered off and the insoluble matter was dissolved in toluene (2 After removing the aqueous layer from the filtrate, the organic layer was concentrated under reduced pressure. Ethyl acetate (100 mL) was added to the mixture, and the mixture was concentrated under reduced pressure again to give 1-bromo- A solution of 3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (approximately 30 mL) was obtained. .

[0289] 1-Bromo-3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (approximately 3 A suspension of 1% platinum-on-carbon catalyst (2.0 g) and ethyl acetate (120 mL) was added to the The atmosphere was replaced with nitrogen and then with hydrogen. The mixture was heated under a hydrogen stream (0.1 MPa) at approximately 60°C for 4 hours. The mixture was stirred and cooled to room temperature. Insoluble matter was removed from the resulting suspension by filtration, and the insoluble matter was dissolved in ethyl acetate ( The filtrate was washed twice with 0.5N aqueous hydrochloric acid (100 mL) to obtain an organic layer. The aqueous layer was extracted with ethyl acetate (50 mL) to obtain an organic layer, which was then combined. Then, 6.5 wt% sodium bicarbonate aqueous solution (50 mL), 5 wt% saline solution (50 The organic layer was concentrated under reduced pressure. The concentrated residue (approximately 30 mL) was dissolved in acetic acid. Ethyl acetate (50 mL) was added and the mixture was concentrated again under reduced pressure to give 3-bromo-5-fluoro-4- A solution of methylaniline in ethyl acetate (approximately 30 mL) was obtained. A solution of 3-bromo-5-fluoro-4-methylaniline in ethyl acetate (approximately 29 mL) with acetic acid To the solution containing ethyl acetate (30 mL), triethylamine (7.2 mL, 51.8 mmol) was added. 1), acetic anhydride (3.3 mL, 34.4 mmol) was added, and the mixture was stirred at room temperature for 6 hours. To the resulting reaction solution, 10 wt% saline (50 mL) was added, and the layers were separated, after which the aqueous layer was removed. The organic layer was concentrated under reduced pressure, and then ethyl acetate (50 mL) was added, followed by concentration again under reduced pressure. Ethyl acetate (80 mL) was added to the concentrated residue (approximately 30 mL), and the mixture was diluted with 4N hydrochloric acid / ethyl acetate solution ( After adding 10.9 mL of HCl (43.7 mmol), the mixture was stirred at room temperature for 1 hour. The insoluble matter was washed with ethyl acetate (40 mL). The filtrate was added with 10 wt% brine (40 mL). After the addition, a 25 wt % aqueous solution of sodium hydroxide (5.6 g) was added to adjust the pH to about 7. After removing the aqueous layer, the organic layer was concentrated under reduced pressure. mL) was added, and the residue (approximately 30 mL) was concentrated under reduced pressure. The residue was stirred at 50°C for 5 hours, and then After stirring at room temperature for 12 hours, the mixture was cooled to 3°C and stirred for 2 hours. The crystals were collected by filtration and diluted with cold toluene (20 mL) and cold 75% aqueous acetonitrile (20 mL). The obtained crystals were dried under reduced pressure at 40°C and N-(3-bromo-5-fluoro- (4-methylphenyl)acetamide was obtained as white crystals (5.7 g, 37% yield).

[0290] 1 H-NMR (500MHz,CDCl3)δ7.41(1H,s),7.39(1H ,d,J=9.2Hz),7.20(1H,brs),2.27(3H,d,J=2.0 Hz), 2.17(3H,s)

[0291] Example 2 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid

[0292] [ka]

[0293] N-(3-bromo-5-fluoro-4-methylphenyl)acetamide (30.0 g, 121.9 mmol), 3-butenoic acid (12.4 mL, 146.3 mmol), diisopropyl Dipropylethylamine (46.0 mL, 268.2 mmol) in tetrahydrofuran (12 A solution of 100 mL of toluene and 100 mL of water was degassed under reduced pressure and replaced with nitrogen. Sphingol (1.1 g, 3.7 mmol) was added. After degassing and replacing with nitrogen under reduced pressure again, acetic acid was added. Palladium(II) dinitrate (0.4 g, 1.8 mmol) was added, and the mixture was degassed under reduced pressure and filled with nitrogen. After the substitution, the mixture was heated under reflux for 5 hours. The reaction mixture was cooled to room temperature, and activated carbon (3.0 g) was added thereto. The mixture was stirred at 40°C for 1 hour. The insoluble matter was filtered off and the insoluble matter was dissolved in 20% aqueous tetrahydrofuran (60 mL The filtrate was washed with 2-methyltetrahydrofuran (300 mL), water (300 mL) and Then, 25 wt% aqueous sodium hydroxide solution (23.4 g, 146.3 mmol) was added. The organic layer was removed, and the aqueous layer was diluted with 2-methyltetrahydrofuran (300 mL) and concentrated hydrochloric acid. (36%, 22.2 g, 219.4 mmol) was added, followed by sodium chloride (30 g). After separation, the aqueous layer was removed and the organic layer was washed with 10 wt% brine (90 mL). The organic layer was concentrated under reduced pressure to give 4-[5-(acetylamino)-3- Fluoro-2-methylphenyl]-3-butenoic acid in 2-methyltetrahydrofuran solution ( Approximately 150 mL) was obtained.

[0294] 4-[5-(acetylamino)-3-fluoro-2-methylphenyl] including geometric isomers ]-3-butenoic acid in 2-methyltetrahydrofuran solution (approximately 140 mL) A suspension of tetrahydrofuran (308 mL) and 5% palladium on carbon (5.6 g) was added. The atmosphere was replaced with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen stream (0.1 MPa) at approximately 40°C for 1 hour. The resulting suspension was stirred and cooled to room temperature. Insoluble matter was filtered off from the resulting suspension. The filtrate was washed with dihydrofuran (112 mL), and water (140 mL) was added to the filtrate, and 1N hydrochloric acid Water was added to adjust the pH to about 2. After separation, the aqueous layer was removed and the resulting organic layer was concentrated under reduced pressure. Ethyl acetate (420 mL) was added to the concentrated residue, and the mixture was concentrated under reduced pressure. The mixture was concentrated under reduced pressure to give a concentrated residue (approximately 170 mL). After stirring for 5 hours, heptane (140 mL) was added and the mixture was cooled to room temperature. The crystals were collected by filtration and washed with ethyl acetate / heptane (3 / 7) (84 mL). Dry under pressure to obtain 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butyl Tannoic acid was obtained as white crystals (26.1 g, 91% yield).

[0295] 1 H-NMR (500MHz,DMSO-d6)δ12.08(1H,brs),9. 97(1H,s),7.42(1H,dd,J=12.5,2.0Hz),7.05(1 H,d,J=1.5Hz),2.59-2.54(2H,m),2.28(2H,t,J =7.3Hz),2.10(3H,d,J=2.0Hz),2.02(3H,s),1. 71(2H,quint,J=7.5Hz)

[0296] (Example 3-1) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene (1-phenyl)acetamide

[0297] [ka]

[0298] 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid (12 A solution of 1.0 g (47.4 mmol) and trifluoroacetic acid (24 mL) was cooled to 4°C and then Add water and trifluoroacetic acid (13.4 mL, 94.8 mmol) dropwise and stir at about 4°C for 4 hours. The resulting reaction solution was added dropwise to 50% aqueous acetonitrile (120 mL) cooled to 5°C. The pH was adjusted to approximately 7 with 25 wt% sodium hydroxide aqueous solution (77.3 g), and then water (5 After that, the mixture was cooled to room temperature and the precipitated crystals were collected by filtration. The crystals were washed with 75% aqueous acetonitrile (60 mL). The obtained crystals were dried under reduced pressure and -(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene (-1-yl)acetamide was obtained as pale yellow-white crystals (10.2 g, 92% yield).

[0299] 1 H-NMR (400MHz, CDCl3)δ12.31(1H,brs),8.43 (1H,d,J=12.8Hz),2.88(2H,t,J=12.0Hz),2.66 (2H,dd,J=7.2,6.0Hz),2.22(3H,s),2.17(3H,d ,J=2.0Hz),2.09(3H,quint,J=6.4Hz)

[0300] (Example 3-2) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene (1-phenyl)acetamide

[0301] [ka]

[0302] 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid (5. A solution of 1000 mg of methyltrifluoroacetic acid (19.7 mmol) and trifluoroacetic acid (10 mL) was cooled to 2°C and then diluted with anhydrous Trifluoroacetic acid (5.6 mL, 39.5 mmol) was added dropwise, and the mixture was stirred at about 5° C. for 3 hours. 50% aqueous acetonitrile (50 mL) was added dropwise to the reaction mixture. The pH was adjusted to about 7 with aqueous sodium solution (33 mL), and then water (17 mL) was added. The precipitated crystals were collected by filtration and mixed with water (25 mL), 75% aqueous acetonitrile (2 The resulting crystals were dried under reduced pressure and washed with N-(3-fluoro-4-methyl-2-methylpropional). (8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide Obtained as pale yellow crystals (4.3 g, 92% yield).

[0303] (Example 3-3) N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene (1-phenyl)acetamide

[0304] [ka]

[0305] 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid (5. A solution of 1000 mg of methyltrifluoroacetic acid (19.7 mmol) and trifluoroacetic acid (10 mL) was cooled to 2°C and then diluted with anhydrous Trifluoroacetic acid (5.6 mL, 39.5 mmol) was added dropwise, and the mixture was stirred at about 5° C. for 4 hours. After adding 17% aqueous acetonitrile (30 mL) dropwise to the reaction mixture, water (20 mL) was added dropwise. After adjusting the pH to approximately 7 with 25 w / v% sodium hydroxide aqueous solution (33 mL), water (17 mL) was added. After that, the mixture was cooled to room temperature and the precipitated crystals were collected by filtration. The resulting crystals were washed with 25 mL of acetonitrile containing 10% water. 3-Fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1 -yl)acetamide was obtained as pale yellow-white crystals (4.3 g, 93% yield).

[0306] (Example 4-1) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) Phthalene-1,7-diyl)diacetamide

[0307] [ka]

[0308] N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) (1-phenyl-1-yl)acetamide (5.0 g, 21.3 mmol) in tetrahydrofuran ( The solution was cooled to 6°C and diluted with amyl nitrite (3.7 mL, 27.6 mmol), potassium chloride (1.2 mL, 27.6 mmol), and Umium tert-butoxide (2.9 g, 25.5 mmol) was added. The mixture was stirred at 3°C ​​for 17 hours. After stirring for 1 hour, acetic acid (25 mL) and acetic anhydride (25 mL) were added, and the temperature was raised to about 20°C. 2% platinum on carbon catalyst (1.5 g) was added, and the atmosphere was replaced with nitrogen and then with hydrogen. The mixture was stirred under a pressure of 0.3 MPa at room temperature for 4 hours. Insoluble matter was removed from the resulting suspension by filtration. The filtrate was washed with ethyl acetate (25 mL), and activated carbon (0.7 g) was added to the filtrate. After stirring at rt for 1 hour, the insoluble matter was filtered off and washed with ethyl acetate (25 mL). The mixture was cooled to °C, and a 5N aqueous solution of sodium hydroxide (50 mL) was added dropwise. The aqueous layer was removed, and the mixture was again A 5N aqueous solution of sodium hydroxide (50 mL) was added, and the aqueous layer was removed. After adding tetrahydrofuran (35 mL) and water (25 mL), 5N aqueous sodium hydroxide was added. The pH was adjusted to about 7 by adding 25 mL of water. After the temperature was raised to room temperature, the aqueous layer was removed and the organic layer was separated. The resulting organic layer was concentrated under reduced pressure to give a concentrated residue. Ethyl acetate (50 mL) was added to the concentrated residue and concentrated under reduced pressure. This procedure was repeated three times. The concentrated residue (approximately 25 mL) was stirred at 40°C for 5 hours, cooled to room temperature, and then stirred at 2°C for 3 hours. The precipitated crystals were collected by filtration and washed with cold ethyl acetate (25 mL) and water (25 mL). The obtained crystals were dried under reduced pressure to give N,N'-(3-fluoro-4-methyl-8- Oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide Obtained as white crystals (3.7 g, 60% yield).

[0309] 1 H-NMR (500MHz,CDCl3)δ11.76(1H,s),8.43(1 H,d,J=13.0Hz),6.53(1H,d,J=4.5Hz),4.62(1H ,dt,J=14.0,5.4Hz),3.08-2.96(2H,m),2.78-2 .72(1H,m),2.23(3H,s),2.15(3H,d,J=1.5Hz), 2.11(3H,s),1.88-1.77(1H,m)

[0310] (Example 4-2) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) Phthalene-1,7-diyl)diacetamide

[0311] [ka]

[0312] N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) (1-phenyl-1-yl)acetamide (35.0 g, 148.8 mmol) of tetrahydrofuran The solution was cooled to 4°C and diluted with amyl nitrite (25.7 mL, 193.4 mmol). l), potassium tert-butoxide (20.0 g, 178.6 mmol) was added. After stirring at 1°C for 17 hours, acetic acid (175 mL) and acetic anhydride (175 mL) were added, and The temperature rose. At about 20°C, 2% platinum on carbon catalyst (11.8 g) was added, and the mixture was purged with nitrogen. The mixture was stirred under a hydrogen atmosphere (0.3 MPa) at room temperature for 3 hours. The insoluble matter was filtered off and washed with ethyl acetate (175 mL). The filtrate was washed with activated carbon (5. After stirring at room temperature for 2 hours, the insoluble matter was filtered off and the insoluble matter was dissolved in ethyl acetate (175 ml) The filtrate was cooled to 1°C and added dropwise with 5N aqueous sodium hydroxide (350 mL). The aqueous layer was removed, and 5N aqueous sodium hydroxide solution (350 mL) was added again. The organic layer was diluted with tetrahydrofuran (245 mL) and water (175 mL). After the addition, 5N aqueous sodium hydroxide solution (150 mL) was added to adjust the pH to about 7. After the temperature was raised to , the aqueous layer was removed and the organic layer was washed with 10 wt% brine (175 mL). The organic layer was concentrated under reduced pressure to give a concentrated residue. Ethyl acetate (350 mL) was added to the concentrated residue. This procedure was repeated three times, and the concentrated residue (approximately 175 mL) was heated at 40°C. After stirring for 5 hours at 4°C, the mixture was cooled to room temperature and stirred at 2°C for 3 hours. The precipitated crystals were collected by filtration and washed with cold vinegar. The resulting crystals were washed with ethyl acetate (175 mL) and water (175 mL). The compound was obtained as white crystals (25.1 g). The obtained crystals (24.0 g, 82.1 mmol) were dissolved in 20% aqueous ethanol (300 mL). The suspension was heated to 65°C. Activated carbon (4.8 g) was added and stirred at 70°C for 30 minutes. The insoluble matter was filtered off and washed with 20% aqueous ethanol (72 mL). After adding water (300 mL) dropwise, the mixture was gradually cooled to 2°C and stirred for 2 hours. The precipitated crystals were collected by filtration. The crystals were then washed with cold 60% aqueous ethanol (120 mL). The obtained crystals were dried under reduced pressure. N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro Naphthalene-1,7-diyl)diacetamide was obtained as white crystals (21.6 g, 52% yield). And got it.

[0313] 1 H-NMR (500MHz,CDCl3)δ11.76(1H,s),8.43(1 H,d,J=13.0Hz),6.53(1H,d,J=4.5Hz),4.62(1H ,dt,J=14.0,5.4Hz),3.08-2.96(2H,m),2.78-2 .72(1H,m),2.23(3H,s),2.15(3H,d,J=1.5Hz), 2.11(3H,s),1.88-1.77(1H,m)

[0314] (Example 4-3) N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) Phthalene-1,7-diyl)diacetamide

[0315] [ka]

[0316] N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) (1-phenyl-1-yl)acetamide (3.0 g, 12.8 mmol) in tetrahydrofuran ( The solution was cooled to 0°C and diluted with amyl nitrite (2.2 mL, 16.6 mmol), potassium chloride (2.2 mL, 16.6 mmol), and Umium tert-butoxide (1.7 g, 15.3 mmol) was added and the mixture was stirred at 3°C ​​for 3 hours. After stirring, acetic acid (15 mL) and acetic anhydride (15 mL) were added, and the temperature was raised to 20°C. The mixture was stirred for 5 hours. At about 3°C, 5% platinum on carbon catalyst (0.4 g) was added, and the atmosphere was replaced with nitrogen. The mixture was purged with hydrogen and stirred under a hydrogen atmosphere (0.6 MPa) at about 5°C for 3 hours. The mixture was stirred at 0°C for 1 hour, and the insoluble matter was filtered off and washed with ethyl acetate (15 mL). Activated carbon (0.5 g) was added to the solution, and after stirring at room temperature for 2 hours, the insoluble matter was filtered off. The filtrate was cooled to about 5°C and washed with 5N aqueous sodium hydroxide ( The aqueous layer was removed and the mixture was diluted with tetrahydrofuran (30 mL) and 5N sodium hydroxide. An aqueous solution of sodium (30 mL) was added, and the aqueous layer was removed. The resulting organic layer was diluted with water (15 mL). After the addition of the above, 5N aqueous sodium hydroxide solution (15 mL) was added to adjust the pH to about 7. After the temperature was raised to 10°C, the aqueous layer was removed and the organic layer was washed with 10 wt% saline (15 mL). The organic layer was concentrated under reduced pressure to give a concentrated residue. Ethyl acetate (30 mL) was added to the concentrated residue, and This procedure was repeated three times, and the concentrated residue (approximately 15 mL) was heated at 40°C for 5 hours. After stirring for 1 hour, the mixture was cooled to room temperature and stirred at 5°C for 2 hours or more. The precipitated crystals were collected by filtration and washed with cold acetic acid. The resulting crystals were washed with ethyl acetate (15 mL) and water (15 mL). N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene) (1,7-diyl) phenyldiacetamide was obtained as white crystals (2.3 g, 62% yield). .

[0317] (Example 5-1) N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydrofuran) Dronaphthalen-2-yl)acetamide

[0318] [ka]

[0319] N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydro Naphthalene-1,7-diyl)diacetamide (3.0 g, 10.3 mmol) and 2N salt The acid / ethanol (30 mL) suspension was stirred at 50° C. for 7 hours. 45 mL) was added and cooled to 1° C. At 1° C., triethylamine (8.6 mL, 61.6 After adding sodium sulfite (26 mg, 0.2 mmol) dropwise, After stirring for 4 hours, the precipitated crystals were filtered and diluted with cold 60% aqueous ethanol (30 mL), water ( The resulting crystals were dried under reduced pressure to give pale green crystals (2.4 g). The obtained pale green crystals (1.8 g) were suspended in acetone (18 mL) at 50°C. After stirring for 5 hours, the mixture was cooled to room temperature, and the precipitated crystals were collected by filtration and washed with acetone (9 mL). The obtained crystals were dried under reduced pressure at 40°C to give N-(8-amino-6-fluoro-5-methyl- ethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide was obtained as pale green crystals (1.6 g, 82% yield).

[0320] 1 H-NMR (400MHz,DMSO-d6)δ8.07(1H,d,J=8.0H z),7.40(2H,brs),6.38(1H,d,J=13.2Hz),4.52 -4.43(1H,m),2.98-2.88(1H,m),2.87-2.76(1H ,m),2.18-2.10(1H,m),1.98(3H,d,J=1.2Hz),1 .90(3H,s),1.88-1.78(1H,m)

[0321] (Example 5-2) N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydrofuran) Dronaphthalen-2-yl)acetamide

[0322] [ka]

[0323] N,N'-(3-fluoro-4-methyl-8-oxo-2-methyl ... 5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide 0.0 g, 17.1 mmol) was added in five portions at room temperature, and the mixture was stirred at 50°C for 5 hours. Water (113 mL) was added to the reaction mixture and cooled to 2° C. Triethylamine (22. 5 mL, 161.4 mmol) was added dropwise, and then sodium sulfite (43 mg, 0.3 mmol) After stirring at 2°C for 3 hours, the precipitated crystals were filtered and dissolved in cold 60% aqueous ethanol. (50 mL) and water (25 mL). The obtained crystals were dried under reduced pressure to give pale blue crystals. (3.9 g). The obtained crystals (1.8 g) were suspended in acetone (18 mL). The solution was stirred at 50°C for 5 hours and then cooled to room temperature. The precipitated crystals were collected by filtration and diluted with acetone (9 ml). The resulting crystals were dried at 40°C under reduced pressure to give N-(8-amino-6-fluoro- 5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl The acetamide was obtained as pale green crystals (1.6 g, 80% yield).

[0324] 1 H-NMR (400MHz,DMSO-d6)δ8.07(1H,d,J=8.0H z),7.40(2H,brs),6.38(1H,d,J=13.2Hz),4.52 -4.43(1H,m),2.98-2.88(1H,m),2.87-2.76(1H ,m),2.18-2.10(1H,m),1.98(3H,d,J=1.2Hz),1 .90(3H,s),1.88-1.78(1H,m)

[0325] Example 6-1 N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-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 Cetoamide

[0326] [ka]

[0327] N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetramethyl-N ... Hydronaphthalen-2-yl)acetamide (170.0 g, 679 mmol) and (4S )-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]ine Triethanolamine-3,6,10(4H)-trione (196.7 g, 747 mmol) A suspension of o-cresol (510 mL) and pyridinium p-toluenesulfonate was added to the suspension. Sulfonate (25.6 g, 102 mmol) was added and refluxed for 32 hours. 00 mL) was added, and the mixture was cooled to room temperature and stirred for an additional 2 hours. The precipitated crystals were filtered and separated. The crystals were washed with acetone (850 mL). The obtained crystals were dried under reduced pressure at 40°C and Colored crystals of N-[(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]acetamide (312.5 g, yield 96%) was obtained.

[0328] 1 H-NMR(400MHz,DMSO-d6)δ0.87(3H,t,J=7.3Hz ),1.79-1.88(2H,m),1.91(3H,s),2.13-2.15(2 H,m),2.39(3H,s),3.13-3.22(2H,m),5.20(2H, dd,J=25.6, 18.9Hz),5.42(2H,s),5.53-5.57( 1H,m),6.52(1H,s),6.65-6.69(0.4H,m),6.75( 0.4H,d,J=7.9Hz),6.95-6.99(0.4H,m),7.03(0 .4H,d,J=7.3Hz),7.13-7.27(0.4H,m).7.30(1H ,s),7.79(1H,d,J=11.0Hz),8.46(1H,d,J=9.2H z),9.19(0.4H,s). 13 C-NMR (100MHz,DMSO-d6)δ7.7, 10.9, 10.9 , 15.9, 22.6, 23.1, 27.7, 30.3, 44.0, 49 .5, 65.2, 72.3, 96.6, 109.7, 109.9, 114. 5, 118.7, 119.1, 121.4, 123.6, 123.7, 12 3.7, 125.3, 125.5, 126.6, 128.2, 128.9, 130.5, 136.2, 136.3, 140.4, 145.2, 147.8 , 147.9, 149.9, 152.3, 155.3, 156.6, 160 .3, 162.8, 169.1, 172.4. MS(ESI)(m / z):478([M+H] + ).

[0329] (Example 6-2) N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-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 Cetoamide

[0330] [ka]

[0331] N-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetramethyl-N ... Hydronaphthalen-2-yl)acetamide (2.5 g, 9.99 mmol) and (4S) -4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indo Lysine-3,6,10(4H)-trione (3.42 g, 12.99 mmol) A suspension of o-cresol (7.5 mL) and pyridinium p-toluenesulfonate (125 mL) was added. The mixture was refluxed for 19 hours (intermediate and and the compound represented by formula (30) and the compound represented by formula (31) are passed through confirmed).

[0332] Compounds represented by formula (30): N-[(2S)-8-{[(4S)-4-ethyl-4-hydroxy-3,10-dioxo-3 ,4,8,10-tetrahydro-1H-pyrano[3,4-f]indolizin-6-yl] Amino}-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene talen-2-yl]acetamide

[0333] [ka]

[0334] 1 H-NMR(500MHz,CDCl3)δ1.04(3H,t,J=7.5Hz), 1.80-1.93(2H,m),2.02-2.18(8H,m),3.80-3.8 5(1H,m),4.62-4.68(1H,m),4.75-4.85(m,2H), 5.20-5.33(m,2H),5.70(1H,d,J=16.0Hz),6.35 (1H,s),6.67(1H,d,J=5.5Hz),6.88(1H,s),6.9 9 (1H, d, J = 12.0 Hz), 11.14 (1H, s). MS(ESI)(m / z):496.5([M+H] + ).

[0335] Compounds represented by formula (31): N-[(2R)-8-{[(4S)-4-ethyl-4-hydroxy-3,10-dioxo-3 ,4,8,10-tetrahydro-1H-pyrano[3,4-f]indolizin-6-yl] Amino}-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene talen-2-yl]acetamide

[0336] [ka]

[0337] 1 H-NMR(500MHz,CDCl3)δ1.03(3H,t,J=7.5Hz), 1.80-1.92(2H,m),2.02-2.18(8H,m),3.79(1H, s),4.60-4.68(1H,m),4.72-4.87(m,2H),5.28( 1H,d,J=16.0Hz),5.70(1H,d,J=16.0Hz),6.35( 1H,s),6.68(1H,d,J=4.5Hz),6.88(1H,s),7.00 (1H,d,J=12.0Hz),11.10(1H,s). MS(ESI)(m / z):496.6([M+H] + ).

[0338] After cooling, the liquid volume was adjusted to 135 mL with toluene and stirred for a further 2 hours. The crystals were filtered and washed with acetone (12.5 mL). Dry at 40°C under reduced pressure to obtain yellow crystals of N-[(9S)-9-ethyl-5-fluoro-9-hydro] Oxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydride 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2 -b]quinolin-1-yl]acetamide (4.58 g, 96% yield).

[0339] The instrumental data was similar to that of the compound described in Example 6-1.

[0340] Example 7-1 Methanesulfonic acid (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 1-Aminium Phosphate Dihydrate

[0341] [ka]

[0342] N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl] Acetamide (300.0 g, 628 mmol) in 2-methoxyethanol (1.5 L) , water (4.5 L), ethylcyclohexane (1.5 L) suspension, methanesulfonic acid (1. After cooling to room temperature, the organic layer was removed and the pressure was reduced to 3 L. The concentrate was heated to 40°C, and methanol (6 L) was added dropwise over 30 minutes. After stirring for a while, the precipitated crystals were filtered and washed with methanol (1.5 L).

[0343] The obtained crystals were dissolved in water (1.2 L), methanol (600 mL), methanesulfonic acid (1. 2 L), activated carbon (15 g) was added, and the mixture was stirred for 30 minutes. ) was added and stirred for 30 minutes, and then the insoluble matter was filtered off. The filtrate was heated to 40°C and washed with methanol (600 mL). After stirring for 2 hours, the precipitated crystals were filtered. The precipitated crystals were washed with methanol (1.5 L).

[0344] The obtained crystals were suspended in ethanol (6 L) and water (600 mL) and refluxed for 1.5 hours. After cooling to room temperature, the mixture was stirred for 30 minutes and the precipitated crystals were filtered. The obtained crystals were dried under reduced pressure at 40°C, and then placed in air at 40% RH for 4 days. By adjusting the humidity, colorless crystals of (1S,9S)-9-ethyl-5-fluoromethanesulfonate are obtained. Oro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13, 15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]ine Dolidino[1,2-b]quinoline-1-aminium dihydrate (152.3 g, yield 43 %) was obtained.

[0345] 1 H-NMR(400MHz,DMSO-d6,D2O)δ0.89(3H,t,J=7 .3Hz),1.90(2H,q,J=7.3Hz),2.35(3H,s),2.38 -2.47(1H,m),2.64(3H,s),3.04-3.11(1H,m),3 .30-3.34(1H,m),5.08(1H,s),5.34(2H,dd,J=1 7.7, 15.9Hz),5.50(2H,dd,J=17.7, 10.4Hz), 7.41(1H,s),7.59(1H,d,J=11.0Hz). 13 C-NMR (125MHz,DMSO-d6)δ7.7, 10.9, 11.0 , 18.5, 20.8, 24.7, 30.2, 39.5, 44.5, 49 .4, 55.9, 65.2, 72.2, 95.3, 96.9, 110.1, 100.3, 119.4, 120.5, 124.6, 124.7, 127. 5, 134.2, 135.2, 135.2, 144.8, 147.8, 14 7.9, 149.9, 152.3, 156.6, 160.6, 162.6, 172.3. MS(ESI)(m / z):436([M+H] + ).

[0346] (Example 7-2) Methanesulfonic acid (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 Phosphorus-1-aminium

[0347] [ka]

[0348] N-[(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,1 3-Dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl] Acetamide (3.5 g, 7.3 mmol) in purified water (53 mL), 2-methoxyethanol A suspension of ethanol (18 mL) and ethylcyclohexane (18 mL) was added to methanesulfonic acid (18 mL). After adding 1 L of the mixture, the mixture was purged with nitrogen under reduced pressure three times (after stirring under reduced pressure at 50 mbar, the mixture was purged with nitrogen under normal pressure three times). The reaction mixture was heated to 85°C and stirred for 11 hours. After checking, the mixture was cooled to 25°C. The mixture was concentrated under reduced pressure to 38.5 mL, and the concentrated solution was heated to 40°C. The mixture was heated, and methanol (18 mL) was added dropwise over 15 minutes. After stirring for 6 hours, methanol (5 mL) was added dropwise. After stirring for another 2 hours, the precipitated crystals were filtered. The crystals were washed with methanol (35 mL).

[0349] The obtained crystals were dissolved in a mixture of purified water (14 mL) and methanesulfonic acid (14 mL). After heating to 37°C, methanol (7 mL), activated carbon (0.35 g), and filter aid (0.7 0g, diatomaceous earth: Celpure C1000) was added, and the vacuum and nitrogen substitution were repeated three times (5 After stirring the suspension for 20 minutes, the insoluble matter was filtered off. The solution was diluted with methanesulfonic acid-purified water-methanol mixture (7 mL, 7 mL, 3.5 mL) and The filtrate was heated to 37°C and washed with methanol (10.5 mL). After stirring for 6 hours, methanol (42 mL) was added dropwise over 1 hour. After stirring for another 2 hours, the precipitated crystals were filtered and the filtered crystals were dissolved in methanol (35 mL). Washed.

[0350] The obtained crystals were suspended in ethanol (70 mL) and water (7 mL) and stirred at 73°C for 2 hours. After cooling to 25°C, the mixture was stirred for 2 hours and the precipitated crystals were filtered. The resulting crystals were dried under reduced pressure at 40°C and purified by filtration using methanesulfonic acid (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]quinoline-1-aminium (1.7 2 g was obtained, yield 44%.

[0351] The instrumental data was similar to that of the compound described in Example 7-1.

[0352] Example 8 N-(3-iodo-5-fluoro-4-methylphenyl)acetamide

[0353] [ka]

[0354] Concentration of 2-fluoro-1-methyl-4-nitrobenzene (5.0 g, 32.3 mmol) A solution of sulfuric acid (≥90%, 25 mL) and heptane (25 mL) was cooled to approximately 1 °C and then heated with N -Iodosuccinimide (10.2 g, 45.1 mmol) was added in six portions. The mixture was stirred at 2°C for 2 hours. The resulting reaction mixture was added to cold water (25 mL). Toluene (25 The organic layer was then diluted with water (25 mL), 6.5 wt % sodium bicarbonate aqueous solution (25 mL), 5 wt% sodium sulfite aqueous solution (25 mL The aqueous layer was removed from the filtrate, and the organic layer was extracted under reduced pressure. Ethyl acetate (50 mL) was added to the concentrated residue (approximately 30 mL), and the mixture was again concentrated under reduced pressure. Concentrated solution of 1-iodo-3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (approximately 15 mL) was obtained.

[0355] 1-Iodo-3-fluoro-2-methyl-5-nitrobenzene in ethyl acetate (approximately 1 A suspension of 1% platinum-on-carbon catalyst (1.1 g) and ethyl acetate (45 mL) was added to 5 mL of ethanol. The atmosphere was replaced with nitrogen and then with hydrogen. The mixture was stirred under a hydrogen stream (0.1 MPa) at approximately 60°C for 5 hours. The resulting suspension was stirred and cooled to room temperature. Insoluble matter was filtered off from the resulting suspension, and the insoluble matter was dissolved in ethyl acetate (1 The filtrate was washed twice with 0.5N aqueous hydrochloric acid (50 mL, 25 mL) to obtain an organic layer. The aqueous layer was extracted with ethyl acetate (25 mL) to obtain an organic layer, which was then combined. Then, 6.5 wt% sodium bicarbonate aqueous solution (25 mL), 5 wt% saline solution (25 mL), and the resulting organic layer was concentrated under reduced pressure to give 3-iodo-5-fluoro A solution of 4-methylaniline in ethyl acetate was obtained.

[0356] A solution of 3-bromo-5-fluoro-4-methylaniline in ethyl acetate was added to ethyl acetate (25 mL) was added to the solution, and triethylamine (3.7 mL, 26.8 mmol), anhydrous acetic acid Acid (1.7 mL, 17.7 mmol) was added and the mixture was stirred at room temperature for 4 hours. 10 wt% brine (25 mL) was added to the mixture, and the layers were separated, after which the aqueous layer was removed. The mixture was concentrated under reduced pressure. Ethyl acetate (50 mL) was added to the concentrated residue, and the mixture was diluted with 4N hydrochloric acid / ethyl acetate. After adding the solution (5.6 mL, 22.6 mmol), the mixture was stirred at room temperature for 15 minutes. The insoluble matter was washed with ethyl acetate (20 mL). The filtrate was diluted with 10 wt% saline (20 mL). After adding the above, 25 w / v% aqueous sodium hydroxide solution (2.5 mL) was added to adjust the pH to approximately 7. After removing the aqueous layer, the organic layer was concentrated under reduced pressure. The concentrated residue was added with acetonitrile (38 mL ), water (38 mL) was added, and the mixture was stirred at 25°C. The precipitated crystals were collected by filtration and The resulting crystals were washed with water and acetonitrile (15 mL). The crystals were dried under reduced pressure at 40°C and N- (3-iodo-5-fluoro-4-methylphenyl)acetamide was obtained as white crystals (2.8g The compound was obtained in a yield of 29%.

[0357] 1 H-NMR (500MHz,CDCl3)δ7.61(1H,s),7.47(1H ,d,J=10.8Hz),7.10(1H,brs),2.30(3H,d,J=2. 3Hz), 2.16(3H,s)

[0358] Example 9 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid

[0359] [ka]

[0360] N-(3-iodo-5-fluoro-4-methylphenyl)acetamide (2.0 g, 6 0.8mmol), 3-butenoic acid (0.7mL, 8.2mmol), diisopropyl ethyl A solution of amine (2.6 mL, 15.0 mmol) in tetrahydrofuran (8 mL), water (2 mL ) solution was degassed under reduced pressure and replaced with nitrogen, and then tri(o-tolyl)phosphine (62.3 mg , 0.2 mmol) was added. After degassing and replacing the atmosphere with nitrogen again under reduced pressure, palladium acetate (II ) (23.0 mg, 0.1 mmol) was added, and the mixture was degassed under reduced pressure, replaced with nitrogen, and then heated for 2 hours. The reaction mixture was heated to reflux, and then cooled to room temperature. To the mixture was added ethanol (10 mL) and water (10 mL), and the mixture was stirred at room temperature for 1 hour. The solution was washed with 20% aqueous tetrahydrofuran (4 mL). Add 10 mL of dorofuran and 10 mL of water to make a 25 w / v% aqueous solution of sodium hydroxide. The organic layer was removed and the aqueous layer was added with 2-methyltetrahydrofuran (1.3 mL, 8.2 mmol). After adding dihydrofuran (20 mL) and concentrated hydrochloric acid (36%, 1.2 g, 12.3 mmol) After separation, the aqueous layer was removed, and the organic layer was washed with 10 wt % sodium chloride. The resulting organic layer was concentrated under reduced pressure to give 4-[5 -(acetylamino)-3-fluoro-2-methylphenyl]-3-butenoic acid residue (1. 9g) was obtained.

[0361] 4-[5-(acetylamino)-3-fluoro-2-methylphenyl] including geometric isomers ]-3-butenoic acid residue (1.9 g) in 2-methyltetrahydrofuran (30 mL), 5% Palladium carbon (1.7 g) was added to the suspension, and the atmosphere was replaced with nitrogen and then with hydrogen. The mixture was stirred under a stream of air (0.3 MPa) at about 40°C for 8 hours and then cooled to room temperature. The insoluble matter was filtered off from the liquid and washed with 2-methyltetrahydrofuran (8 mL). Water (10 mL) was added to the filtrate, and the pH was adjusted to about 2 with 1N hydrochloric acid. After separation, the aqueous layer was removed. The resulting organic layer was concentrated under reduced pressure. Ethyl acetate (10 mL) was added to the concentrated residue, and After heating to about 50°C, heptane (10 mL) was added and the mixture was cooled to room temperature. The crystals were collected by filtration and washed with ethyl acetate / heptane (3 / 7) (6 mL). Dry under reduced pressure to obtain 4-[5-(acetylamino)-3-fluoro-2-methylphenyl] Butanoic acid was obtained as white crystals (1.4 g, 81% yield).

[0362] Example 10 N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene (1-phenyl)acetamide

[0363] [ka]

[0364] 4-[5-(acetylamino)-3-fluoro-2-methylphenyl]butanoic acid (20 0mg, 0.79mmol), thionyl chloride (86μL, 1.18mmol), methyl chloride A solution of aluminum chloride (263 mg, 1.97 m mol) was added and stirred at room temperature for 2 hours. The aqueous layer was removed, and the air layer was diluted with water (10 ml), 6.5 ml of ethyl acetate, and the mixture was stirred for 1 hour. Wash with wt% aqueous sodium bicarbonate solution (10 ml) and water (10 ml), and the obtained organic layer The layer was dried over sodium sulfate. After filtering off the insoluble matter, the solvent was distilled off under reduced pressure to obtain a residue. was purified by preparative thin layer chromatography (hexane:ethyl acetate=2:1) ​​to obtain N-(3- Fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl The obtained product was acetamide as pale yellowish white crystals (125 mg, 67% yield).

[0365] The instrumental data was similar to that of the compound described in Example 3-1. [Sequence List Free Text]

[0366] SEQ ID NO: 1: Amino acid sequence of the anti-HER2 antibody heavy chain SEQ ID NO: 2: Amino acid sequence of the anti-HER2 antibody light chain SEQ ID NO: 3: Amino acid sequence of the heavy chain of the anti-HER3 antibody SEQ ID NO: 4: Amino acid sequence of the light chain of the anti-HER3 antibody SEQ ID NO: 5: Amino acid sequence of the anti-TROP2 antibody heavy chain SEQ ID NO: 6: Amino acid sequence of the anti-TROP2 antibody light chain SEQ ID NO: 7: Amino acid sequence of the anti-B7-H3 antibody heavy chain SEQ ID NO: 8: Amino acid sequence of the anti-B7-H3 antibody light chain SEQ ID NO: 9: Amino acid sequence of the anti-GPR20 antibody heavy chain SEQ ID NO: 10: Amino acid sequence of the anti-GPR20 antibody light chain SEQUENCE LISTING <110> DAIICHI SANKYO COMPANY, LIMITED <120> NOVEL METHOD FOR PRODUCING ANTIBODY-DRUG CONJUGATE <130> PD20A-0131 <150> JP2017-167690 <151> 2017-08-31 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-HER2 antibody <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-HER2 antibody <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-HER3 antibody <400> 3 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Lys Trp Thr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 4 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-HER3 antibody <400> 4 Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 5 <211> 470 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-TROP2 antibody <400> 5 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Thr Ala Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala 65 70 75 80 Glu Asp Phe Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr Ser 85 90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp 115 120 125 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> 6 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-TROP2 antibody <400> 6 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Ile Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 7 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-B7-H3 antibody <400> 7 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 8 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-B7-H3 antibody <400> 8 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 9 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of anti-GPR20 antibody <400> 9 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Tyr Ile Ser Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Lys Tyr Met Gly Phe Ile Asn Pro Gly Ser Gly His Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Thr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Ala Gly Gly Phe Leu Arg Ile Ile Thr Lys 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 10 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Light chain of anti-GPR20 antibody <400> 10 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Asp Thr Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser 35 40 45 Val Ser Thr Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Lys Gln Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Gly Asn Leu Glu Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Asn Tyr Tyr Cys Gln Gln Ile Asn 100 105 110 Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230

Claims

[Claim 1] The invention described herein.

Images