Methods for selective manufacturing of antibody-drug conjugates

Abstract

This invention relates to a method for selectively manufacturing antibody-drug conjugates, i.e., a method for producing antibody-drug conjugate compositions, comprising: (i) reacting an antibody with a reducing agent in a buffer solution to reduce interchain disulfide; and (ii) reacting a drug linker intermediate with an antibody having a thiol group obtained in step (i), wherein the reaction temperature in step (i) is from -10°C to 10°C, and the average number of bound drugs in the resulting antibody-drug conjugate composition is 3.5 to 4.5, and the content of antibody-drug conjugates in the resulting antibody-drug conjugate composition having four drug linkers bound to heavy-light interchain thiols is 50% or more; and an antibody-drug conjugate composition wherein the content of antibody-drug conjugates having an average number of bound drugs of 3.5 to 4.5 and the content of antibody-drug conjugates having four drug linkers bound to heavy-light interchain thiols is 50% or more.

Description

[0001] This application is a divisional application of the invention patent application filed on June 28, 2016, with application number 201680038178.2 and entitled "Method for Selectively Manufacturing Antibody-Drug Conjugates". Technical Field

[0002] The present invention relates to a method for producing an antibody-drug conjugate composition wherein the number of drugs bound and the binding sites are controlled, and to an antibody-drug conjugate composition wherein the number of drugs bound and the binding sites are controlled. Background Technology

[0003] It is anticipated that antibody-drug conjugates (hereinafter also referred to as "ADCs") formed by binding a cytotoxic drug to an antibody that binds to an antigen expressed on the surface of cancer cells and then internalizing it into the cells can selectively deliver the drug to cancer cells, causing the drug to accumulate within the cancer cells and kill them (see Non-Patent Literature 1 to 3). As such antibody-drug conjugates, antibody-drug conjugates formed by binding eczemab, a camptothecin derivative, to an anti-B7-H3 antibody, etc., are known (Patent Literature 1).

[0004] Antibodies possess four interchain disulfides. These interchain disulfides are more readily accessible to solvents and easier to reduce than other disulfides. Therefore, these interchain disulfides can serve as binding sites for drugs (or drug linkers) in antibody-drug conjugates. The interchain disulfides in the antibody are reduced, and then the drug binds to the resulting thiol groups, thereby producing antibody-drug conjugates in which 2 to 8 drug molecules bind to a single antibody molecule. Furthermore, methods for selectively generating antibody-drug conjugates having four drug linkers binding to heavy-heavy interchain thiols are known, wherein the method involves first completely reducing the interchain disulfides in the antibody, re-oxidizing some of the resulting interchain thiols to return them to disulfides, and then binding the drug to the remaining interchain thiols (Patent Document 2). However, methods for selectively generating antibody-drug conjugates having four drug linkers binding to heavy-light interchain thiols are still unknown.

[0005] Citation List

[0006] Patent documents

[0007] Patent Document 1: International Publication No. WO2014 / 057687

[0008] Patent Document 2: International Publication No. WO2005 / 084390

[0009] Non-patent literature

[0010] Non-patent literature 1: Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13.

[0011] Non-patent literature 2: Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537.

[0012] Non-patent literature 3: Damle NK, Expert Opin. Biol. Ther. (2004) 4, 1445-1452. Invention Overview

[0013] Technical issues

[0014] Antibody-drug conjugates containing eight drugs bound to a single antibody molecule are excellent in antitumor efficacy, but can cause safety issues such as side effects and toxicity in certain situations. Therefore, to reduce side effects or toxicity while maintaining therapeutic efficacy, antibody-drug conjugates with an average number of bound drugs less than eight are used. Such conjugates with an average number of bound drugs less than eight can be obtained, for example, by reacting the drug with the antibody while controlling the amount of drug per antibody molecule. The reaction products are antibody-drug conjugate compositions in which the number of bound drugs is 2, 4, 6, and 8. Therefore, it is possible that even if antibody-drug conjugate compositions have the same average number of bound drugs as each other, their efficacy and toxicity may differ if they each have a different distribution of the number of bound drugs. That is, when the content of antibody-drug conjugates with an average number of bound drugs of 4 is high, the therapeutic effect may be reduced and strong toxicity may be exhibited compared to the case where the content of antibody-drug conjugates with an average number of bound drugs of 4 is high. Furthermore, there may be situations where, even if antibody-drug conjugates have the same number of bound drugs as each other, their therapeutic efficacy and toxicity differ due to differences in the drug binding sites. Therefore, it is desirable to develop methods for generating antibody-drug conjugate compositions in which the number of bound drugs and binding sites are controlled during the generation of the antibody-drug conjugate composition.

[0015] Solution to the problem

[0016] As a result of in-depth research aimed at achieving the above objectives, the inventors have discovered a simpler method for producing antibody-drug conjugate compositions in which the number of bound drugs and binding sites are controlled. Specifically, the inventors have discovered that antibody-drug conjugate compositions can be produced by reducing an antibody in a buffer solution at a temperature of -10°C to 10°C using a reducing agent and then reacting a drug linker intermediate with the obtained antibody having thiol groups, wherein the content of the antibody-drug conjugate (where the average number of bound drugs is 3.5 to 4.5 and four drug linkers are bound to heavy-light chain inter-thiols) is 50% or more. Furthermore, the inventors have also discovered that the antibody-drug conjugate compositions of the present invention have superior safety compared to antibody-drug conjugate compositions produced by conventional production methods (i.e., antibody-drug conjugate compositions in which the content of the antibody-drug conjugate (where the average number of bound drugs is 3.5 to 4.5 and four drug linkers are bound to heavy-light chain inter-thiols) is 35% or less, thus completing the present invention.

[0017] Specifically, the invention of this application relates to the following (1) to (77): (1)

[0019] A method for generating an antibody-drug conjugate composition, comprising:

[0020] (i) The step of reacting the antibody with a reducing agent in a buffer solution to reduce interchain disulfide; and

[0021] (ii) The step of reacting the drug linker intermediate with the thiol-containing antibody obtained in step (i), wherein

[0022] The reaction temperature in step (i) is from -10°C to 10°C, and

[0023] The average number of bound drugs in the resulting antibody-drug conjugate composition is 3.5 to 4.5, and the content of antibody-drug conjugates in which four drug linkers are bound to heavy-light interchain thiols is 50% or more. (2)

[0025] According to the production method described in (1) above, the average number of bound drugs in the antibody-drug conjugate composition produced is 4.0 to 4.1. (3)

[0027] According to the production method described in (1) or (2) above, the content of the antibody-drug conjugate composition in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 90%. (4)

[0029] According to the production method described in (3) above, the content of the antibody-drug conjugate composition in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 80%. (5)

[0031] According to the production method described in (4) above, the content of the antibody-drug conjugate composition in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 70%. (6)

[0033] According to the production method described in (5) above, the content of the antibody-drug conjugate composition in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 60%. (7)

[0035] According to any one of (1) to (6) above, in the produced antibody-drug conjugate composition, the content of the antibody-drug conjugate in which the four drug linkers are bound to heavy-heavy chain thiols is 5% or less. (8)

[0037] According to the production method described in (7) above, the content of the antibody-drug conjugate composition in which the four drug linkers are bound to heavy-heavy chain thiols is 1% or less. (9)

[0039] According to any one of the production methods in (1) to (8) above, the content of the antibody-drug conjugate composition in which two drug linkers are bound to heavy-heavy interchain thiols and two drug linkers are bound to heavy-light interchain thiols is 5% or less. (10)

[0041] According to any one of the production methods in (1) to (8) above, the content of the antibody-drug conjugate composition in which two drug linkers are bound to heavy-heavy interchain thiols and two drug linkers are bound to heavy-light interchain thiols is 1% or less. (11)

[0043] According to any one of (1) to (10) above, the production method wherein the reaction temperature in step (i) is -5°C to 5°C. (12)

[0045] According to the production method described in (11) above, the reaction temperature in step (i) is -3°C to 3°C. (13)

[0047] According to the production method described in (12) above, the reaction temperature in step (i) is 0°C to 2°C. (14)

[0049] According to the production method described in (13) above, the reaction temperature in step (i) is 0°C to 1°C. (15)

[0051] According to any one of (1) to (14) above, the production method wherein the reaction temperature in step (ii) is 0°C to 2°C. (16)

[0053] According to any one of the production methods (1) to (15) above, the reducing agent is used in an amount of 2 to 3 molar equivalents per antibody molecule. (17)

[0055] The production method according to any one of (1) to (16) above, wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof. (18)

[0057] According to the production method described in (17) above, the salt of tris(2-carboxyethyl)phosphine is tris(2-carboxyethyl)phosphine hydrochloride. (19)

[0059] The production method according to any one of (1) to (18) above, wherein the buffer is a histidine buffer. (20)

[0061] The production method according to any one of (1) to (19) above, wherein the buffer solution contains a chelating agent. (twenty one)

[0063] According to the production method described above (20), the chelating agent is ethylenediaminetetraacetic acid. (twenty two)

[0065] The production method according to any one of (1) to (21) above, wherein the antibody is an anti-TROP2 antibody, an anti-CD98 antibody, an anti-B7-H3 antibody or an anti-HER2 antibody. (twenty three)

[0067] According to the production method described above (22), the antibody is an anti-TROP2 antibody. (twenty four)

[0069] According to the production method described above (22), the antibody is an anti-CD98 antibody. (25)

[0071] According to the production method described above (22), the antibody is an anti-B7-H3 antibody. (26)

[0073] According to the production method described in (22) above, the antibody is an anti-HER2 antibody. (27)

[0075] The production method according to any one of (1) to (26) above, wherein the pharmaceutical linker intermediate has an N-substituted maleimide group. (28)

[0077] According to the production method described above (27), wherein

[0078] The drug connector intermediate is

[0079] [Formula 1]

[0080]

[0081] [Equation 2]

[0082]

[0083] ,or

[0084] [Formula 3]

[0085]

[0086] Wherein -GGFG- represents a tetrapeptide residue composed of glycine-glycine-phenylalanine-glycine. (29)

[0088] According to the production method described above (28), wherein

[0089] The drug connector intermediate is

[0090] [Formula 4]

[0091]

[0092] Wherein -GGFG- represents a tetrapeptide residue composed of glycine-glycine-phenylalanine-glycine. (30)

[0094] The antibody-drug conjugate composition produced by any one of the production methods described in (1) to (29) above. (31)

[0096] An antibody-drug conjugate composition wherein the average number of bound drugs is 3.5 to 4.5 and the content of the antibody-drug conjugate wherein 4 drug linkers are bound to heavy-light interchain thiols is 50% or more. (32)

[0098] According to the antibody-drug conjugate composition described in (31) above, the average number of bound drugs is 4.0 to 4.1. (33)

[0100] According to the antibody-drug conjugate composition described in (31) or (32) above, the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light interchain thiol is in the range of 50% to 90%. (34)

[0102] According to the antibody-drug conjugate composition described above (33), the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light chain inter-thiol is in the range of 50% to 80%. (35)

[0104] According to the antibody-drug conjugate composition described above (34), the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light interchain thiol is in the range of 50% to 70%. (36)

[0106] According to the antibody-drug conjugate composition described above (35), the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 60%. (37)

[0108] The antibody-drug conjugate composition according to any one of (31) to (36) above, wherein the content of the antibody-drug conjugate in which the four drug linkers are bound to heavy-heavy chain thiols is 5% or less. (38)

[0110] According to the antibody-drug conjugate composition described above (37), the content of the antibody-drug conjugate in which the four drug linkers are bound to heavy-heavy chain thiols is 1% or less. (39)

[0112] The antibody-drug conjugate composition according to any one of (31) to (38) above, wherein the content of the antibody-drug conjugate in which two drug linkers are bound to heavy-heavy interchain thiols and two drug linkers are bound to heavy-light interchain thiols is 5% or less. (40)

[0114] According to the antibody-drug conjugate composition described above (39), the content of the antibody-drug conjugate in which two drug linkers are bound to heavy-heavy interchain thiols and two drug linkers are bound to heavy-light interchain thiols is 1% or less. (41)

[0116] The antibody-drug conjugate composition according to any one of (31) to (40) above, wherein the antibody is an anti-TROP2 antibody, an anti-CD98 antibody, an anti-B7-H3 antibody or an anti-HER2 antibody. (42)

[0118] According to the antibody-drug conjugate composition described in (41) above, wherein the antibody is an anti-TROP2 antibody. (43)

[0120] According to the antibody-drug conjugate composition described above (41), wherein the antibody is an anti-CD98 antibody. (44)

[0122] According to the antibody-drug conjugate composition described in (41) above, wherein the antibody is an anti-B7-H3 antibody. (45)

[0124] According to the antibody-drug conjugate composition described in (41) above, wherein the antibody is an anti-HER2 antibody. (46)

[0126] The antibody-drug conjugate composition according to any one of (31) to (45) above, wherein

[0127] The drug connector is

[0128] [Formula 5]

[0129]

[0130] [Formula 6]

[0131]

[0132] ,or

[0133] [Formula 7]

[0134]

[0135] Where A represents the binding site with the antibody, and -GGFG- represents a tetrapeptide residue composed of glycine-glycine-phenylalanine-glycine. (47)

[0137] According to the antibody-drug conjugate composition described above (46), wherein

[0138] The drug connector is

[0139] [Formula 8]

[0140]

[0141] Where A represents the binding site with the antibody, and -GGFG- represents a tetrapeptide residue composed of glycine-glycine-phenylalanine-glycine. (48)

[0143] A pharmaceutical composition comprising an antibody-drug conjugate composition according to any one of (30) to (47) above. (49)

[0145] The pharmaceutical composition described in (48) above is used to treat tumors and / or cancer. (50)

[0147] The pharmaceutical composition described in (49) above is used to treat lung cancer, kidney cancer, urothelial carcinoma, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, bile duct cancer, thyroid cancer, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia and / or multiple myeloma. (51)

[0149] Methods for treating tumors and / or cancer, comprising administering an antibody-drug conjugate composition according to any one of (30) to (47) above. (52)

[0151] According to the treatment method described above (51), it is a method for treating lung cancer, kidney cancer, urothelial carcinoma, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, bile duct cancer, thyroid cancer, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia and / or multiple myeloma. (53)

[0153] A method for generating antibodies with thiol groups includes the step of reacting the antibody with a reducing agent in a buffer solution to reduce interchain disulfides, wherein...

[0154] The reaction temperature is from -10℃ to 10℃, and

[0155] The resulting thiol-containing antibodies are used to generate antibody-drug conjugate compositions, wherein the average number of bound drugs is 3.5 to 4.5, and the content of antibody-drug conjugates in which 4 drug linkers are bound to heavy-light chain thiols is 50% or more. (54)

[0157] According to the production method described above (53), the antibodies with thiol groups produced are used to produce antibody-drug conjugate compositions, wherein the average number of drugs bound is 4.0 to 4.1. (55)

[0159] According to the production method described in (53) or (54) above, the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 90%. (56)

[0161] According to the production method described above (55), the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 80%. (57)

[0163] According to the production method described above (56), the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 70%. (58)

[0165] According to the production method described above (57), the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which the four drug linkers are bound to the heavy-light chain thiol is in the range of 50% to 60%. (59)

[0167] According to any one of the production methods described in (53) to (58) above, the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which four drug linkers are bound to heavy-heavy chain thiols is in the range of 5% or less. (60)

[0169] According to the production method described above (59), the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which the four drug linkers are bound to heavy-heavy chain thiols is in the range of 1% or less. (61)

[0171] According to any one of the production methods (53) to (60) above, the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which two drug linkers are bound to heavy-heavy chain thiols and two drug linkers are bound to heavy-light chain thiols is 5% or less. (62)

[0173] According to the production method described above (61), the antibody with thiol groups produced is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate in which two drug linkers are bound to heavy-heavy chain thiols and two drug linkers are bound to heavy-light chain thiols is 1% or less. (63)

[0175] The production method according to any one of (53) to (62) above, wherein the reaction temperature is -5°C to 5°C. (64)

[0177] According to the production method described above (63), the reaction temperature is -3°C to 3°C. (65)

[0179] According to the production method described above (64), the reaction temperature is 0°C to 2°C. (66)

[0181] According to the production method described above (65), the reaction temperature is 0°C to 1°C. (67)

[0183] According to any one of the production methods described in (53) to (66) above, the reducing agent is used in an amount of 2 to 3 molar equivalents per antibody molecule. (68)

[0185] The production method according to any one of (53) to (67) above, wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof. (69)

[0187] According to the production method described above (68), the salt of tris(2-carboxyethyl)phosphine is tris(2-carboxyethyl)phosphine hydrochloride. (70)

[0189] The production method according to any one of (53) to (69) above, wherein the buffer is a histidine buffer. (71)

[0191] The production method according to any one of (53) to (70) above, wherein the buffer solution contains a chelating agent. (72)

[0193] According to the production method described above (71), the chelating agent is ethylenediaminetetraacetic acid. (73)

[0195] The production method according to any one of (53) to (72) above, wherein the antibody is an anti-TROP2 antibody, an anti-CD98 antibody, an anti-B7-H3 antibody or an anti-HER2 antibody. (74)

[0197] According to the production method described above (73), the antibody is an anti-TROP2 antibody. (75)

[0199] According to the production method described above (73), the antibody is an anti-CD98 antibody. (76)

[0201] According to the production method described above (73), the antibody is an anti-B7-H3 antibody. (77)

[0203] According to the production method described above (73), the antibody is an anti-HER2 antibody.

[0204] Advantages of the present invention

[0205] According to the present invention, a method for producing an antibody-drug conjugate composition wherein the number of bound drugs and binding sites are controlled, and an antibody-drug conjugate composition wherein the number of bound drugs and binding sites are controlled, are provided. The antibody-drug conjugate compositions of the present invention are excellent in terms of safety and can be used as drugs for treating tumors and / or cancers. Furthermore, they are preferred because antibody-drug conjugate compositions wherein the number of bound drugs and binding sites are controlled to be constant can be obtained, as they also exhibit excellent quality control. Brief description of the attached diagram

[0206] [ Figure 1 ] Figure 1 This is a view showing the nucleotide and amino acid sequences of the humanized anti-TROP2 antibody heavy chain (hTINA1-H1).

[0207] [ Figure 2 ] Figure 2 This is a view showing the nucleotide and amino acid sequences of the humanized anti-TROP2 antibody light chain (hTINA1-L1).

[0208] [ Figure 3 ] Figure 3 This is a view showing the nucleotide and amino acid sequences of the humanized anti-CD98 antibody heavy chain (h23M-H1).

[0209] [ Figure 4 ] Figure 4 This is a view showing the nucleotide and amino acid sequences of the humanized anti-CD98 antibody light chain (h23M-L1).

[0210] [ Figure 5 ] Figure 5 This is a graph showing the peak area ratio (%) of each chain in the humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition produced by conventional methods.

[0211] [ Figure 6 ] Figure 6 This is a graph showing the distribution (%) of the respective numbers of drugs bound in a humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition produced by conventional methods.

[0212] [ Figure 7 ] Figure 7 This is a graph showing the peak area ratio (%) of each chain in the humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition produced by the method of the present invention.

[0213] [ Figure 8 ] Figure 8 This is a graph showing the distribution (%) of the respective numbers of bound drugs in the humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition produced by the method of the present invention.

[0214] [ Figure 9 ] Figure 9 This is a graph showing the peak area ratio (%) of each chain in the humanized anti-CD98 antibody (hM23-H1L1) ADC composition produced by conventional methods.

[0215] [ Figure 10 ] Figure 10 This is a graph showing the distribution (%) of the respective numbers of drugs bound in a humanized anti-CD98 antibody (hM23-H1L1) ADC composition produced by conventional methods.

[0216] [ Figure 11 ] Figure 11This is a graph showing the peak area ratio (%) of each chain in the humanized anti-CD98 antibody (hM23-H1L1) ADC composition produced by the method of the present invention.

[0217] [ Figure 12 ] Figure 12 This is a graph showing the distribution (%) of the respective numbers of bound drugs in the humanized anti-CD98 antibody (hM23-H1L1) ADC composition produced by the method of the present invention.

[0218] [ Figure 13 ] Figure 13 This is a view showing the amino acid sequence of the humanized anti-B7-H3 antibody heavy chain (M30-H1).

[0219] [ Figure 14 ] Figure 14 This is a view showing the amino acid sequence of the humanized anti-B7-H3 antibody light chain (M30-L4).

[0220] [ Figure 15 ] Figure 15 This is a graph showing the peak area ratio (%) of each chain in the humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition produced by conventional methods.

[0221] [ Figure 16 ] Figure 16 This is a graph showing the distribution (%) of the respective numbers of drugs bound in a humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition produced by conventional methods.

[0222] [ Figure 17 ] Figure 17 This is a graph showing the peak area ratio (%) of each chain in the humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition produced by the method of the present invention.

[0223] [ Figure 18 ] Figure 18 This is a graph showing the distribution (%) of the respective numbers of bound drugs in the humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition produced by the method of the present invention.

[0224] [ Figure 19 ] Figure 19 This is a view showing the amino acid sequence of the humanized anti-HER2 antibody heavy chain.

[0225] [ Figure 20 ] Figure 20 This is a view showing the amino acid sequence of the humanized anti-HER2 antibody light chain.

[0226] [ Figure 21 ] Figure 21 This is a graph showing the peak area ratio (%) of each chain in a humanized anti-HER2 antibody ADC composition produced by conventional methods.

[0227] [ Figure 22 ] Figure 22 This is a graph showing the distribution (%) of the respective numbers of drugs bound in a humanized anti-HER2 antibody ADC composition produced by conventional methods.

[0228] [ Figure 23 ] Figure 23 This is a graph showing the peak area ratio (%) of each chain in the humanized anti-HER2 antibody ADC composition produced by the method of the present invention.

[0229] [ Figure 24 ] Figure 24 This is a graph showing the distribution (%) of the respective numbers of bound drugs in the humanized anti-HER2 antibody ADC composition produced by the method of the present invention.

[0230] [ Figure 25 ] Figure 25 This is a graph showing the tumor growth inhibition effect of the humanized anti-TROP2 antibody ADC composition produced by conventional methods and the tumor growth inhibition effect of the humanized anti-TROP2 antibody ADC composition produced by the method of the present invention.

[0231] Description of the implementation plan

[0232] In this specification, the term "cancer" is used to have the same meaning as the term "tumor".

[0233] In this specification, the term "gene" is used to include not only DNA, but also its mRNA and cDNA and its cRNA.

[0234] In this specification, the term "polynucleotide" is used to have the same meaning as nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers.

[0235] In this specification, the term "polypeptide" is used to have the same meaning as the term "protein".

[0236] In this specification, the term "cell" includes cells in an individual animal and cultured cells.

[0237] In this specification, the term "interchain disulfide" is used to refer to a disulfide located between two heavy chains in an antibody (heavy-heavy chain intersulfide) or a disulfide located between a heavy chain and a light chain in an antibody (heavy-light chain intersulfide).

[0238] In this specification, the term "interchain thiol" is used to refer to the thiol group obtained by reducing the interchain disulfide of the antibody.

[0239] In this specification, the term "heavy-heavy chain thiol" is used to refer to the thiol group obtained by reducing the heavy-heavy chain disulfide of the antibody.

[0240] In this specification, the term "heavy-light interchain thiol" is used to refer to the thiol group obtained by reducing the heavy-light interchain disulfide of the antibody.

[0241] In this specification, the term "tumor-associated antigen (TAA)" is used to refer to an antigen that is expressed in both normal cells and tumor cells, but whose expression is relatively limited to tumor cells.

[0242] In this specification, the term "tumor-specific antigen (TSA)" is used to refer to an antigen that is specific to tumor cells.

[0243] In this specification, the term "TROP2" is used to have the same meaning as the TROP2 protein.

[0244] In this specification, the term "CD98" is used to have the same meaning as the CD98 protein. Since CD98 consists of a heavy chain and a light chain, the terms "CD98 heavy chain" and "CD98 light chain" are used to have the same meaning as the CD98 heavy chain protein and the CD98 light chain protein, respectively. Furthermore, in this specification, unless otherwise stated, the term "CD98" is used interchangeably with "CD98 heavy chain" and "CD98 light chain," or "CD98 heavy chain" or "CD98 light chain."

[0245] In this specification, the term "anti-TROP2 antibody" is used to refer to an antibody that can bind to TROP2.

[0246] In this specification, the term "anti-CD98 antibody" is used to refer to an antibody that can bind to CD98.

[0247] In this specification, the term "anti-B7-H3 antibody" is used to refer to an antibody that can bind to B7-H3.

[0248] In this specification, the term "anti-HER2 antibody" is used to refer to an antibody that can bind to HER2.

[0249] In this specification, the term "cytotoxic" is used to mean any pathological change in cells caused by any given means. It refers not only to direct external damage, but also to all types of structural or functional damage to cells, such as DNA cleavage, base dimer formation, chromosome cleavage, damage to the cell's mitotic apparatus, and reduced activity of various types of enzymes.

[0250] In this specification, the term "cytotoxicity" is used to refer to the effects that cause the aforementioned cytotoxic phenomena.

[0251] In this specification, “antibody-dependent cytotoxicity” is used to mean “antibody-dependent cytotoxic (ADCC) activity”, and this activity means the activity of antibody-mediated NK cells to inflict damage on target cells such as tumor cells.

[0252] In this specification, the term "complement-dependent cytotoxicity" is used to mean "complement-dependent cytotoxic (CDC) activity," and this activity means the activity of antibody-mediated complement to cause damage to target cells such as tumor cells.

[0253] In this specification, the term "epitope" is used to refer to a partial peptide or partial three-dimensional structure of an antigen that binds to a specific antibody. This epitope, as a partial peptide of an antigen, can be determined by methods well-known to those skilled in the art, such as immunoassays, for example, by the following method: First, various partial structures of the antigen are generated. To generate such partial structures, known oligopeptide synthesis techniques can be applied. For example, a series of peptides are generated using gene recombination techniques well-known to those skilled in the art, wherein the antigen is sequentially cleaved at an appropriate length from its C-terminus or N-terminus, and subsequently, the reactivity of the antibody with such peptides is studied, and the recognition site is roughly determined. Subsequently, further shorter peptides are synthesized, and their reactivity with the aforementioned peptides is then studied to determine the epitope. Alternatively, the epitope, as a partial three-dimensional structure of an antigen that binds to a specific antibody, can be determined by specifying the amino acid residues of the antigen adjacent to the aforementioned antibody via X-ray structural analysis.

[0254] In this specification, the phrase "antibodies binding to the same epitope" refers to different antibodies that bind to a common epitope. If a second antibody binds a portion of the peptide or a portion of the three-dimensional structure bound by a first antibody, then the first and second antibodies can be identified as binding to the same epitope. Furthermore, by demonstrating that the second antibody competes with the first antibody for binding to the antigen (i.e., the second antibody prevents the first antibody from binding to the antigen), the first and second antibodies can be identified as binding to the same epitope, even if the specific sequence or structure of the epitope has not yet been determined. Additionally, when the first and second antibodies bind to the same epitope and the first antibody further exhibits specific effects such as antitumor activity, the second antibody can be expected to have the same activity as the first antibody.

[0255] In this specification, the term "CDR" is used to refer to the complementarity-determining region (CDR). It is known that both the heavy and light chains of an antibody molecule possess three CDRs. These CDRs are also known as hypervariable regions and are present in the variable regions of both the heavy and light chains of the antibody, where primary structure mutations are particularly high. On the primary structure of the polypeptide chain in both the heavy and light chains, the CDR is divided into three sites. In this specification, regarding the antibody's CDRs, the heavy chain CDRs are designated CDRH1, CDRH2, and CDRH3, starting from the N-terminus of the heavy chain's amino acid sequence, while the light chain CDRs are designated CDRL1, CDRL2, and CDRL3, starting from the N-terminus of the light chain's amino acid sequence. These sites are located close to each other in the three-dimensional structure and determine the antibody's specificity for the antigen to which it binds.

[0256] In this specification, the term "several / types" is used to refer to a number from 2 to 10. The number is preferably 2 to 9, more preferably 2 to 8, even more preferably 2 to 7, further preferably 2 to 6, even more preferably 2 to 5, even more preferably 2 to 4, even more preferably 2 to 3, and even more preferably 2.

[0257] In this specification, the term "antibody-drug conjugate composition" is used to mean a composition comprising, in any given ratio, an antibody-drug conjugate bound by two drug linkers, an antibody-drug conjugate bound by four drug linkers, an antibody-drug conjugate bound by six drug linkers, an antibody-drug conjugate bound by eight drug linkers, and an antibody not bound by any drug linkers. In this specification, "antibody-drug conjugate composition" is also referred to as "ADC composition".

[0258] In this specification, the "average number of bound drugs" is also referred to as the drug:antibody ratio (DAR), and the average number of bound drugs means the average number of drugs that bind a single antibody molecule in the antibody-drug conjugate composition.

[0259] In this specification, the term "content" is used to refer to the content (based on the mol% of antibody) of an antibody-drug conjugate composition having a specific number of binding drugs and specific binding sites.

[0260] In this specification, the term "identity" is used to have the same meaning as the term "homology".

[0261] 1. Antibody

[0262] The antibodies used in this invention can be generated against target antigens, such as tumor-specific antigens (TAAs) or tumor-associated antigens (TSAs). These antibodies possess the ability to recognize tumor cells, bind to such tumor cells, and be incorporated into and internalized within them.

[0263] There are no particular restrictions on the type of target antigen, as long as it is a tumor cell-associated antigen. Examples of target antigens include B7-H3, CD3, CD30, CD33, CD37, CD56, CD98, DR5, EGFR, EPHA2, FGFR2, FGFR4, FOLR1 (folate receptor 1), HER2, HER3, TROP2, and VEGF.

[0264] The antibodies used in this invention can be obtained by methods described, for example, in WO2009 / 091048, WO2011 / 027808, or WO2012 / 133572. Specifically, a non-human animal is immunized with a target antigen, and then lymph, lymphoid tissue, blood cell samples, or bone marrow-derived cells are collected from the immunized animal. Subsequently, plasma cells and / or plasmablasts of the non-human animal that specifically bind to the target antigen are selected. Antibody genes responding to the target antigen are collected from the obtained plasma cells and / or plasmablasts, and then the nucleotide sequence of the antibody genes is identified. Subsequently, the aforementioned antibody or antibody fragment thereof can be obtained based on the identified gene nucleotide sequence. The antibodies thus obtained are examined for their binding activity with the target antigen, so that antibodies suitable for human diseases can be selected.

[0265] Alternatively, according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp.495-497; Kennet, R. ed., Monoclonal Antibodies, pp.365-367, Plenum Press, NY (1980)), antibody-producing cells that generate antibodies against a target antigen are fused with myeloma cells to create a hybridoma in order to obtain monoclonal antibodies. Specific examples of such methods are described in WO2009 / 048072 (published April 16, 2009) and WO2010 / 117011 (published October 14, 2010).

[0266] The antibodies used in this invention include recombinant antibodies that are artificially modified for the purpose of reducing heteroantigenicity against humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced according to known methods.

[0267] Chimeric antibodies are, for example, antibodies whose variable and constant regions are heterologous to each other, such as chimeric antibodies formed by conjugating the variable region of a mouse or rat-derived antibody with a constant region derived from a human (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0268] Examples of humanized antibodies include antibodies formed by incorporating only the CDR into a human-derived antibody (see Nature (1986) 321, pp.522-525), and antibodies formed by grafting some amino acid residues in the framework and the CDR into a human antibody according to the CDR grafting method (International Publication No. WO90 / 07861).

[0269] Preferred examples of antibodies of the present invention include anti-TROP2 antibody, anti-CD98 antibody, anti-B7-H3 antibody, and anti-HER2 antibody.

[0270] A real-world example of a humanized anti-TROP2 antibody may be any given combination of the following: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of any of the following: (1) an amino acid sequence consisting of amino acid residues at positions 20 to 140 of SEQ ID NO:2, (2) an amino acid sequence having at least 95% homology to the amino acid sequence in (1) above, and (3) an amino acid sequence comprising the deletion, substitution, or addition of one or more amino acids of the amino acid sequence in (1) above; and the light chain comprises a light chain variable region consisting of any of the following: (4) an amino acid sequence consisting of amino acid residues at positions 21 to 129 of SEQ ID NO:4, (5) an amino acid sequence having at least 95% homology to the amino acid sequence in (4) above, and (6) an amino acid sequence comprising the deletion, substitution, or addition of one or more amino acids of the amino acid sequence in (4) above.

[0271] An example of the antibody described above, which comprises a preferred combination of a heavy chain and a light chain, may be antibody (hTINA1-H1L1), which consists of a heavy chain and a light chain, wherein the heavy chain consists of an amino acid sequence consisting of amino acid residues at positions 20 to 470 of SEQ ID NO:2, and the light chain consists of an amino acid sequence consisting of amino acid residues at positions 21 to 234 of SEQ ID NO:4.

[0272] Humanized anti-TROP2 antibodies are not limited to specific humanized antibodies, as long as they retain the amino acid sequence shown in SEQ ID NO:5 (TAGMQ) for CDRH1, the amino acid sequence shown in SEQ ID NO:6 (WINTHSGVPKYAEDFKG) for CDRH2, the amino acid sequence shown in SEQ ID NO:7 (SGFGSSYWYFDV) for CDRH3, the amino acid sequence shown in SEQ ID NO:8 (KASQDVSTAVA) for CDRL1, the amino acid sequence shown in SEQ ID NO:9 (SASYRYT) for CDRL2, and the amino acid sequence shown in SEQ ID NO:10 (QQHYITPLT) for CDRL3, as shown in the sequence listing.

[0273] Examples of humanized anti-CD98 antibodies may be any given combination of the following: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of any of the following: (1) an amino acid sequence consisting of amino acid residues at positions 20 to 135 of SEQ ID NO:12, (2) an amino acid sequence having at least 95% identity with the amino acid sequence in (1) above, and (3) an amino acid sequence comprising the deletion, substitution, or addition of one or more amino acids of the amino acid sequence in (1) above; and the light chain comprises a light chain variable region consisting of any of the following: (4) an amino acid sequence consisting of amino acid residues at positions 21 to 135 of SEQ ID NO:14, (5) an amino acid sequence having at least 95% identity with the amino acid sequence in (4) above, and (6) an amino acid sequence comprising the deletion, substitution, or addition of one or more amino acids of the amino acid sequence in (4) above.

[0274] An example of the antibody described above, which comprises a preferred combination of a heavy chain and a light chain, may be antibody (hM23-H1L1), which consists of a heavy chain and a light chain, wherein the heavy chain consists of an amino acid sequence consisting of amino acid residues at positions 20 to 465 of SEQ ID NO:12, and the light chain consists of an amino acid sequence consisting of amino acid residues at positions 21 to 240 of SEQ ID NO:14.

[0275] Humanized anti-CD98 antibodies are not limited to specific humanized antibodies, as long as they retain the amino acid sequence shown in SEQ ID NO:15 (NYLIE) for CDRH1, the amino acid sequence shown in SEQ ID NO:16 (VINPGSGVTNYNEKFKG) for CDRH2, the amino acid sequence shown in SEQ ID NO:17 (AEAWFAY) for CDRH3, the amino acid sequence shown in SEQ ID NO:18 (KSSQSLLYSSNQKNYLA) for CDRL1, the amino acid sequence shown in SEQ ID NO:19 (WASTRES) for CDRL2, and the amino acid sequence shown in SEQ ID NO:20 (QRYYGYPWT) for CDRL3, as shown in the sequence listing.

[0276] Examples of humanized anti-B7-H3 antibodies may be any given combination of the following: a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region consisting of any of the following: (1) an amino acid sequence consisting of amino acid residues at positions 20 to 141 of SEQ ID NO:25, (2) an amino acid sequence having at least 95% identity with the amino acid sequence in (1) above, and (3) an amino acid sequence comprising the deletion, substitution, or addition of one or more amino acids of the amino acid sequence in (1) above; and wherein the light chain comprises a light chain variable region consisting of any of the following: (4) an amino acid sequence consisting of amino acid residues at positions 21 to 128 of SEQ ID NO:26, (5) an amino acid sequence having at least 95% identity with the amino acid sequence in (4) above, and (6) an amino acid sequence comprising the deletion, substitution, or addition of one or more amino acids of the amino acid sequence in (4) above.

[0277] An example of the antibody described above, which comprises a preferred combination of a heavy chain and a light chain, may be antibody (M30-H1-L4), which consists of a heavy chain and a light chain, wherein the heavy chain consists of an amino acid sequence consisting of amino acid residues at positions 20 to 471 of SEQ ID NO:25, and the light chain consists of an amino acid sequence consisting of amino acid residues at positions 21 to 233 of SEQ ID NO:26.

[0278] Humanized anti-B7-H3 antibodies are not limited to specific humanized antibodies, as long as they retain the amino acid sequence shown in SEQ ID NO:27 (NYVMH) for CDRH1, the amino acid sequence shown in SEQ ID NO:28 (YINPYNDDVKYNEKFKG) for CDRH2, the amino acid sequence shown in SEQ ID NO:29 (WGYYGSPLYYFDY) for CDRH3, the amino acid sequence shown in SEQ ID NO:30 (RASSRLIYMH) for CDRL1, the amino acid sequence shown in SEQ ID NO:31 (ATSNLAS) for CDRL2, and the amino acid sequence shown in SEQ ID NO:32 (QQWNSNPPT) for CDRL3, as shown in the sequence listing.

[0279] An example of a humanized anti-HER2 antibody is an antibody (trastuzumab; US Patent No. 5821337) that consists of a heavy chain and a light chain, wherein the heavy chain consists of an amino acid sequence consisting of amino acid residues at positions 1 to 449 of SEQ ID NO:33, and the light chain consists of an amino acid sequence consisting of amino acid residues at positions 1 to 214 of SEQ ID NO:34.

[0280] Furthermore, the antibodies used in this invention also include CDR-modified humanized antibodies (where 1-3 amino acid residues in each CDR are replaced by other amino acid residues), as long as the CDR-modified humanized antibodies have binding activity with tumor cells.

[0281] The antibodies used in this invention also include human antibodies. These human antibodies can be obtained by generating mice using human antibodies, the mice of which possess human chromosomal segments containing the heavy and light chain genes of the human antibody (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (edited by Kitagawa, Y., Matsuda, T. and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727; etc.).

[0282] Moreover, there are known methods for obtaining human antibodies derived from phage displays selected from human antibody libraries (see Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43(7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1(2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109(3), pp. 427-431; etc.).

[0283] For example, a phage display method can be used, which involves expressing the variable region of a human antibody as a single-chain antibody (scFv) on the surface of a phage, and then selecting phages that bind to the antigen (Nature Biotechnology (2005), 23,(9), pp.1105-1116). By analyzing the genes of phages selected based on their antigen-binding activity, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of the scFv that binds to the antigen is elucidated, it will become possible to obtain human antibodies by generating an expression vector with that DNA sequence and then introducing the expression vector into a suitable host (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0284] The binding activity of the antibody to the target antigen is evaluated to select the preferred antibody. The dissociation constant between the antibody and the antigen can be measured using the Biacore T200 (GE Healthcare Bioscience), which utilizes surface plasmon resonance (SPR) as the detection principle. For example, an antibody adjusted to have an appropriate concentration relative to the antigen immobilized as a ligand is reacted with the analyte, and then their binding and dissociation are measured to obtain the association rate constant ka1, the dissociation rate constant kd1, and the dissociation constant (KD; KD = kd1 / ka1). Binding activity with the target antigen can be evaluated not only using the Biacore T200, but also using devices utilizing surface plasmon resonance (SPR) as the detection principle, the KinExA (Sapidyne Instruments) utilizing size exclusion assays as the detection principle, the BLItz System (Pall) utilizing biolayer interferometry as the detection principle, and ELISA (enzyme-linked immunosorbent assay) methods.

[0285] The activity of internalization in cells can be confirmed by the following methods: (1) a fluorescence microscopy assay using a secondary antibody (fluorescently labeled) bound to a therapeutic antibody to visualize the antibody incorporated into the cell (Cell Death and Differentiation (2008) 15, 751-761); (2) a fluorescence assay using a secondary antibody (fluorescently labeled) bound to a therapeutic antibody to measure the amount of fluorescence incorporated into the cell; or (3) the Mab-ZAP assay, in which an immunotoxin bound to a therapeutic antibody is used, and when the immunotoxin is incorporated into the cell, the toxin is released and cell growth is inhibited (BioTechniques 28:162-165, January 2000). As this immunotoxin, a recombinant protein consisting of the catalytic region of diphtheria toxin and protein G can also be used.

[0286] Another example of an indicator used to compare antibody properties is antibody stability. Differential scanning calorimetry (DSC) is a method that can rapidly and accurately measure the thermal denaturation midpoint (Tm), which serves as a good indicator of the relative structural stability of a protein. By measuring such Tm values ​​using DSC and then comparing the obtained values, differences in thermal stability can be compared. It is known that the storage stability of antibodies shows some degree of correlation with the thermal stability of antibodies (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and using thermal stability as an indicator, preferred antibodies can be selected. Examples of other indicators used for antibody selection include high yield in suitable host cells and low adhesion in aqueous solutions. For example, since the antibody with the highest yield does not always exhibit the highest thermal stability, a comprehensive judgment based on the above indicators is necessary to select the antibody most suitable for human administration.

[0287] Furthermore, antibody-dependent cytotoxicity can be enhanced by regulating the glycan modification that binds to antibodies. Techniques for regulating antibody glycan modification, as described in WO99 / 54342, WO2000 / 61739, WO2002 / 31140, etc., are known, but this technique is not limited to these.

[0288] When an antibody gene has been isolated and subsequently introduced into a suitable host to produce antibodies, a suitable combination of host and expression vector can be used. A specific example of an antibody gene may be a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence of the antibody described herein. For transformation of host cells, the heavy chain sequence gene and the light chain sequence gene can be inserted into a single expression vector, or these genes can be inserted into separate expression vectors. When eukaryotic cells are used as the host, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells such as COS cells (Gluzman, Y., Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), and a dihydrofolate reductase-deficient cell line from Chinese hamster ovaries (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220). On the other hand, when prokaryotic cells are used as the host, for example, *Escherichia coli* or *Bacillus subtilis* can be used. The target antibody gene is introduced into these cells for transformation, and the transformed cells are then cultured in vitro to obtain antibodies. In the above culture methods, the yield varies depending on the antibody sequence, and therefore the yield can be used as an indicator to select antibodies that are easily produced as drugs from those with equivalent binding activity.

[0289] The antibody isotypes used in this invention are not limited, and examples of antibody isotypes include IgG (IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE. IgG or IgM is preferred, and IgG1, IgG2, or IgG3 is more preferred.

[0290] Examples of common antibody functions include antigen-binding activity, activity to neutralize antigen activity, activity to enhance antigen activity, antibody-dependent cytotoxicity, complement-dependent cytotoxicity, complement-dependent cellular cytotoxicity, and internalization activity.

[0291] Furthermore, the antibodies used in this invention can be multispecific antibodies that are specific to at least two different types of antigens. Typically, this molecule binds to two types of antigens (i.e., bispecific antibodies). However, the "multispecific antibody" in this invention includes antibodies that are specific to a wider range of antigens (e.g., three antigens).

[0292] The antibodies used in this invention can be antibodies with 80% to 99% identity (or homology) with the heavy chain and / or light chain of the aforementioned antibodies. By combining sequences with high homology to the aforementioned heavy chain and light chain amino acid sequences, antibodies having the same antigen-binding and internalizing activities as those of the aforementioned antibodies can be selected. This homology is typically 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining amino acid sequences comprising substitutions, deletions, and / or additions of one or more amino acid residues relative to the heavy chain and / or light chain amino acid sequences, antibodies having various types of activities equivalent to those of the aforementioned antibodies can be selected. The number of amino acid residues to be replaced, deleted, and / or added is typically 10 or fewer amino acid residues, preferably 9 or fewer amino acid residues, more preferably 8 or fewer amino acid residues, more preferably 7 or fewer amino acid residues, even more preferably 6 or fewer amino acid residues, further preferably 5 or fewer amino acid residues, even more preferably 4 or fewer amino acid residues, even more preferably 3 or fewer amino acid residues, even more preferably 2 or fewer amino acid residues, and most preferably 1 amino acid residue.

[0293] It is known that antibodies produced in cultured mammalian cells have a lysine residue deletion at the carboxyl terminus of the heavy chain (Journal of Chromatography A, 705:129-134 (1995)). Furthermore, it is known that two amino acid residues, glycine and lysine, are deleted at the carboxyl terminus of the heavy chain, and that the proline residue at the carboxyl terminus is neoamiditized (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding activity and effector functions of the antibody (complement activation, antibody-dependent cytotoxicity, etc.). Therefore, the present invention also includes antibodies that have undergone the above modifications, and specific examples of such antibodies include deletion mutants containing one or two amino acid deletions at the carboxyl terminus of the heavy chain, and deletion mutants formed by amidation of the above deletion mutants (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, deletion mutants of the heavy chains of the antibodies according to the invention are not limited to the aforementioned deletion mutants, as long as they retain antigen-binding activity and effector function. The two heavy chains constituting the antibodies according to the invention can be any type of heavy chain selected from full-length antibodies and the aforementioned deletion mutants, or a combination of any two types selected from the above group. The ratio of individual deletion mutants can be influenced by the type of mammalian cells cultured to produce the antibodies according to the invention and the culture conditions. The principal component of the antibodies according to the invention can be a case in which one amino acid residue is missing from the carboxyl terminus of each of the two heavy chains.

[0294] Homology between two types of amino acid sequences can be determined using the default parameters of the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," NucleicAcids Res. 25: 3389-3402). The Blast algorithm is also available at www.ncbi.nlm.nih.gov / blast on the Internet. It should be noted that using the aforementioned Blast algorithm, two types of percentage values ​​can be calculated: Identity (or Identities) and Positivity (or Positivities). The former is the value obtained when the amino acid residues between the two types of amino acid sequences for which homology should be obtained are identical. The latter is a value that also considers amino acid residues with similar chemical structures. In this specification, the identity value when amino acid residues are identical to each other is defined as the homology value.

[0295] The obtained antibody can be purified until it becomes homogeneous. For the separation and purification of antibodies, common separation and purification methods applied to proteins can be used. Antibodies can be separated and purified by, for example, column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing, and then by combining the selected methods (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al., eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but the separation and purification methods are not limited to these.

[0296] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography.

[0297] These chromatographic methods can be performed using liquid chromatography such as HPLC or FPLC.

[0298] Examples of columns used in affinity chromatography include protein A columns and protein G columns.

[0299] 2. Medications

[0300] The drug used in this invention is not particularly limited, as long as it is a compound with antitumor activity and also has substituents or a portion of the structure capable of binding to the linker structure. With this drug, the linker is partially or entirely cleaved in tumor cells, releasing the antitumor compound portion, thereby exhibiting antitumor activity. If the linker is cleaved at the portion that binds to the drug, the antitumor compound is released while retaining its original structure, thereby exhibiting the original antitumor activity. The drug is allowed to bind to the antibody via the linker portion having a specific structure. In this specification, the drug linker comprising the drug and the linker portion is also referred to as a "drug".

[0301] Examples of antitumor compounds include calichimycin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin or its derivatives, alistatin or its derivatives, maytansine or its derivatives, paclitaxel or its derivatives, and camptothecin or its derivatives. Among these compounds, eczema or monomethylalistatin E are preferred.

[0302] Ecinotecan ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13(9H,15H)-dione), a camptothecin derivative, is a compound represented by the following formula.

[0303] [Formula 9]

[0304]

[0305] Monomethyl arithmetic amine E ((2R,3R)-N-[(1R,2S)-1-methyl-2-hydroxy-2-phenylethyl]-2-methyl-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-4-[(N-methyl-L-Val-L-Val-)(methyl)amino]-5-methylheptanoyl]-2-pyrrolidinyl]-3-methoxypropionamide) is a compound represented by the following formula.

[0306] [Formula 10]

[0307]

[0308] 3. Connector

[0309] The drug used in this invention can bind to an antibody via a linker. The linker used in this invention preferably has an N-substituted maleimide group. Examples of linkers include cleavable and non-cleavable linkers. Examples of cleavable linkers are peptide linkers that are cleaved by intracellular proteases, such as lysosomal proteases or endosomal proteases.

[0310] The connector used in this invention preferably has a structure induced by any of the following formulas:

[0311] [Equation 11]

[0312]

[0313] [Equation 12]

[0314]

[0315] ,and

[0316] [Equation 13]

[0317]

[0318] And preferably, it has a structure induced by the following formula:

[0319] [Formula 14]

[0320]

[0321] The term "GGFG" refers to a tetrapeptide residue consisting of glycine-glycine-phenylalanine-glycine.

[0322] The connector used in this invention can be prepared, for example, according to the method described in Example 58 of WO2014 / 057687.

[0323] 4. Drug connector intermediates

[0324] The drug linker intermediate used in this invention can be generated by reacting the carboxyl group of the linker compound with the amino group of the antitumor compound using a condensing agent or the like.

[0325] The drug linker intermediate used in the method of the present invention is not particularly limited, as long as it is a compound capable of reacting with the interchain thiol of the antibody. The drug linker intermediate is preferably a compound having an N-substituted maleimide group, and more preferably...

[0326] [Formula 15]

[0327]

[0328] [Formula 16]

[0329]

[0330] ,or

[0331] [Equation 17]

[0332]

[0333] And its further preferred form

[0334] [Formula 18]

[0335]

[0336] The drug linker intermediate used in this invention can be prepared, for example, according to the methods described in Examples 58, 43 and 14 of WO2014 / 057687.

[0337] If the drug linker intermediate is an N-substituted maleimide group, it can react with interchain thiols generated by the reduction of the interchain disulfide of the antibody, thereby allowing the antibody to bind to the drug via the linker. Therefore, the drug linker intermediate is preferably a compound with an N-substituted maleimide group. However, the drug linker intermediate used herein is not limited to this compound, and all types of drug linker intermediates can be used in the production method of the present invention, provided they have functional groups for promoting the reaction with the interchain thiols of the antibody.

[0338] 5. Antibody-drug conjugates

[0339] Regarding antibody-drug conjugates, the number of drugs binding to a single antibody molecule is an important factor affecting efficacy and safety. Because antibodies have four interchain disulfides, and these disulfides are composed of two thiol groups, the number of drugs binding to a single antibody molecule can be 2, 4, 6, or 8.

[0340] Examples of antibody-drug conjugates (hereinafter also referred to as "D2") in which two drug linkers bind to a single antibody molecule include an antibody-drug conjugate (hereinafter also referred to as "D2-1") in which two drug linkers bind to a heavy-light chain thiol and an antibody-drug conjugate (hereinafter also referred to as "D2-2") in which two drug linkers bind to a heavy-heavy chain thiol.

[0341] [Formula 19]

[0342]

[0343] Examples of antibody-drug conjugates (hereinafter also referred to as "D4") in which four drug linkers bind to a single antibody molecule include an antibody-drug conjugate in which four drug linkers bind to a heavy-light chain thiol (hereinafter also referred to as "D4-1"), an antibody-drug conjugate in which four drug linkers bind to a heavy-heavy chain thiol (hereinafter also referred to as "D4-2"), and an antibody-drug conjugate in which two drug linkers bind to a heavy-light chain thiol and two drug linkers bind to a heavy-heavy chain thiol (hereinafter also referred to as "D4-3").

[0344] [Formula 20]

[0345]

[0346] Examples of antibody-drug conjugates (hereinafter also referred to as "D6") in which six drug linkers bind to a single antibody molecule include an antibody-drug conjugate (hereinafter also referred to as "D6-1") in which four drug linkers bind to the heavy-light chain thiol and two drug linkers bind to the heavy-heavy chain thiol, and an antibody-drug conjugate (hereinafter also referred to as "D6-2") in which two drug linkers bind to the heavy-light chain thiol and four drug linkers bind to the heavy-heavy chain thiol.

[0347] [Equation 21]

[0348]

[0349] An example of an antibody-drug conjugate (hereinafter also referred to as "D8") in which four drug linkers bind to a heavy-light chain thiol and four drug linkers bind to a heavy-heavy chain thiol is an antibody-drug conjugate in which four drug linkers bind to a heavy-heavy chain thiol.

[0350] [Equation 22]

[0351]

[0352] The interchain thiol of the antibody, for example, forms a thioether at the 3-position of the N-substituted maleimide group in the drug linker intermediate and binds thereto. That is, the binding portion of the antibody to the drug linker is represented, for example, by the following formula:

[0353] [Equation 23]

[0354]

[0355] "Antibody-S-" is derived from antibody.

[0356] The preferred drug connector is,

[0357] [Equation 24]

[0358]

[0359] [Equation 25]

[0360]

[0361] ,or

[0362] [Equation 26]

[0363]

[0364] Where A represents the binding site with the antibody, and more preferably, is...

[0365] [Equation 27]

[0366]

[0367] Where A represents the binding site with the antibody.

[0368] Antibody-drug conjugates are produced by determining reaction conditions, such as the amounts of raw materials and / or reagents used in the reaction, allowing control over the number of drugs bound. Unlike the chemical reactions of low molecular weight compounds, antibody-drug conjugates are typically obtained as mixtures of varying numbers of drugs bound. The number of drugs bound to a single antibody molecule is specified and expressed as an average, i.e., the average number of drugs bound.

[0369] The production method of the present invention is a method for producing antibody-drug conjugate compositions, wherein the number of bound drugs and binding sites are controlled, and the production method comprises: a first step of selectively reducing the heavy-light chain disulfides of the antibody to convert them into thiol groups, and a second step of reacting a drug linker intermediate with an antibody having thiol groups to produce an antibody-drug conjugate composition (wherein the number of bound drugs and binding sites are controlled). Each step will then be described in detail.

[0370] (First step) Antibody reduction

[0371] Antibodies with thiol groups can be produced by reacting the antibody with a reducing agent in a buffer solution at a temperature of -10°C to 10°C.

[0372] The reaction temperature is preferably -5°C to 5°C, more preferably -3°C to 3°C, even more preferably 0°C to 2°C, and even more preferably 0°C to 1°C.

[0373] The amount of reducing agent is 1 to 4 molar equivalents, preferably 2 to 3 molar equivalents, depending on the amount of a single antibody molecule.

[0374] As a reducing agent, for example, tris(2-carboxyethyl)phosphine or a salt thereof, dithiothreitol, or 2-mercaptoethanol can be used. The reducing agent is preferably tris(2-carboxyethyl)phosphine or a salt thereof, and more preferably tris(2-carboxyethyl)phosphine hydrochloride.

[0375] As a buffer, histidine buffer, phosphate buffer, borate buffer, acetate buffer, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer, etc. can be used, and histidine buffer is preferred.

[0376] The buffer solution preferably contains a chelating agent. Examples of chelating agents that may be used herein include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid. EDTA is preferred. This buffer solution can be used at concentrations from 1 to 20 mM.

[0377] The reaction time is preferably 1 to 15 hours, more preferably 3 to 10 hours, and even more preferably 5 to 7 hours.

[0378] The pH used in this reaction is from pH 5 to 9, preferably from pH 6 to 8, and more preferably from pH 6.8 to 7.2.

[0379] (Second step) Conjugation of antibody to drug linker intermediate

[0380] The drug linker intermediate is reacted with the thiol-containing antibody obtained in the first step to produce an antibody-drug conjugate composition. The drug linker intermediate is used in amounts of 2 to 10 molar equivalents, preferably 4 to 6 molar equivalents, based on the amount of a single antibody molecule.

[0381] Specifically, a solution in which the drug linker intermediate has been dissolved is added to a buffer containing the thiol-containing antibody obtained in the first step, so that they can react with each other.

[0382] Examples of solvents that can be used herein to dissolve drug linker intermediates include organic solvents such as 50% aqueous acetone, 80% aqueous ethanol, 80% aqueous methanol, 80% aqueous isopropanol, 80% aqueous dimethyl sulfoxide, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP). Among these solvents, 50% aqueous acetone or 80% aqueous dimethyl sulfoxide is preferred.

[0383] An organic solvent solution containing dissolved drug linker intermediates is added at a rate of 1% to 20% v / v to a buffer solution containing antibodies with thiol groups, so that they can react with each other.

[0384] The reaction temperature is preferably -10°C to 10°C, more preferably -5°C to 5°C, and even more preferably 0°C to 2°C.

[0385] The preferred reaction time is 0.5 to 2 hours.

[0386] The conjugation reaction can be terminated by using a thiol-containing reagent to deactivate the reactivity of the unreacted drug linker intermediate.

[0387] As a thiol-containing reagent, cysteine ​​or N-acetyl-L-cysteine ​​can be used, for example. More specifically, the conjugation reaction can be terminated by adding N-acetyl-L-cysteine ​​in an amount of 1 to 2 molar equivalents to the drug linker intermediate used, and then allowing them to react at room temperature for 10 to 30 minutes.

[0388] After the conjugation reaction is complete, purification can be performed using commercially available ultrafiltration membranes. While adding acetate buffer, histidine buffer, phosphate buffer, etc., to the reaction product, the low molecular weight fraction can be removed using an ultrafiltration membrane. Suitable ultrafiltration membranes can be used. Ultrafiltration membranes with a molecular weight of 1 kDa to 100 kDa can be used, and those with a molecular weight of 30 kDa are preferred.

[0389] 6. Identification of antibody-drug conjugate compositions

[0390] The resulting antibody-drug conjugate composition was concentrated, buffer-exchanged, purified, and the antibody concentration and the average number of bound drugs were determined according to the following procedures, so that the antibody-drug conjugate composition could be identified.

[0391] (1) Concentration of aqueous solutions of antibodies or antibody-drug conjugates

[0392] The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (50,000 MWCO, Millipore Corporation) container and then centrifuged using an Allegra X-15R (Beckman Coulter, Inc.) for 5 to 20 minutes at 2000 G to 3800 G to concentrate the antibody or antibody-drug conjugate solution.

[0393] (2) Measurement of antibody concentration

[0394] The concentration of the antibody can be measured using a UV measurement device (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's method. During the measurement, the absorbance at 280 nm (1.3 mL mg) is used.-1 cm -1 Up to 1.8 mL mg -1 cm -1 The specific values ​​vary depending on the individual antibody.

[0395] (3) Antibody buffer exchange

[0396] According to the manufacturer's instructions, a NAP-25 column (catalog number 17-0852-02, GE Healthcare Japan Corporation) using a Sephadex G-25 carrier was equilibrated with phosphate buffer (10 mM, pH 6.0; hereinafter also referred to as "PBS6.0 / EDTA") containing sodium chloride (137 mM) and ethylenediaminetetraacetic acid (5 mM). Subsequently, 2.5 mL of antibody aqueous solution was applied to a single NAP-25 column, and the fraction eluted with 3.5 mL of PBS6.0 / EDTA was fractionated. This fraction was concentrated using the same method as described in (1) above, and the antibody concentration was then measured using the same method as described in (2) above. The antibody concentration could then be adjusted using PBS6.0 / EDTA.

[0397] (4) Purification of antibody-drug conjugate composition

[0398] The NAP-25 column was equilibrated with acetate buffer (10 mM, pH 5.5; hereinafter also referred to as “ABS”) containing sorbitol (5%). An aqueous solution of the antibody-drug conjugate reaction (2.5 mL) was added to the NAP-25 column, and then eluted with buffer at the manufacturer’s specified amount to fractionate the antibody fraction. This fraction was added back to the NAP-25 column, and the gel filtration purification operation involving elution with buffer was repeated a total of two or three times to obtain the antibody-drug conjugate composition in which unbound drug linker intermediates or low molecular weight compounds (tris(2-carboxyethyl)phosphonic acid hydrochloride, N-acetyl-L-cysteine, and dimethyl sulfoxide) had been removed.

[0399] (5) Separation method using hydrophobic column chromatography for antibody-drug conjugate compositions

[0400] (5-1) HPLC Measurement Method

[0401] HPLC analysis was performed under the following measurement conditions.

[0402] HPLC System: Shimadzu Science HPLC System

[0403] Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm)

[0404] Column: TSKgel Butyl-NPR (4.6 × 100 mm, 2.5 μm; TOSOH CORPORATION)

[0405] Column temperature: 30℃

[0406] Mobile phase A: 25 mM phosphate buffer (pH 7.0) aqueous solution containing 1.5 M ammonium sulfate.

[0407] Mobile phase B: A mixed solution containing 75% 25mM phosphate buffer (pH 7.0) and 25% isopropanol.

[0408] Gradient program: 20% - 60% (0 min - 20 min), 20% - 80% (20 min - 20.1 min), 80% - 80% (20.1 min - 23 min), 80% - 20% (23 min - 23.1min), 20% - 20% (23.1min - 40 min)

[0409] Injected sample volume: 2 μL.

[0410] (5-2) Data Analysis

[0411] Regarding the current data, due to the column characteristics, antibody-drug conjugates are eluted in order of increasing number of bound drugs based on differences in salt concentration. Therefore, the number of bonds can be assumed by measuring individual area values. The peaks, in elution order, are D0 (antibody not bound by any drug linker), D2, D4-1, D4-2, D6, and D8, and thus, the distribution can be determined.

[0412] The antibody-drug conjugate composition produced by the production method of the present invention contains 50% or more of antibody-drug conjugates with a number of 4 bound drugs.

[0413] The D4-1 content in the antibody-drug conjugate composition produced by the production method of the present invention is 50% or more, or in the range of 50% to 90%, 50% to 80%, 50% to 70%, or 50% to 60%.

[0414] The content of D4-2 in the antibody-drug conjugate composition produced by the production method of the present invention is preferably 5% or less, and more preferably 1% or less.

[0415] The content of D4-3 in the antibody-drug conjugate composition produced by the production method of the present invention is preferably 5% or less, and more preferably 1% or less.

[0416] (6) Measurement of antibody concentration and average number of bound drugs in antibody-drug conjugate compositions (UV method)

[0417] The concentration of the bound drug in the antibody-drug conjugate composition can be calculated as follows: measure the UV absorbance of the antibody-drug conjugate aqueous solution at two wavelengths (280 nm and 370 nm), and then perform the following calculation.

[0418] Since the total absorbance at a specific wavelength is equal to the sum of the absorbances of all absorbing chemicals in the system [additivity of absorbance], if we assume that the molar absorptivity of the antibody and drug does not change before and after the antibody-drug conjugate, the antibody concentration and drug concentration in the antibody-drug conjugate composition are represented by the following relationship expression.

[0419] A 280 = A D,280 + A A,280 = ε D,280 C D + ε A,280 C A Expression (1)

[0420] A 370 = A D,370 + A A,370 = ε D,370 C D + ε A,370 C A Expression (2)

[0421] In the above expression, A 280 A represents the absorbance of the antibody-drug conjugate aqueous solution at 280 nm. 370 A represents the absorbance of the antibody-drug conjugate aqueous solution at 370 nm. A,280 A represents the absorbance of the antibody at 280 nm. A,370 A represents the absorbance of the antibody at 370 nm. D,280 A represents the absorbance of the conjugate precursor at 280 nm. D,370 ε represents the absorbance of the conjugate precursor at 370 nm. A,280 ε represents the molar absorptivity of the antibody at 280 nm. A,370 ε represents the molar absorptivity of the antibody at 370 nm. D,280 ε represents the molar absorptivity of the conjugate precursor at 280 nm. D,370 C represents the molar absorptivity of the conjugate precursor at 370 nm. A Represents the antibody concentration in the antibody-drug conjugate composition, and CD This represents the drug concentration in the antibody-drug conjugate composition.

[0422] In this article, for ε A,280 ε A,370 ε D,280 and ε D,370 The value represented is a pre-prepared value (an estimated calculated value or a measurement obtained through UV measurement of the compound). For example, the value ε can be assumed from the amino acid sequence of the antibody according to a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A,280 Value ε A,370 It is usually zero. According to the Lambert-Beer law (absorbance = molar concentration × molar absorptivity × optical path length), the value ε can be obtained by measuring the absorbance of the solution (in which the conjugate precursor is dissolved at a specific concentration). D,280 and ε D,370 Measure the value A of antibody-drug conjugate aqueous solution. 280 and A 370 Then, the obtained value is substituted into formulas (1) and (2) to solve the simultaneous equations, thereby obtaining C. A and C D In addition, C D Divide by C A To obtain the average number of drugs bound to each antibody.

[0423] (7) Measurement of the average number of drugs bound to each single antibody molecule in an antibody-drug conjugate composition (RPC method)

[0424] Alternatively, the average number of drug molecules bound to each individual antibody molecule in the antibody-drug conjugate composition can be obtained by high-performance liquid chromatography (HPLC) using reversed-phase chromatography (RPC) as described below, instead of the UV method.

[0425] (7-1) Sample preparation for HPLC analysis (reduction of antibody-drug conjugates)

[0426] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes to obtain a sample in which the interchain disulfides of the antibody-drug conjugate had been cleaved, and the obtained sample was subsequently used for HPLC analysis.

[0427] (7-2) HPLC analysis

[0428] HPLC analysis was performed under the following measurement conditions.

[0429] HPLC System: Agilent 1290 HPLC System (Agilent Technologies)

[0430] Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm)

[0431] Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Agilent Technologies, P / NPL1912-1802)

[0432] Column temperature: 80℃

[0433] Mobile phase A: 0.04% trifluoroacetic acid (TFA) aqueous solution

[0434] Mobile phase B: Acetonitrile solution containing 0.04% TFA

[0435] Gradient program: 29% - 36% (0 min - 12.5 min), 36% - 42% (12.5 min - 15 min), 42% - 29% (15 min - 15.1 min), 29% - 29% (15.1 min - 25 min)

[0436] Injected sample volume: 15 μL.

[0437] (7-3) Data Analysis

[0438] (7-3-1) When comparing the light chain (L0) and heavy chain (H0) of an antibody that does not bind to any drug, the hydrophobicity increases proportionally to the number of drugs bound, and the retention time is prolonged in the cases of drug-bound light chains (one drug-bound light chain: L1) and drug-bound heavy chains (one drug-bound heavy chain: H1, two drug-bound heavy chains: H2, and three drug-bound heavy chains: H3). Therefore, elution occurs in the order of L0, L1, H0, H1, H2, and H3. As a result of comparing the retention times between L0 and H0, the detection peak can be assigned to any one of L0, L1, H0, H1, H2, and H3.

[0439] [Equation 28]

[0440]

[0441] (7-3-2) Since the drug linker absorbs UV, the peak area value is corrected according to the number of drug linkers bound, using the molar absorptivity of the light chain, heavy chain and drug linker, according to the following expression.

[0442] [Expression 1]

[0443] .

[0444] [Expression 2]

[0445] .

[0446] In this paper, the molar absorptivity (280 nm) of the light and heavy chains of each antibody can be assumed from the amino acid sequences of the light and heavy chains of each antibody using known calculation methods (Protein Science, 1995, vol. 4, 2411-2423). Furthermore, the molar absorptivity (280 nm) of the drug linker can be obtained from actual measurements of the molar absorptivity (280 nm) of compounds prepared by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine, followed by conversion of the N-substituted maleimide group to succinimide sulfide.

[0447] (7-3-3) Calculate the ratio (%) of the peak area of ​​each chain to the total peak area correction value according to the following expression.

[0448] [Expression 3]

[0449]

[0450] A Li A Hi Each peak area correction value: L i H i

[0451] If L1 and H1 have been preferentially produced, it can be assumed that the heavy-light interchain disulfide has been selectively reduced. On the other hand, if L0 and H2 have been preferentially produced, it can be assumed that the heavy-heavy interchain disulfide has been selectively reduced.

[0452] (7-3-4) Calculate the average number of bound drugs in the antibody-drug conjugate composition according to the following expression.

[0453] The average number of bound drugs = (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100 × 2

[0454] The average number of bound drugs in the antibody-drug conjugate composition produced by the production method of the present invention is preferably 3.5 to 4.5, and more preferably 4.0 to 4.1.

[0455] 7. Pharmaceutical formulations comprising antibody-drug conjugate compositions.

[0456] After the antibody-drug conjugate composition obtained by the present invention has been transferred into tumor cells, its linker portion is cleaved and the drug is released into the tumor cells.

[0457] In the case of antibody-drug conjugates represented by the following formula:

[0458] [Equation 29]

[0459]

[0460] The compound represented by the following formula is released:

[0461] [Formula 30]

[0462] .

[0463] Because this compound has an unstable acetal-amine structure, it can be further hydrolyzed to produce a compound represented by the following formula:

[0464] [Equation 31]

[0465]

[0466] In the case of antibody-drug conjugates represented by the following formula:

[0467] [Equation 32]

[0468]

[0469] The compound represented by the following formula is released:

[0470] [Equation 33]

[0471] .

[0472] In the case of antibody-drug conjugates represented by the following formula:

[0473] [Formula 34]

[0474]

[0475] The compound represented by the following formula is released:

[0476] [Formula 35]

[0477] .

[0478] Since the antibody-drug conjugate compositions obtained by the present invention exhibit cytotoxicity against cancer cells, they can be used as active ingredients in pharmaceutical compositions for the treatment and / or prevention of cancer.

[0479] In other words, the antibody-drug conjugate composition obtained by this invention can be selected and used as a chemotherapeutic agent as a primary treatment in cancer therapy. Due to its use, cancer cell growth can be slowed, proliferation can be inhibited, and cancer cells can be further destroyed. By doing so, cancer patients can experience relief from cancer-related symptoms or improved quality of life (QOL), and their lives can be prolonged, thus achieving therapeutic effects. Even in cases where cancer cell destruction cannot be achieved, higher QOL can be achieved by inhibiting or controlling cancer cell growth, leading to longer survival for the patient.

[0480] The antibody-drug conjugate compositions obtained by this invention can be used not only as standalone drugs in such drug therapies, but also as agents in combination with other therapies in adjuvant therapies. These antibody-drug conjugate compositions can be combined with surgery, radiotherapy, hormone therapy, etc. Furthermore, these antibody-drug conjugate compositions can also be used as agents in neoadjuvant therapies.

[0481] In addition to the aforementioned therapeutic uses, the antibody-drug conjugate compositions obtained by this invention are expected to have the effect of inhibiting the growth of very small metastatic cancer cells and further destroying such metastatic cancer cells. For example, the antibody-drug conjugate compositions are expected to have the effect of inhibiting and destroying cancer cells in body fluids during metastasis, or inhibiting and destroying very small cancer cells that have just attached to any tissue. Therefore, the antibody-drug conjugate compositions are expected to have the effect of inhibiting and preventing cancer metastasis, especially after cancer has been removed by surgical procedures.

[0482] The antibody-drug conjugate compositions obtained by this invention can not only be administered to patients as a systemic therapy, but are also expected to be applied locally to cancerous tissues and exhibit therapeutic effects.

[0483] Examples of cancer types include, but are not limited to, lung cancer, kidney cancer, urothelial carcinoma, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, bile duct cancer, thyroid cancer, lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and multiple myeloma.

[0484] The antibody-drug conjugate compositions obtained by the present invention can be used as active ingredients in pharmaceutical compositions for treating autoimmune diseases or for inhibiting rejection reactions to transplantation.

[0485] When a pharmaceutical composition comprising an antibody-drug conjugate composition obtained by the present invention is administered to mammals (e.g., humans, horses, cattle, pigs, etc., preferably humans), it may be administered systemically or locally, and preferably by parenteral administration.

[0486] Examples of routes of administration for parenteral application include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Examples of methods of administration include injection and bolus injection, with injection being the preferred method.

[0487] The pharmaceutical compositions of the present invention can be prepared by selecting a suitable form according to the method of administration and then using commonly used methods for preparing various types of preparations. For example, the antibody-drug conjugate composition obtained by the present invention is mixed with solvents such as sterile liquids (including water and oils (derived from petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), saline, dextran aqueous solution, or glycerol aqueous solution, and additives such as humectants, emulsifiers, or pH buffers (described in "Remington's Pharmaceutical Sciences" by EW Martin et al.) to prepare the pharmaceutical compositions of the present invention.

[0488] The pharmaceutical compositions of the present invention may contain solubilizers, local anesthetics (e.g., lidocaine) for relieving pain at the injection site, etc. In one aspect, the pharmaceutical compositions of the present invention may be provided in which the active ingredient, solvent, etc., are each placed in separate containers. Furthermore, when the pharmaceutical compositions of the present invention are administered by injection, they may be administered, for example, in the form of an injection vial containing the active ingredient and sterile pharmaceutical-grade water or saline. When the pharmaceutical compositions of the present invention are administered by injection, the active ingredient may be mixed with sterile water for injection or saline prior to administration.

[0489] The pharmaceutical compositions of the present invention may comprise an antibody-drug conjugate composition and at least one cancer therapeutic agent other than the aforementioned antibody-drug conjugate composition. The antibody-drug conjugate compositions obtained by the present invention may also be administered together with other cancer therapeutic agents, thereby enhancing the anticancer effect. Other anticancer agents for this purpose may be administered simultaneously, separately, or sequentially to individual subjects with the antibody-drug conjugate composition. Alternatively, such other anticancer agents may be administered at different intervals than the antibody-drug conjugate compositions of the present invention. Examples of such cancer therapeutic agents include carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, agents described in International Publication No. WO2003 / 038043 and further LH-RH analogs (leuprorelin, goserelin, etc.), estradiol nitrogen mustard-phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), and aromatase inhibitors (anastrozole, letrozole, exemestane, etc.). However, there are no restrictions on the types of cancer treatment agents, as long as they are drugs with anti-tumor activity.

[0490] The pharmaceutical compositions of the present invention can also be provided in the form of lyophilized formulations or liquid formulations. When the pharmaceutical compositions of the present invention are provided as lyophilized formulations, they may be formulations containing suitable preparative additives used in the relevant technical field. Furthermore, when the pharmaceutical compositions of the present invention are provided as liquid formulations, they may be formulations containing suitable preparative additives used in the relevant technical field.

[0491] The composition and concentration of the active ingredient in the pharmaceutical compositions of the present invention vary depending on the method of administration. In the case of antibody-drug conjugate compositions included in the pharmaceutical compositions of the present invention, because the affinity of the antibody-drug conjugate for the antigen is increased, that is, the affinity is increased with respect to the dissociation constant (Kd value) of the antibody-drug conjugate for the antigen (i.e., because the Kd value is decreased), the antibody-drug conjugate composition can exhibit efficacy even when administered in small amounts. Therefore, in order to determine the application dose of the antibody-drug conjugate composition, the dose can be determined based on the conditions of the affinity of the antibody-drug conjugate for the antigen. When the antibody-drug conjugate composition produced by the production method of the present invention is administered to a human, for example, at a dose of about 0.001 to 100 mg / kg, either as a single dose or in several doses at intervals of 1 to 180 days.

[0492] The invention will be specifically described in the following examples. However, these examples are not intended to limit the scope of the invention. Furthermore, the following examples are not intended to be limiting in any way. In addition, unless otherwise stated, the reagents, solvents, and starting materials described in this specification are readily available from commercially available sources. Example

[0493] (Example 1) Construction of humanized anti-TROP2 antibody expression vector and antibody production

[0494] (i) Construction of a humanized anti-TROP2 antibody heavy chain (hTINA1-H1) expression vector

[0495] A DNA fragment containing the variable region encoding the humanized anti-TROP2 antibody heavy chain (hTINA1-H1) was synthesized (Artificial Gene Synthesis Service, GENEART). This DNA fragment is represented by nucleotide numbers 36 to 437 in the nucleotide sequence of the humanized anti-TROP2 antibody heavy chain (hTINA1-H1), as shown in SEQ ID NO:2. Using the synthesized DNA fragment as a template, the DNA fragment containing the variable region encoding the humanized anti-TROP2 antibody heavy chain (hTINA1-H1) was amplified using KOD-Plus- (TOYOBO) and the following primer set. Subsequently, the expression vector pCMA-G1 containing the chimeric and humanized antibody IgG1 type heavy chain was cleaved with the restriction enzyme BlpI, and the DNA fragment was then inserted into the cleavage site using the In-Fusion HDPCR Cloning Kit (CLONTECH) to construct the humanized anti-TROP2 antibody heavy chain (hTINA1-H1) expression vector. The obtained expression vector was named "pCMA-G1 / hTINA1-H1".

[0496] Primer set:

[0497] 5'-agctcccagatgggtgctgagc-3' (SEQ ID NO:21: Primer EG-Inf-F)

[0498] 5'-gggcccttggtggaggctgagc-3' (SEQ ID NO:22: Primer EG1-Inf-R)

[0499] (ii) Construction of humanized anti-TROP2 antibody light chain (hTINA1-L1) expression vector

[0500] A DNA fragment containing the variable region encoding the humanized anti-TROP2 antibody light chain (hTINA1-L1) was synthesized (Artificial Gene Synthesis Service, GENEART). This DNA fragment is represented by nucleotide numbers 38 to 402 in the nucleotide sequence of the humanized anti-TROP2 antibody light chain (hTINA1-L1), as shown in SEQ ID NO:4 in the sequence listing. Using the synthesized DNA fragment as a template, the DNA fragment containing the variable region encoding the humanized anti-TROP2 antibody light chain (hTINA1-L1) was amplified using KOD-Plus- (TOYOBO) and the following primer set. Subsequently, the expression vector pCMA-LK containing the chimeric and humanized antibody light chain was cleaved with the restriction enzyme BsiWI, and the DNA fragment was then inserted into the cleavage site using the In-FusionHD PCR Cloning Kit (CLONTECH) to construct the humanized anti-TROP2 antibody light chain (hTINA1-L1) expression vector. The obtained expression vector was named "pCMA-LK / hTINA1-L1".

[0501] Primer set:

[0502] 5'-ctgtggatctccggcgcgtacggc-3' (SEQ ID NO:23: Primer CM-LKF)

[0503] 5'-ggagggggcggccaccgtacg-3' (SEQ ID NO:24: Primer KCL-Inf-R)

[0504] (iii) Production of humanized anti-TROP2 antibody (hTINA1-H1L1)

[0505] FreeStyle 293F cells (Invitrogen) were subcultured and cultured according to the instruction manual. Specifically, 1.2 × 10⁶ cells in the logarithmic growth phase were subcultured and cultured. 9 One FreeStyle 293F cell (Invitrogen) was seeded in a 3L Fernbach Erlenmeyer flask (CORNING) and then diluted with FreeStyle293 expression medium (Invitrogen) to adjust to 1.0 × 10⁶ cells / year. 6Cells / mL. Cells were then cultured in an 8% CO2 incubator at 37°C with shaking at 90 rpm for 1 hour. Polyethyleneimine (Polyscience #24765; 3.6 mg) was then dissolved in Opti-Pro SFM (Invitrogen; 20 mL), followed by the addition of light chain expression vectors (0.8 mg) and heavy chain expression vectors (0.4 mg) prepared using the PureLink HiPure plasmid kit (Invitrogen) to Opti-Pro SFM (Invitrogen; 20 mL). The expression vector / Opti-Pro SFM mixture (20 mL) was added to the polyethyleneimine / Opti-Pro SFM mixture (20 mL), and the resulting mixture was gently stirred and then incubated for 5 minutes. FreeStyle 293F cells were then added to the reaction mixture. The resulting mixture was cultured in an 8% CO2 incubator at 37°C with shaking at 90 rpm for 7 days, and then the culture supernatant was filtered through a disposable capsule filter (ADVANTEC #CCS-045-E1H).

[0506] The humanized anti-TROP2 antibody obtained by combining pCMA-G1 / hTINA1-H1 and pCMA-LK / hTINA1-L1 was named "hTINA1-H1L1".

[0507] (iv) Purification of humanized anti-TROP2 antibody (hTINA1-H1L1)

[0508] The antibody was purified from the culture supernatant obtained in (iii) above through a two-step process: rProtein A affinity chromatography (4°C–6°C) and ceramic hydroxyapatite (room temperature). Following both rProtein A affinity chromatography and ceramic hydroxyapatite purification, a buffer exchange step was performed at 4°C–6°C. First, the culture supernatant was applied to a MabSelect SuRe (HiTrap column, manufactured by GE Healthcare Bioscience) that had been equilibrated with PBS. After all the culture supernatant had been placed in the column, the column was washed with PBS at a volume two or more times the column volume. Subsequently, elution was performed using 2M arginine hydrochloride solution (pH 4.0) to collect the fraction containing the antibody. The fraction was replaced with PBS using dialysis (Slide-A-Lyzer dialysis kit, Thermo Scientific), and then an antibody solution, diluted five-fold with a buffer consisting of 5 mM sodium phosphate and 50 mM MES (pH 7.0), was applied to a ceramic hydroxyapatite column (Bio-Scale CHTType-I hydroxyapatite column, JAPAN Bio-Rad Laboratories KK) equilibrated with a buffer consisting of 5 mM NaPi, 50 mM MES, and 30 mM NaCl (pH 7.0). A linear concentration gradient elution was then performed using sodium chloride, and the fraction containing the antibody was collected. The fraction was then replaced with HBsor (25 mM histidine / 5% sorbitol, pH 6.0) using dialysis (Slide-A-Lyzer dialysis kit, Thermo Scientific). Finally, the extract was concentrated using a centrifugal UF filtration device VIVASPIN20 (cutoff molecular weight: UF10K, Sartorius, 4°C), and the IgG concentration was adjusted to 20 mg / mL or higher to prepare a purified sample.

[0509] (v) Buffer exchange and concentration adjustment of humanized anti-TROP2 antibody (hTINA1-H1L1)

[0510] Following the manufacturer's instructions, equilibrate a NAP-25 column (catalog number 17-0852-02, GE Healthcare Japan Corporation) using the Sephadex G-25 carrier with phosphate buffer (10 mM, pH 6.0; hereinafter also referred to as "PBS6.0 / EDTA") containing sodium chloride (137 mM) and ethylenediaminetetraacetic acid (5 mM). Apply 2.5 mL of the antibody aqueous solution containing the humanized anti-TROP2 antibody (hTINA1-H1L1) generated in (iv) above to the aforementioned single NAP-25 column and collect the fraction diluted with 3.5 mL of PBS6.0 / EDTA. The fraction was aliquoted into Amicon Ultra (50,000 MWCO, Millipore Corporation) containers and then centrifuged using an Allegra X-15R centrifuge (2000 G to 3800 G for 5 to 20 minutes) to concentrate the antibody solution. The antibody concentration was measured using a UV measuring device (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. For measurement, an absorbance of 280 nm (1.54 mL mg) was used. -1 cm -1 The antibody concentration was measured, and then adjusted to 21.8 mg / mL using PBS6.0 / EDTA.

[0511] (Example 2) Generation of drug linker intermediates

[0512] N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl]glycyl-glycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycamide, represented by the following formula, was synthesized by the method described in Example 58 of WO2014 / 057687.

[0513] [Formula 36]

[0514]

[0515] (Example 3) Production of humanized anti-TROP2 antibody ADC composition

[0516] (Example 3-1) Humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition was generated according to conventional methods.

[0517] (i) Antibody reduction

[0518] Humanized anti-TROP2 antibody (hTINA1-H1L1) (15 mL: corresponding to 327 mg, concentration: 21.8 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and 18 mL of 25 mM histidine buffer (pH 5.0) was added. 0.5 M EDTA aqueous solution (0.027 mL; 6 equivalents based on antibody) was added to this reaction solution, followed by 0.1 g / mL polysorbate 20 aqueous solution (0.033 mL; 0.01% based on antibody). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.12. While stirring at 24°C, 1.00 mg / mL of tris(2-carboxyethyl)phosphonic acid hydrochloride aqueous solution (1.58 mL; 2.45 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then heated for 3 hours to achieve an internal temperature of 35°C to 36°C, which reduced the interchain disulfides of the antibody.

[0519] (ii) Conjugation of antibody to drug linker intermediate

[0520] The solution obtained in (i) above was cooled, and then a 50% aqueous solution of acetone (1.71 mL; 5.2 equivalents per single antibody molecule) of the compound obtained in Example 2 at an internal temperature of 16°C to 17°C was added to the reaction solution with stirring for 60 minutes. The resulting mixture was stirred for 20 minutes at the same temperature as above to allow the drug linker intermediate to bind to the antibody. Subsequently, a 50 mM aqueous solution of N-acetylcysteine ​​(0.135 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred for 20 minutes at the same temperature as above. The reaction of the drug linker intermediate was terminated, and then the pH of the reaction mixture was adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0521] (iii) Purification

[0522] Using Pellicon XL (Millipore Japan, 50 cm) 2The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 500 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 17.6 mL of a solution containing the humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition.

[0523] (iv) Evaluation of characteristics

[0524] [Common Operation A] Measurement of the average number of drugs bound to each individual antibody molecule in an antibody-drug conjugate composition.

[0525] The average number of drug molecules bound to each individual antibody molecule in the antibody-drug conjugate composition was obtained by high performance liquid chromatography (HPLC) analysis using the following methods.

[0526] 1. Sample preparation for HPLC analysis (reduction of antibody-drug conjugates)

[0527] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was mixed with a dithiothreitol (DTT) aqueous solution (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes to prepare a sample in which disulfides were cleaved between the heavy and light chains and between heavy chains of the antibody-drug conjugate. The obtained sample was used for HPLC analysis.

[0528] 2. HPLC analysis

[0529] HPLC analysis was performed under the following measurement conditions.

[0530] HPLC System: Shimadzu Science HPLC System

[0531] Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm)

[0532] Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Agilent Technologies)

[0533] Column temperature: 80℃

[0534] Mobile phase A: 0.05% trifluoroacetic acid (TFA) aqueous solution

[0535] Mobile phase B: Acetonitrile solution containing 0.04% TFA

[0536] Gradient program: 29% - 36% (0 min - 12.5 min), 36% - 42% (12.5-15 min), 42% -29% (15 min - 15.1 min), 29% - 29% (15.1 min - 25 min)

[0537] Injected sample volume: 15 μL.

[0538] 4. Data Analysis

[0539] When comparing the light chain (L0) and heavy chain (H0) of the antibody that does not bind to the drug, the hydrophobicity increases proportionally to the number of drug-bound light chains (one drug-bound light chain: L1) and drug-bound heavy chains (one drug-bound heavy chain: H1, two drug-bound heavy chains: H2, and three drug-bound heavy chains: H3), and the retention time is prolonged. Therefore, elution occurs in the order of L0, L1, H0, H1, H2, and H3. As a result of comparing the retention times between L0 and H0, the detection peak is assigned to any one of L0, L1, H0, H1, H2, and H3.

[0540] Since drug linkers absorb UV, the peak area value is corrected based on the number of drug linkers bound, using the molar absorptivity of the light chain, heavy chain, and drug linker, according to the following expression.

[0541] [Expression 4]

[0542] .

[0543] [Expression 5]

[0544] .

[0545] In this paper, the molar absorptivity (280 nm) of the light and heavy chains of each antibody is estimated from the amino acid sequences of the light and heavy chains using known calculation methods (Protein Science, 1995, vol. 4, 2411-2423). In the case of the humanized anti-TROP2 antibody (hTINA1-H1L1), based on its amino acid sequence, the value 27640 is used as the estimate of the molar absorptivity of the light chain, and the value 83810 is used as the estimate of the molar absorptivity of the heavy chain. Furthermore, the molar absorptivity (280 nm) of the drug linker is used based on actual measured molar absorptivity (280 nm) of compounds prepared by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine, followed by conversion of the N-substituted maleimide group to succinimide sulfide.

[0546] Calculate the ratio (%) of the peak area of ​​each chain to the total peak area correction value using the following expression.

[0547] [Expression 6]

[0548]

[0549] A Li A Hi Each peak area correction value: L i H i

[0550] The average number of drugs bound to each individual antibody molecule in an antibody-drug conjugate composition is calculated using the following expression.

[0551] The average number of bound drugs = (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100 × 2

[0552] The antibody concentration was found to be 16.49 mg / mL, the antibody yield was found to be 290 mg (86%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 4.4. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 5 middle.

[0553] [Common Operation B] Separation methods involving hydrophobic column chromatography for antibody-drug conjugate compositions.

[0554] 1. HPLC Measurement Method

[0555] HPLC analysis was performed under the following measurement conditions.

[0556] HPLC System: Shimadzu Science HPLC System

[0557] Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm)

[0558] Column: TSKgel Butyl-NPR (4.6 × 100 mm, 2.5 μm; TOSOH CORPORATION)

[0559] Column temperature: 30℃

[0560] Mobile phase A: 25 mM phosphate buffer (pH 7.0) aqueous solution containing 1.5 M ammonium sulfate.

[0561] Mobile phase B: A mixed solution containing 75% 25mM phosphate buffer (pH 7.0) and 25% isopropanol.

[0562] Gradient program: 20% - 60% (0 min - 20 min), 20% - 80% (20 min - 20.1 min), 80% - 80% (20.1 min - 23 min), 80% - 20% (23 min - 23.1min), 20% - 20% (23.1min - 40 min)

[0563] Injected sample volume: 2 μL.

[0564] 2. Data Analysis

[0565] Regarding the current data, due to column characteristics and differences in salt concentration, antibody-drug conjugates are eluted in order of increasing number of bound drugs. Therefore, the distribution of bond number is assumed by measuring individual area values. The peaks, in elution order, are D0 (antibody not bound by any drug linker), D2, D4-1, D4-2, D6, and D8, and the distribution is as follows: D0: 4.8%, D2: 16.8%, D4-1: 24.6%, D4-2: 13.1%, D6: 24.8%, and D8: 12.6%. Figure 6 ).

[0566] (Example 3-2) Production of humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition according to the method of the present invention

[0567] (i) Antibody reduction

[0568] Humanized anti-TROP2 antibody (hTINA1-H1L1) (22.9 mL: corresponding to 500 mg, concentration: 21.8 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (22 mL, pH 5.0) was further added. 0.5 M EDTA aqueous solution (0.0344 mL; 5 equivalents based on antibody) was added to this reaction solution, followed by 0.1 g / mL polysorbate 20 aqueous solution (0.050 mL; 0.01% based on antibody). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.12, and the mixture was then cooled. At an internal temperature of 0°C to 1°C, with stirring, 1.00 mg / mL of tris(2-carboxyethyl)phosphonic acid hydrochloride aqueous solution (2.54 mL; 2.58 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then stirred at an internal temperature of 0°C to 1°C for 6 hours to reduce the interchain disulfides of the antibody.

[0569] (ii) Conjugation of antibody to drug linker intermediate

[0570] Under stirring at an internal temperature of 0°C to 2°C, a 50% aqueous solution of acetone (2.99 mL; 5.1 equivalents per single antibody molecule) of the compound obtained in Example 2 at a concentration of 6.03 mg / mL was added to the solution obtained in (i) above for 10 minutes. The resulting mixture was stirred at the same temperature as above for 40 minutes to allow the drug linker intermediate to bind to the antibody. Subsequently, an aqueous solution of 50 mM N-acetylcysteine ​​(0.206 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was stirred further at the same temperature as above for 10 minutes. The reaction of the drug linker intermediate was terminated, and the pH of the reaction mixture was then adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0571] (iii) Purification

[0572] Using Pellicon XL (Millipore Japan, 50 cm) 2 The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 700 mL, thus removing low molecular weight substances. The remaining solution is then concentrated to obtain 23.6 mL of a solution containing the humanized anti-TROP2 antibody (hTINA1-H1L1) ADC composition.

[0573] (iv) Evaluation of characteristics

[0574] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-TROP2 antibody (hTINA1-H1L1), based on its amino acid sequence, the value 27640 was used as an estimate of the molar absorptivity of the light chain, and the value 83810 was used as an estimate of the molar absorptivity of the heavy chain.

[0575] The antibody concentration was found to be 19.63 mg / mL, the antibody yield was found to be 463 mg (92%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 4.1. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 7 middle.

[0576] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 1.9%, D2: 19.5%, D4-1: 53.3%, D6: 18.5% and D8: 5.5%. Figure 8 ).

[0577] (v) Results

[0578] The humanized anti-TROP2 antibody ADC composition produced by a common method (Example 3-1) has an average number of bound drugs of 4.4 and a D4-1 content of 24.6%. On the other hand, the humanized anti-TROP2 antibody ADC composition produced by the method of the present invention (Example 3-2) has an average number of bound drugs of 4.1 and a D4-1 content of 53.3%.

[0579] (Example 4) Construction of humanized anti-CD98 antibody expression vector and antibody production

[0580] (i) Construction of humanized anti-CD98 antibody heavy chain (hM23-H1) expression vector

[0581] A DNA fragment (nucleotide numbers 36 to 422) containing the DNA sequence encoding the variable region of the humanized anti-CD98 antibody heavy chain (hM23-H1) was synthesized (Artificial Gene Synthesis Service, GENEART), represented by nucleotide numbers 58 to 405 in the nucleotide sequence of the humanized anti-CD98 antibody heavy chain (hM23-H1) shown in SEQ ID NO:11. Using the synthesized DNA fragment as a template, the DNA fragment containing the DNA sequence encoding the variable region of hM23-H1 was amplified using KOD-Plus- (TOYOBO) and the following primer set. Subsequently, the expression vector pCMA-G1 containing the chimeric and humanized antibody IgG1 type heavy chain was cleaved with the restriction enzyme BlpI, and the DNA fragment was then inserted into the cleavage site using the In-Fusion HD PCR Cloning Kit (CLONTECH) to construct the humanized anti-CD98 antibody heavy chain (hM23-H1) expression vector. The obtained expression vector was named "pCMA-G1 / hM23-H1".

[0582] Primer set:

[0583] 5'-AGCTCCCAGATGGTGCTGAGC-3' (SEQ ID NO:21: Primer EG-Inf-F)

[0584] 5'-GGGCCCTTGGTGGAGGCTGAGC-3' (SEQ ID NO:22: Primer EG1-Inf-R)

[0585] (ii) Construction of humanized anti-CD98 antibody light chain (hM23-L1) expression vector

[0586] A DNA fragment (nucleotide numbers 38 to 420) containing the DNA sequence encoding the variable region of the humanized anti-CD98 antibody light chain (hM23-L1) was synthesized (Artificial Gene Synthesis Service, GENEART), the DNA fragment being represented by nucleotide numbers 61 to 405 in the nucleotide sequence of the humanized anti-CD98 antibody light chain (hM23-L1) shown in SEQ ID NO:13. Using the synthesized DNA fragment as a template, the DNA fragment containing the DNA sequence encoding the variable region of the humanized anti-CD98 antibody light chain (hM23-L1) was amplified using KOD-Plus- (TOYOBO) and the following primer set. Subsequently, the expression vector pCMA-LK containing the chimeric and humanized antibody light chain was cleaved with the restriction enzyme BsiWI, and then the DNA fragment was inserted into the cleavage site using the In-FusionHD PCR Cloning Kit (CLONTECH) to construct the humanized anti-CD98 antibody light chain (hM23-L1) expression vector. The obtained expression vector was named "pCMA-LK / hM23-L1".

[0587] Primer set:

[0588] 5'-CTGTGGATCTCCGGCGCGTACGGC-3' (SEQ ID NO:23: Primer CM-LKF)

[0589] 5'-GGAGGGGGCGGCCACCGTACG-3' (SEQ ID NO:24: Primer KCL-Inf-R)

[0590] (iii) Generation of humanized anti-CD98 antibody (hM23-H1L1)

[0591] The humanized anti-CD98 antibody was produced using the same method as applied in Example 1, (iii). The humanized anti-CD98 antibody obtained by combining pCMA-G1 / hM23-H1 and pCMA-LK / hM23-L1 was named "hM23-H1L1".

[0592] (iv) Purification of humanized anti-CD98 antibody (hM23-H1L1)

[0593] The antibody was purified from the culture supernatant obtained in (iii) above using the same method as that used in Example 1, (iv).

[0594] (v) Buffer exchange and concentration adjustment of humanized anti-CD98 antibody (hM23-H1L1)

[0595] The purified humanized anti-CD98 antibody (hM23-H1L1) from (iv) was subjected to buffer exchange and concentration adjustment using the same method as applied in Example 1, (v). During the operation, an absorbance of 280 nm (1.65 mL mg) was used. -1 cm -1 The antibody concentration was measured and then adjusted to 40 mg / mL using PBS6.0 / EDTA.

[0596] (Example 5) Production of humanized anti-CD98 antibody ADC composition

[0597] (Example 5-1) Humanized anti-CD98 antibody (hM23-H1L1) ADC composition was generated according to conventional methods.

[0598] (i) Antibody reduction

[0599] Humanized anti-CD98 antibody (hM23-H1L1) (12 mL: corresponding to 480 mg, concentration: 40 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (36 mL, pH 5.0) was further added. 0.5 M EDTA aqueous solution (CALBIOCHEM; 0.0394 mL; antibody-based, 6 equivalents) was added to this reaction solution, followed by 0.1 g / mL polysorbate 20 (NOF CORPORATION) aqueous solution (0.048 mL; antibody-based, 0.01%). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.10. While stirring at 21°C, 2.17 mL of 1.00 mg / mL tris(2-carboxyethyl)phosphonic acid hydrochloride (Nacalai Tesque, Inc.) aqueous solution (2.31 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then heated for 3 hours to achieve an internal temperature of 35°C to 36°C, which reduced the interchain disulfides of the antibody.

[0600] (ii) Conjugation of antibody to drug linker intermediate

[0601] The solution obtained in (i) above was cooled, and then a 50% aqueous solution of acetone (2.74 mL; 5.2 equivalents per single antibody molecule) of the compound obtained in Example 2 at an internal temperature of 17°C to 18°C ​​was added to the reaction solution for 7 minutes with stirring. The resulting mixture was stirred at the same temperature as above for 40 minutes to allow the drug linker intermediate to bind to the antibody. Subsequently, an aqueous solution of 50 mM acetylcysteine ​​(Kishida Chemical Co., Ltd.) (0.197 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred at the same temperature as above for 30 minutes. The reaction of the drug linker intermediate was terminated, and then the pH of the reaction mixture was adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0602] (iii) Purification

[0603] Using Pellicon XL (Millipore Japan, 50 cm) 2 The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 600 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 21.6 mL of a solution containing the humanized anti-CD98 antibody (hM23-H1L1) ADC composition.

[0604] (iv) Evaluation of characteristics

[0605] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-CD98 antibody (hM23-H1L1), based on its amino acid sequence, the value 41370 was used as an estimate of the molar absorptivity of the light chain, and the value 77810 was used as an estimate of the molar absorptivity of the heavy chain.

[0606] The antibody concentration was found to be 20.8 mg / mL, the antibody yield was found to be 449 mg (91%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 4.0. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 9 middle.

[0607] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 4.2%, D2: 24.2%, D4-1: 27.8%, D4-2: 13.3%, D6: 20.8%, and D8: 7.6%. Figure 10 ).

[0608] (Example 5-2) Production of a humanized anti-CD98 antibody (hM23-H1L1) ADC composition according to the method of the present invention

[0609] (i) Antibody reduction

[0610] Humanized anti-CD98 antibody (hM23-H1L1) (12.5 mL: corresponding to 500 mg, concentration: 40 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (27.5 mL, pH 5.0) was further added. 0.5 M EDTA aqueous solution (CALBIOCHEM; 0.041 mL; antibody-based, 6 equivalents) was added to this reaction solution, followed by 0.1 g / mL polysorbate 20 (NOF CORPORATION) aqueous solution (0.050 mL; antibody-based, 0.01%). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.10. The reaction solution was cooled, and 2.75 mL of 1.00 mg / mL tris(2-carboxyethyl)phosphonium hydrochloride (Nacalai Tesque, Inc.) aqueous solution (2.80 equivalents per single antibody molecule) was added to the reaction mixture under stirring at an internal temperature of 0°C to 1°C. The resulting mixture was then stirred for 6 hours to achieve an internal temperature of 0°C to 1°C, which allowed the interchain disulfides of the antibody to be reduced.

[0611] (ii) Conjugation of antibody to drug linker intermediate

[0612] Under stirring at an internal temperature of 0.7°C to 1.2°C, a 50% aqueous solution of acetone (3.14 mL; 5.4 equivalents per single antibody molecule) of the compound obtained in Example 2 at a concentration of 6.08 mg / mL was added to the solution obtained in (i) above for 10 minutes. The resulting mixture was stirred at the same temperature as above for 50 minutes to allow the drug linker intermediate to bind to the antibody. Subsequently, an aqueous solution of 50 mM N-acetylcysteine ​​(Kishida Chemical Co., Ltd.) (0.205 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred at the same temperature as above for 30 minutes. The reaction of the drug linker intermediate was terminated, and the pH of the reaction mixture was then adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0613] (iii) Purification

[0614] Using Pellicon XL (Millipore Japan, 50 cm) 2 The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 600 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 23.6 mL of a solution containing the humanized anti-CD98 antibody (hM23-H1L1) ADC composition.

[0615] (iv) Evaluation of characteristics

[0616] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-CD98 antibody (hM23-H1L1), based on its amino acid sequence, the value 41370 was used as an estimate of the molar absorptivity of the light chain, and the value 77810 was used as an estimate of the molar absorptivity of the heavy chain.

[0617] The antibody concentration was found to be 19.91 mg / mL, the antibody yield was found to be 470 mg (94%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 4.1. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 11 middle.

[0618] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 2.2%, D2: 18.1%, D4-1: 51.0%, D6: 20.6% and D8: 7.6%. Figure 12 ).

[0619] (v) Results

[0620] The humanized anti-CD98 antibody ADC composition produced by a common method (Example 5-1) has an average number of bound drugs of 4.0 and a D4-1 content of 27.8%. On the other hand, the humanized anti-CD98 antibody ADC composition produced by the method of the present invention (Example 5-2) has an average number of bound drugs of 4.1 and a D4-1 content of 51.0%.

[0621] (Example 6) Production of humanized anti-B7-H3 antibody ADC composition

[0622] (Example 6-1) Humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition was generated according to conventional methods.

[0623] (i) Antibody reduction

[0624] Humanized anti-B7-H3 antibody (M30-H1-L4) (produced according to the method described in Reference Example 1 of WO2014 / 057687, 12.4 mL: corresponding to 250 mg, concentration: 20.1 mg / mL; 25 mM citrate buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (18 mL, pH 7.5) was further added thereto. 0.5 M EDTA aqueous solution (CALBIOCHEM; 0.018 mL; 5 equivalents based on antibody) was added to this reaction solution, followed by 0.1 g / mL polysorbate 80 (NOF CORPORATION) aqueous solution (0.013 mL; 0.01% based on antibody). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.02. While stirring at 35°C, 1.00 mg / mL of tris(2-carboxyethyl)phosphonic acid hydrochloride (Nacalai Tesque, Inc.) aqueous solution (1.05 mL; 2.15 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then heated for 2 hours to achieve an internal temperature of 35°C to 36°C, which reduced the interchain disulfides of the antibody.

[0625] (ii) Conjugation of antibody to drug linker intermediate

[0626] The solution obtained in (i) above was cooled, and then a 50% aqueous solution of acetone (1.36 mL; 4.8 equivalents per single antibody molecule) of the compound obtained in Example 2 at an internal temperature of 15°C to 16°C was added to the reaction solution with stirring for 4 minutes. The resulting mixture was stirred for 20 minutes at the same temperature as above to allow the drug linker intermediate to bind to the antibody. Subsequently, an aqueous solution of 50 mM N-acetylcysteine ​​(Kishida Chemical Co., Ltd.) (0.102 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred for 20 minutes at the same temperature as above. The reaction of the drug linker intermediate was terminated, and then the pH of the reaction mixture was adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0627] (iii) Purification

[0628] Using Pellicon XL (Millipore Japan, 50 cm) 2 The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 300 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 13.1 mL of a solution containing the humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition.

[0629] (iv) Evaluation of characteristics

[0630] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-B7-H3 antibody (M30-H1-L4), based on its amino acid sequence, the value 30160 was used as an estimate of the molar absorptivity of the light chain, and the value 87250 was used as an estimate of the molar absorptivity of the heavy chain.

[0631] The antibody concentration was found to be 18.4 mg / mL, the antibody yield was found to be 241 mg (94%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 3.8. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 15 middle.

[0632] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 6.5%, D2: 30.5%, D4-1: 27.9%, D4-2: 12.3%, D6: 17.7% and D8: 4.9% ( Figure 16 ).

[0633] (Example 6-2) Production of a humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition according to the method of the present invention.

[0634] (i) Antibody reduction

[0635] Humanized anti-B7-H3 antibody (M30-H1-L4) (produced according to the method described in Reference Example 1 of WO2014 / 057687, 27.1 mL: corresponding to 500 mg, concentration: 18.5 mg / mL; 10 mM histidine buffer) was placed in a glass reaction vessel, and 25 mL of 10 mM histidine buffer was further added. Sucrose (MERCK; 1.25 g) and 0.5 M EDTA aqueous solution (CALBIOCHEM; 0.041 mL; 6 equivalents based on antibody) were added to this reaction solution, followed by the addition of 0.1 g / mL polysorbate 80 (NOFCORPORATION) aqueous solution (0.050 mL; 0.01% based on antibody). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.08. The reaction solution was cooled and stirred at an internal temperature of 0°C to 1°C. 1.00 mg / mL of tris(2-carboxyethyl)phosphonic acid hydrochloride (Nacalai Tesque, Inc.) aqueous solution (2.08 mL; 2.13 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then stirred for 5.5 hours to achieve an internal temperature of 0°C to 1°C, which allowed the interchain disulfides of the antibody to be reduced.

[0636] (ii) Conjugation of antibody to drug linker intermediate

[0637] Under stirring at an internal temperature of 0°C to 1°C, a 50% aqueous solution of acetone (2.82 mL; 4.8 equivalents per single antibody molecule) of the compound obtained in Example 2 at a concentration of 6.04 mg / mL was added to the solution obtained in (i) above for 20 minutes. The resulting mixture was stirred at the same temperature as above for 20 minutes to allow the drug linker intermediate to bind to the antibody. Subsequently, an aqueous solution of 50 mM N-acetylcysteine ​​(Kishida Chemical Co., Ltd.) (0.205 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred at the same temperature as above for 20 minutes. The reaction of the drug linker intermediate was terminated, and the pH of the reaction mixture was then adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0638] (iii) Purification

[0639] Using Pellicon XL (Millipore Japan, 50 cm) 2 The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 800 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 23.6 mL of a solution containing the humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition.

[0640] (iv) Evaluation of characteristics

[0641] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-B7-H3 antibody (M30-H1-L4), based on its amino acid sequence, the value 30160 was used as an estimate of the molar absorptivity of the light chain, and the value 87250 was used as an estimate of the molar absorptivity of the heavy chain.

[0642] The antibody concentration was found to be 19.4 mg / mL, the antibody yield was found to be 455 mg (89%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 4.1. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 17 middle.

[0643] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 2.7%, D2: 22.3%, D4-1: 58.4%, D6: 14.1% and D8: 2.4%. Figure 18 ).

[0644] (v) Results

[0645] The average number of bound drugs in the humanized anti-B7-H3 antibody ADC composition produced by the conventional method (Example 6-1) is 3.8, and the content of D4-1 is 27.9%. On the other hand, the average number of bound drugs in the humanized anti-B7-H3 antibody ADC composition produced by the method of the present invention (Example 6-2) is 4.1, and the content of D4-1 is 58.4%.

[0646] (Example 7) Production of humanized anti-HER2 antibody ADC composition

[0647] (Example 7-1) Production of humanized anti-HER2 antibody ADC composition according to conventional methods

[0648] (i) Antibody reduction

[0649] Humanized anti-HER2 antibody (trastuzumab; US Patent No. 5821337) (22.3 mL: corresponding to 500 mg, concentration: 22.4 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (27 mL, pH 5.0) was further added. 0.5 M EDTA aqueous solution (0.034 mL; 5 equivalents based on antibody) was added to this reaction solution, followed by 0.1 g / mL polysorbate 20 aqueous solution (0.050 mL; 0.01% based on antibody). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.12. While stirring at 22°C, 1.00 mg / mL of tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (2.12 mL; 2.15 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then stirred for 3 hours to achieve an internal temperature of 22°C to 25°C, which allowed the interchain disulfides of the antibody to be reduced.

[0650] (ii) Conjugation of antibody to drug linker intermediate

[0651] The solution obtained in (i) above was cooled, and then a 50% aqueous solution of acetone (2.77 mL; 4.8 equivalents per single antibody molecule) of the compound obtained in Example 2 at an internal temperature of 11°C to 13°C was added to the reaction solution over 20 minutes with stirring. The resulting mixture was stirred for 20 minutes at the same temperature as above to allow the drug linker intermediate to bind to the antibody. Subsequently, a 50 mM aqueous solution of N-acetylcysteine ​​(0.206 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred for 20 minutes at the same temperature as above. The reaction of the drug linker intermediate was terminated, and the pH of the reaction mixture was then adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0652] (iii) Purification

[0653] Using Pellicon XL (Millipore Japan, 50 cm) 2 The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 600 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 22.7 mL of a solution containing the humanized anti-HER2 antibody ADC composition.

[0654] (iv) Evaluation of characteristics

[0655] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-HER2 antibody (trastuzumab), based on its amino acid sequence, the value 26150 was used as an estimate of the molar absorptivity of the light chain, and the value 81290 was used as an estimate of the molar absorptivity of the heavy chain.

[0656] The antibody concentration was found to be 20.39 mg / mL, the antibody yield was found to be 462 mg (90%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 3.9. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 21 middle.

[0657] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 3.6%, D2: 26.1%, D4-1: 34.1%, D4-2: 13.6%, D6: 17.6%, and D8: 5.0%. Figure 22 ).

[0658] (Example 7-2) Production of humanized anti-HER2 antibody ADC composition according to the method of the present invention

[0659] (i) Antibody reduction

[0660] Humanized anti-HER2 antibody (trastuzumab; US Patent No. 5821337) (22.3 mL: corresponding to 500 mg, concentration: 22.4 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (25 mL, pH 5.0) was further added. 0.5 M EDTA aqueous solution (0.034 mL; 5 equivalents based on antibody) was added to this reaction solution, followed by 0.1 g / mL polysorbate 20 aqueous solution (0.050 mL; 0.01% based on antibody). Subsequently, 0.3 M disodium hydrogen phosphate aqueous solution was added to the reaction mixture to adjust the pH to 7.13, and the mixture was then cooled. At an internal temperature of 0°C to 1°C, with stirring, 1.00 mg / mL of tris(2-carboxyethyl)phosphine hydrochloride aqueous solution (2.37 mL; 2.40 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was then stirred at an internal temperature of 0°C to 1°C for 6 hours to reduce the interchain disulfides of the antibody.

[0661] (ii) Conjugation of antibody to drug linker intermediate

[0662] Under stirring at an internal temperature of 0°C to 2°C, a 50% aqueous solution of acetone (2.84 mL; 4.9 equivalents per single antibody molecule) of the compound obtained in Example 2 at a concentration of 6.14 mg / mL was added to the solution obtained in (i) above for 10 minutes. The resulting mixture was stirred at the same temperature as above for 40 minutes to allow the drug linker intermediate to bind to the antibody. Subsequently, a 50 mM aqueous solution of N-acetylcysteine ​​(0.206 mL; 3 equivalents per single antibody molecule) was added to the reaction mixture, and the resulting mixture was further stirred at the same temperature as above for 50 minutes. The reaction of the drug linker intermediate was terminated, and the pH of the reaction mixture was then adjusted to pH 5.0 using a 10% aqueous solution of acetic acid.

[0663] (iii) Purification

[0664] Using Pellicon XL (Millipore Japan, 50 cm) 2The solution obtained in step (ii) above is circulated while 10 mM histidine buffer (pH 5.0) is added using a roller pump, and a washing operation is performed until the discharged water volume is 600 mL to remove low molecular weight substances. The remaining solution is then concentrated to obtain 21.7 mL of a solution containing the humanized anti-HER2 antibody ADC composition.

[0665] (iv) Evaluation of characteristics

[0666] The average number of bound drugs was measured in the same manner as the commonly used procedure A described in Example 3-1, (iv). In the case of the humanized anti-HER2 antibody (trastuzumab), based on its amino acid sequence, the value 26150 was used as an estimate of the molar absorptivity of the light chain, and the value 81290 was used as an estimate of the molar absorptivity of the heavy chain.

[0667] The antibody concentration was found to be 21.2 mg / mL, the antibody yield was found to be 459 mg (89%), and the average number (n) of drug bound per single antibody molecule, measured by common procedure A, was found to be 4.0. HPLC chromatograms representing the peak area ratio (%) of each chain are shown below. Figure 23 middle.

[0668] The area values ​​of the number of bound drugs were measured in the same manner as the commonly used operation B described in Example 3-1, (iv). The distribution of the number of bound drugs was as follows: D0: 2.8%, D2: 23.8%, D4-1: 55.2%, D6: 15.0% and D8: 3.3%. Figure 24 ).

[0669] (v) Results

[0670] The average number of bound drugs in the humanized anti-HER2 antibody ADC composition produced by the conventional method (Example 7-1) is 3.9, and the content of D4-1 is 34.1%. On the other hand, the average number of bound drugs in the humanized anti-HER2 antibody ADC composition produced by the method of the present invention (Example 7-2) is 4.0, and the content of D4-1 is 55.2%.

[0671] (Test Example 1) The therapeutic effect of antibody-drug conjugate combinations

[0672] Mice: 5-6 week old female BALB / c-nu / nu mice (Charles River Laboratories International, Inc.) were acclimatized to SPF conditions for 4 to 7 days prior to use in experiments. Mice were fed sterile solid feed (FR-2, Funabashi Farms Co., Ltd) and sterile tap water (prepared by adding 5-15 ppm sodium hypochlorite solution to tap water).

[0673] Measurement and Calculation: Measure the long and short axes of the tumor two or more times per week using electronic digital calipers (CD-15C, Mitutoyo Corp.), then calculate the tumor volume (mm). 3 The calculation expression used is as follows.

[0674] Tumor volume (mm) 3 = 1 / 2 × major axis (mm) × [minor axis (mm)] 2

[0675] The human pancreatic cancer cell line CFPAC-1 (4×10⁻¹¹) already purchased from ATCC was used. 6 (Number of cells) were suspended in physiological saline. The obtained solution was then subcutaneously implanted into female BALB / c-nu / nu mice (day 0), and the mice were then randomly assigned to groups on day 11. After grouping, the humanized anti-TROP2 antibody ADC composition produced according to conventional methods (Example 3-1) and the humanized anti-TROP2 antibody ADC composition produced according to the method of the present invention (Example 3-2) were administered to mice via tail vein at doses of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, respectively. The antibody-drug conjugate compositions were diluted with acetate-buffered saline (pH 5.5) (Nacalai Tesque, Inc.), and the resulting solution was then administered to each mouse at a volume of 10 mL / kg. The minimum dose that caused tumor volume reduction (reduction dose) was used as an indicator to determine efficacy. The reduction dose of the humanized anti-TROP2 antibody ADC composition produced by conventional methods was 1 mg / kg, and the reduction dose of the humanized anti-TROP2 antibody ADC composition produced by the method of the present invention was also 1 mg / kg. Figure 25 Therefore, it has been demonstrated that the antibody-drug conjugate compositions produced by the production method of the present invention have therapeutic effects equivalent to those of antibody-drug conjugate compositions produced by conventional production methods.

[0676] (Test Example 2) Safety of Antibody-Drug Conjugate Combinations

[0677] Humanized anti-TROP2 antibody ADC compositions produced according to conventional methods (Example 3-1) and those produced according to the method of the present invention (Example 3-2) were each administered to cynomolgus monkeys of different species at three-week intervals for a total of three times. The monkeys were observed until the day after the last administration, and the maximum dose (HNSTD) without serious toxicity was analyzed. As a result, the HNSTD of the humanized anti-TROP2 antibody ADC composition produced by conventional methods was 10 mg / kg, while the HNSTD of the humanized anti-TROP2 antibody ADC composition produced by the method of the present invention was 30 mg / kg. Therefore, the antibody-drug conjugate compositions produced by the production method of the present invention demonstrate superior safety compared to those produced by conventional production methods.

[0678] (Consideration 1)

[0679] The results from Examples 3, 4, 6, and 7 show that the average number of bound drugs in antibody-drug conjugate compositions produced by conventional methods and the average number of bound drugs in antibody-drug conjugate compositions produced by the method of the present invention are both 3.5 to 4.5. On the other hand, the content of antibody-drug conjugates with four drug linkers bound to heavy-light interchain thiols in antibody-drug conjugate compositions produced by conventional methods is 35% or less, while the same content in antibody-drug conjugate compositions produced by the method of the present invention is 50% or more. Therefore, it has been demonstrated that antibody-drug conjugate compositions with an average number of bound drugs of 3.5 to 4.5 and a content of antibody-drug conjugates with four drug linkers bound to heavy-light interchain thiols of 50% or more can be selectively produced using the method of the present invention.

[0680] (Consideration 2)

[0681] The results of Example 2 demonstrate that the antibody-drug conjugate composition produced by the production method of the present invention has a superior safety profile compared to antibody-drug conjugate compositions produced by conventional production methods.

[0682] The foregoing results demonstrate that the antibody-drug conjugate composition of the present invention (an antibody-drug conjugate composition wherein the average number of bound drugs is 3.5 to 4.5, and the content of antibody-drug conjugates in which 4 drug linkers are bound to heavy-light interchain thiols is 50% or more) has a superior safety profile compared to antibody-drug conjugate compositions produced by conventional manufacturing methods (an antibody-drug conjugate composition wherein the average number of bound drugs is 3.5 to 4.5, and the content of antibody-drug conjugates in which 4 drug linkers are bound to heavy-light interchain thiols is 35% or less).

[0683] Sequence List Free Text

[0684] SEQ ID NO:1: Nucleotide sequence of humanized anti-TROP2 antibody heavy chain (hTINA1-H1)

[0685] SEQ ID NO:2: Amino acid sequence of humanized anti-TROP2 antibody heavy chain (hTINA1-H1)

[0686] SEQ ID NO:3: Nucleotide sequence of humanized anti-TROP2 antibody light chain (hTINA1-L1)

[0687] SEQ ID NO:4: Amino acid sequence of humanized anti-TROP2 antibody light chain (hTINA1-L1)

[0688] SEQ ID NO:5: Amino acid sequence of anti-TROP2 antibody (TINA1) CDRH1

[0689] SEQ ID NO:6: Amino acid sequence of anti-TROP2 antibody (TINA1) CDRH2

[0690] SEQ ID NO:7: Amino acid sequence of anti-TROP2 antibody (TINA1) CDRH3

[0691] SEQ ID NO:8: Amino acid sequence of anti-TROP2 antibody (TINA1) CDRL1

[0692] SEQ ID NO:9: Amino acid sequence of anti-TROP2 antibody (TINA1) CDRL2

[0693] SEQ ID NO:10: Amino acid sequence of anti-TROP2 antibody (TINA1) CDRL3

[0694] SEQ ID NO:11: Nucleotide sequence of humanized anti-CD98 antibody heavy chain (hM23-H1)

[0695] SEQ ID NO:12: Amino acid sequence of humanized anti-CD98 antibody heavy chain (hM23-H1)

[0696] SEQ ID NO:13: Nucleotide sequence of humanized anti-CD98 antibody light chain (hM23-L1)

[0697] SEQ ID NO:14: Amino acid sequence of humanized anti-CD98 antibody light chain (hM23-L1)

[0698] SEQ ID NO:15: Amino acid sequence of anti-CD98 antibody (M23) CDRH1

[0699] SEQ ID NO:16: Amino acid sequence of anti-CD98 antibody (M23) CDRH2

[0700] SEQ ID NO:17: Amino acid sequence of anti-CD98 antibody (M23) CDRH3

[0701] SEQ ID NO:18: Amino acid sequence of anti-CD98 antibody (M23) CDRL1

[0702] SEQ ID NO:19: Amino acid sequence of anti-CD98 antibody (M23) CDRL2

[0703] SEQ ID NO:20: Amino acid sequence of anti-CD98 antibody (M23) CDRL3

[0704] SEQ ID NO:21: Nucleotide sequence of primer EG-Inf-F

[0705] SEQ ID NO:22: Nucleotide sequence of primer EG1-Inf-R

[0706] SEQ ID NO:23: Nucleotide sequence of primer CM-LKF

[0707] SEQ ID NO:24: Nucleotide sequence of primer KCL-Inf-R

[0708] SEQ ID NO:25: Amino acid sequence of humanized anti-B7-H3 antibody heavy chain (M30-H1)

[0709] SEQ ID NO:26: Amino acid sequence of humanized anti-B7-H3 antibody light chain (M30-L4)

[0710] SEQ ID NO:27: Amino acid sequence of anti-B7-H3 antibody (M30) CDRH1

[0711] SEQ ID NO:28: Amino acid sequence of anti-B7-H3 antibody (M30) CDRH2

[0712] SEQ ID NO:29: Amino acid sequence of anti-B7-H3 antibody (M30) CDRH3

[0713] SEQ ID NO:30: Amino acid sequence of anti-B7-H3 antibody (M30) CDRL1

[0714] SEQ ID NO:31: Amino acid sequence of anti-B7-H3 antibody (M30) CDRL2

[0715] SEQ ID NO:32: Amino acid sequence of anti-B7-H3 antibody (M30) CDRL3

[0716] SEQ ID NO:33: Amino acid sequence of the humanized anti-HER2 antibody heavy chain

[0717] SEQ ID NO:34: Amino acid sequence of the humanized anti-HER2 antibody light chain.

Claims

1. A method for producing an antibody-drug conjugate composition, comprising a method for producing an antibody having a thiol group, wherein the method includes a step of reacting the antibody with a reducing agent in a buffer solution to reduce interchain disulfide, wherein... The isotype of the antibody is IgG1. The reducing agent is tris(2-carboxyethyl)phosphine hydrochloride. The buffer solution contains a chelating agent, wherein the buffer solution is a histidine buffer, and the chelating agent is ethylenediaminetetraacetic acid (EDTA), and the reaction temperature is 0°C to 1°C. The reaction time is 5 to 7 hours. The reducing agent is used in amounts of 2 to 3 molar equivalents per antibody molecule to obtain antibodies with thiol groups, and The produced thiol-containing antibodies are reacted with drug linker intermediates having N-substituted maleimide groups to produce antibody-drug conjugate compositions, wherein the average number of bound drugs is 4.0 to 4.1, and the content of antibody-drug conjugates in which four drug linkers are bound to heavy-light chain thiols is 50% to 60%. The drug linker intermediate having an N-substituted maleimide group is: ， -GGFG- represents a tetrapeptide residue composed of glycine-glycine-phenylalanine-glycine; and The coupling reaction temperature is 0℃ to 2℃.

2. The method of claim 1, wherein the produced thiol-containing antibody is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate with four drug linkers bound to heavy-heavy chain thiols is in the range of 5% or less.

3. The method of claim 2, wherein the produced thiol-containing antibody is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate with four drug linkers bound to heavy-heavy chain thiols is in the range of 1% or less.

4. The method according to any one of claims 1 to 3, wherein the produced thiol-containing antibody is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate having two drug linkers bound to heavy-heavy chain thiols and two drug linkers bound to heavy-light chain thiols is 5% or less.

5. The method of claim 4, wherein the produced thiol-containing antibody is used to produce an antibody-drug conjugate composition, wherein the content of the antibody-drug conjugate having two drug linkers bound to heavy-heavy chain thiols and two drug linkers bound to heavy-light chain thiols is 1% or less.

6. The method according to any one of claims 1 to 3, wherein the antibody is an anti-TROP2 antibody.

7. The method according to claim 6, wherein the anti-TROP2 antibody retains CDRH1 having the amino acid sequence shown in SEQ ID NO:5 (TAGMQ), CDRH2 having the amino acid sequence shown in SEQ ID NO:6 (WINTHSGVPKYAEDFKG), CDRH3 having the amino acid sequence shown in SEQ ID NO:7 (SGFGSSYWYFDV), CDRL1 having the amino acid sequence shown in SEQ ID NO:8 (KASQDVSTAVA), CDRL2 having the amino acid sequence shown in SEQ ID NO:9 (SASYRYT), and CDRL3 having the amino acid sequence shown in SEQ ID NO:10 (QQHYITPLT).

8. The method of claim 6, wherein the anti-TROP2 antibody consists of a heavy chain and a light chain, the heavy chain consisting of: The light chain consists of an amino acid sequence composed of amino acid residues at positions 20 to 470 of SEQ ID NO:2, and the light chain consists of an amino acid sequence composed of amino acid residues at positions 21 to 234 of SEQ ID NO:

4.

9. The method of claim 8, wherein a lysine residue is missing at the carboxyl terminus of the heavy chain of the anti-TROP2 antibody.

10. The method according to any one of claims 1 to 3, wherein the antibody is an anti-CD98 antibody.

11. The method according to any one of claims 1 to 3, wherein the antibody is an anti-B7-H3 antibody.

12. The method according to claim 11, wherein the anti-B7-H3 antibody retains CDRH1 having the amino acid sequence shown in SEQ ID NO:27 (NYVMH), CDRH2 having the amino acid sequence shown in SEQ ID NO:28 (YINPYNDDVKYNEKFKG), CDRH3 having the amino acid sequence shown in SEQ ID NO:29 (WGYYGSPLYYFDY), CDRL1 having the amino acid sequence shown in SEQ ID NO:30 (RASSRLIYMH), CDRL2 having the amino acid sequence shown in SEQ ID NO:31 (ATSNLAS), and CDRL3 having the amino acid sequence shown in SEQ ID NO:32 (QQWNSNPPT).

13. The method of claim 11, wherein the anti-B7-H3 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises: The amino acid sequence consisting of amino acid residues at positions 20 to 471 of SEQ ID NO:25, and the light chain consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 233 of SEQ ID NO:

26.

14. The method of claim 13, wherein a lysine residue is deleted at the carboxyl terminus of the heavy chain of the anti-B7-H3 antibody.

15. The method according to any one of claims 1 to 3, wherein the antibody is an anti-HER2 antibody.

16. The method of claim 15, wherein the anti-HER2 antibody comprises a heavy chain and a light chain, the heavy chain comprising: An amino acid sequence consisting of amino acid residues at positions 1 to 449 of SEQ ID NO:33, and the light chain consisting of an amino acid sequence consisting of amino acid residues at positions 1 to 214 of SEQ ID NO:

34.

17. The method of claim 15, wherein the anti-HER2 antibody comprises a heavy chain and a light chain, the heavy chain having the amino acid residues shown in SEQ ID NO:33, and the light chain having the amino acid sequence shown in SEQ ID NO:34.

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