Method for producing antibody-drug conjugates

Abstract

This invention provides a method for stably producing antibody-drug conjugate compositions containing each isomer in a fixed ratio. [Solution] A method for producing an antibody-drug conjugate composition, comprising the steps of (i) reacting an antibody with a reducing agent to obtain an antibody having a thiol group; and then (ii) reacting the antibody having a thiol group obtained in (i) with a drug linker intermediate, wherein step (i) is carried out until the composition ratio of the antibody having four heavy-chain-light-chain thiols and the composition ratio of the antibody having four heavy-chain-heavy-chain thiols reach a steady value.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for stably producing an antibody-drug conjugate composition comprising, at a constant ratio, isomers of an antibody-drug conjugate that differ in drug binding position. BACKGROUND ART

[0002] An Antibody-Drug Conjugate (ADC), which is prepared by conjugating a cytotoxic drug to an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into cells, can selectively deliver the drug to cancer cells, thereby accumulating the drug in cancer cells and is expected to kill cancer cells (Non-Patent Documents 1 to 5).

[0003] An antibody has four interchain disulfides, which are more accessible to solvents than other disulfides and thus are easily reduced, and can be used as binding sites for drugs (or drug linkers) in antibody-drug conjugates. By reducing the interchain disulfides of an antibody and conjugating a drug to the resulting thiol groups, an antibody-drug conjugate in which a maximum of 8 drugs are conjugated per antibody molecule is produced.

[0004] Antibody-drug conjugates, in which eight drug molecules are bound to each antibody molecule, may exhibit excellent antitumor effects but may have low safety tolerance. Therefore, to improve safety tolerance while maintaining therapeutic efficacy, antibody-drug conjugates with an average drug binding count of less than eight are sometimes used. Antibody-drug conjugates with an average drug binding count of less than eight can be obtained, for example, by controlling the amount of drug per antibody molecule during the reaction. The reaction products are compositions of antibody-drug conjugates with drug binding counts of 2, 4, 6, and 8. Consequently, even with antibody-drug conjugate compositions having the same average drug binding count, differences in the distribution of each drug binding count can lead to differences in therapeutic efficacy and safety tolerance. For example, in an antibody-drug conjugate composition with an average drug binding count of approximately 4, a high content of antibody-drug conjugates with 0 and 8 drug binding counts may result in decreased therapeutic efficacy and reduced safety tolerance compared to a composition with a high content of antibody-drug conjugates with 4 drug binding counts. Furthermore, even antibody-drug conjugates with the same number of drug bindings may exhibit different therapeutic efficacy and safety tolerances depending on the drug binding site. Therefore, in the production of antibody-drug conjugate compositions, a method for producing antibody-drug conjugate compositions in which the number and binding site of drugs are controlled is required.

[0005] Methods for producing antibody-drug conjugate compositions in which the number and position of drug bindings are controlled include a method (Patent Document 1) in which the interchain disulfide of the antibody is completely reduced, a portion of the resulting interchain thiol is reoxidized back to disulfide, and the drug is bound to the remaining interchain thiol, thereby selectively producing an antibody-drug conjugate in which four drug linkers are bound to the heavy-chain-heavy-chain interchain thiol, and a method (Patent Document 2) in which the interchain disulfide of the antibody is reduced at a low temperature and the drug is bound to the resulting interchain thiol, thereby selectively producing an antibody-drug conjugate in which four drug linkers are bound to the heavy-chain-light-chain interchain thiol. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2005 / 084390 [Patent Document 2] International Publication No. 2017 / 002776 [Non-patent literature]

[0007] [Non-Patent Document 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13. [Non-Patent Document 2] Alley, SC, et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [Non-Patent Document 3] Damle NK Expert Opin. Biol. Ther. (2004) 4, 1445-1452. [Non-Patent Document 4] Senter PD, et al., Nature Biotechnology (2012) 30, 631-637. [Non-Patent Document 5] Howard A. et al., J Clin Oncol 29: 398-405. [Overview of the initiative] [Problems that the invention aims to solve]

[0008] Methods for producing antibody-drug conjugate compositions containing a specific isomer in a high proportion among the various isomers of antibody-drug conjugates with different drug binding sites are known (International Publication Nos. 2005 / 084390 and 2017 / 002776). However, from an industrial perspective, there is sometimes a need for a method to stably produce antibody-drug conjugate compositions containing each isomer in a constant proportion, rather than containing a specific isomer in a high proportion. No such method for producing antibody-drug conjugate compositions is known, and there is a need for the development of an industrially superior production method. [Means for solving the problem]

[0009] The inventors of the present invention conducted diligent research to solve the above problems and discovered that by performing the process of reacting an antibody with a reducing agent to obtain an antibody having thiol groups over a long period of time, the composition ratio of antibodies having four heavy-chain-light-chain thiols and the composition ratio of antibodies having four heavy-chain-heavy-chain thiols reach a steady state (hereinafter also referred to as "equilibriumization of the reduction reaction"). Furthermore, they discovered that by reacting the thiol-group-containing antibody obtained in this way with a drug linker intermediate, an antibody-drug conjugate composition containing various isomers of antibody-drug conjugates with different drug binding positions in a fixed ratio can be stably produced, thus completing the present invention.

[0010] In other words, the present invention provides the following [1] to

[56] . [1] (i) A step of reacting an antibody with a reducing agent to obtain an antibody having a thiol group; then, A method for producing an antibody-drug conjugate composition, comprising the step of reacting the antibody having a thiol group obtained in (i) with a drug linker intermediate, A manufacturing method characterized in that step (i) is carried out until the composition ratio of antibodies having four heavy-chain-light-chain thiols and the composition ratio of antibodies having four heavy-chain-heavy-chain thiols reach a steady state. [2] The manufacturing method described in [1], wherein step (i) is carried out over a period of 4 hours or more. [3] The manufacturing method according to [1], wherein step (i) is carried out over a period of 12 hours or more. [4] The manufacturing method described in [1], wherein step (i) is carried out over a period of 16 hours or more. [5] The manufacturing method described in [1], wherein step (i) is carried out over a period of 20 hours or more. [6] The manufacturing method according to [1], wherein step (i) is carried out over a period of 31 hours or more. [7] The manufacturing method according to any one of items [1] to [6], wherein step (i) is performed at 0 to 20°C. [8] The manufacturing method according to any one of items [1] to [6], wherein step (i) is performed at 5 to 20°C. [9] The manufacturing method according to any one of items [1] to [6], wherein step (i) is performed at 5 to 10°C.

[10] The manufacturing method according to any one of items [1] to [6], wherein step (i) is performed at approximately 10°C.

[11] The manufacturing method according to any one of [1] to

[10] , wherein the average number of drug conjugates in the antibody-drug conjugate composition produced is 3.5 to 4.5.

[12] The manufacturing method according to any one of [1] to

[11] , wherein the antibody-drug conjugate composition produced contains 50% or more of an antibody-drug conjugate to which four drug linkers are attached.

[13] The manufacturing method according to any one of [1] to

[12] , wherein the proportion of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain inter-light-chain thiols in the manufactured antibody-drug conjugate composition is 1.5 to 2.5 times the proportion of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain inter-heavy-chain thiols.

[14] A manufacturing method according to any one of items [1] to

[13] , wherein the reducing agent is used in an amount of 1.9 to 2.5 equivalents per molecule of antibody.

[15] The production method according to any one of [1] to

[14] , wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof.

[16] The production method according to any one of [1] to

[15] , wherein the drug-linker intermediate has an N-substituted maleimidyl group.

[17] The production method according to any one of [1] to

[15] , wherein the drug-linker intermediate is a compound represented by formula

[0011]

Chemical Structure

[0012] .

[18] In the antibody-drug conjugate composition produced, the drug-linker is represented by formula

[0013]

Chemical Structure

[0014] (wherein, A represents the binding position to an antibody, and the drug-linker is bound to the antibody via a thioether bond) The production method according to

[17] , which is represented by said formula.

[19] The production method according to any one of [1] to

[18] , wherein the antibody is an anti-TROP2 antibody or an anti-B7-H3 antibody.

[20] The production method according to

[19] , wherein the antibody is an anti-TROP2 antibody.

[21] The method for producing an anti-TROP2 antibody according to

[20] , wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 5, CDRH2 having the amino acid sequence described in SEQ ID NO: 6, and CDRH3 having the amino acid sequence described in SEQ ID NO: 7, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 8, CDRL2 having the amino acid sequence described in SEQ ID NO: 9, and CDRL3 having the amino acid sequence described in SEQ ID NO: 10. [twenty two] The method for producing an anti-TROP2 antibody according to

[20] , wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 1, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 2, [twenty three] The method for producing an anti-TROP2 antibody according to

[20] , wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234). [twenty four] A method for producing an anti-TROP2 antibody, wherein the lysine residue at the heavy chain carboxyl terminus is deleted, as described in

[23] . [twenty five] The manufacturing method described in

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

[26] The method for producing an anti-B7-H3 antibody according to

[25] , wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 11, CDRH2 having the amino acid sequence described in SEQ ID NO: 12, and CDRH3 having the amino acid sequence described in SEQ ID NO: 13, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 14, CDRL2 having the amino acid sequence described in SEQ ID NO: 15, and CDRL3 having the amino acid sequence described in SEQ ID NO: 16.

[27] The method for producing an anti-B7-H3 antibody according to

[25] , wherein the antibody comprises a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 3, and a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 4,

[28] The method for producing an anti-B7-H3 antibody according to

[25] , wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 to 21 to 233.

[29] A method for producing an anti-B7-H3 antibody, wherein the lysine residue at the heavy chain carboxyl terminus is deleted, as described in

[28] .

[30] An antibody-drug conjugate composition manufactured by the manufacturing method described in any one of items [1] to

[29] .

[31] (i) A step of reacting an antibody with a reducing agent to obtain an antibody having a thiol group; then, A method for producing an antibody-drug conjugate composition, comprising the step of reacting the antibody having a thiol group obtained in (i) with a drug linker intermediate, (i) The process is carried out at 0-20°C for 4 hours or more. A method for producing an antibody-drug conjugate composition characterized in that the content of antibody-drug conjugates with four drug linkers attached is 50% or more, and the proportion of antibody-drug conjugates with four drug linkers attached to the heavy-chain interchain thiol is 1.5 to 2.5 times the proportion of antibody-drug conjugates with four drug linkers attached to the heavy-chain interchain thiol.

[32] The manufacturing method according to

[31] , wherein step (i) is carried out over a period of 12 hours or more.

[33] The manufacturing method according to

[31] , wherein step (i) is carried out over a period of 16 hours or more.

[34] The manufacturing method described in

[31] , wherein step (i) is carried out over a period of 20 hours or more.

[35] The manufacturing method according to

[31] , wherein step (i) is carried out over a period of 31 hours or more.

[36] The manufacturing method according to any one of items

[31] to

[35] , wherein step (i) is performed at 5 to 20°C.

[37] The manufacturing method according to any one of items

[31] to

[35] , wherein step (i) is performed at 5 to 10°C.

[38] The manufacturing method according to any one of items

[31] to

[35] , wherein step (i) is performed at approximately 10°C.

[39] The method for producing an antibody-drug conjugate composition, wherein the average number of drug-bound components in the antibody-drug conjugate composition produced is 3.5 to 4.5, according to any one of the claims

[31] to

[38] .

[40] A manufacturing method according to any one of the items

[31] to

[39] , wherein the reducing agent is used in an amount of 1.9 to 2.5 equivalents per molecule of antibody.

[41] The method for producing a product according to any one of

[31] to

[40] , wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof.

[42] A method for producing a drug linker intermediate having an N-substituted maleimidyl group, according to any one of

[31] to

[41] .

[43] The drug linker intermediate is,

[0015] [ka]

[0016] A method for producing a compound represented by

[31] to

[41] as described in any one of the following paragraphs.

[44] The drug linker of the antibody-drug conjugate composition to be manufactured is of the formula

[0017] [ka]

[0018] (In the formula, A indicates the binding site with the antibody, and the drug linker is bound to the antibody by a thioether bond.) The manufacturing method described in

[43] , as shown in

[43] .

[45] The manufacturing method according to any one of the items

[31] to

[44] , wherein the antibody is an anti-TROP2 antibody or an anti-B7-H3 antibody.

[46] The manufacturing method according to

[45] , wherein the antibody is an anti-TROP2 antibody.

[47] The method for producing an anti-TROP2 antibody according to

[46] , wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 5, CDRH2 having the amino acid sequence described in SEQ ID NO: 6, and CDRH3 having the amino acid sequence described in SEQ ID NO: 7, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 8, CDRL2 having the amino acid sequence described in SEQ ID NO: 9, and CDRL3 having the amino acid sequence described in SEQ ID NO: 10.

[48] The method for producing an anti-TROP2 antibody according to

[46] , wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 1, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 2,

[49] The method for producing an anti-TROP2 antibody according to

[46] , wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234).

[50] A method for producing an anti-TROP2 antibody, wherein the lysine residue at the heavy chain carboxyl terminus is deleted, as described in

[49] .

[51] The manufacturing method described in

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

[52] The method for producing an anti-B7-H3 antibody according to

[51] , wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 11, CDRH2 having the amino acid sequence described in SEQ ID NO: 12, and CDRH3 having the amino acid sequence described in SEQ ID NO: 13, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 14, CDRL2 having the amino acid sequence described in SEQ ID NO: 15, and CDRL3 having the amino acid sequence described in SEQ ID NO: 16.

[53] The method for producing an anti-B7-H3 antibody according to

[51] , wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 3 to 20141, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 4 to 20128.

[54] The method for producing an anti-B7-H3 antibody according to

[51] , wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 to 21 to 233.

[55] A method for producing an anti-B7-H3 antibody, wherein the lysine residue at the heavy chain carboxyl terminus is deleted, as described in

[54] .

[56] An antibody-drug conjugate composition manufactured by the manufacturing method described in any one of items

[31] to

[55] . [Effects of the Invention]

[0019] The present invention provides a method for stably producing antibody-drug conjugate compositions containing isomers of antibody-drug conjugates with different drug binding sites in a fixed ratio. [Brief explanation of the drawing]

[0020] [Figure 1] This figure shows the amino acid sequence (SEQ ID NO: 1) of the anti-TROP2 antibody heavy chain. [Figure 2]This figure shows the amino acid sequence (SEQ ID NO: 2) of the light chain of the anti-TROP2 antibody. [Figure 3] This figure shows the amino acid sequence (SEQ ID NO: 3) of the anti-B7-H3 antibody heavy chain. [Figure 4] This figure shows the amino acid sequence (SEQ ID NO: 4) of the light chain of the anti-B7-H3 antibody. [Figure 5] This figure shows the change over time in the content of D4 among D0 to D8 in the production of an anti-TROP2 antibody-drug conjugate composition. [Figure 6] This figure shows the change over time in the composition ratio of D4-1 and D4-2 in D4 during the production of an anti-TROP2 antibody-drug conjugate composition. [Figure 7] This figure shows the change over time in the content of D4 among D0 to D8 in the production of an anti-B7-H3 antibody-drug conjugate composition. [Figure 8] This figure shows the change over time in the composition ratio of D4-1 and D4-2 in D4 during the production of an anti-B7-H3 antibody-drug conjugate composition. [Modes for carrying out the invention]

[0021] The following describes preferred embodiments for carrying out the present invention. The embodiments described below are merely examples of typical embodiments of the present invention, and this should not be interpreted as narrowing the scope of the invention.

[0022] 1. Antibody-drug conjugates In the present invention, "antibody-drug conjugate" refers to a complex in which an antibody is bound to a cytotoxic drug via a linker. Examples of antibody-drug conjugates include U.S. Patent No. 6,214,345, International Publication No. 2002 / 083067, International Publication No. 2003 / 026577, International Publication No. 2004 / 054622, International Publication No. 2005 / 112919, International Publication No. 2006 / 135371, International Publication No. 2007112193, International Publication No. 2008 / 033891, International Publication No. 2009 / 100194, International Publication No. 2009 / 134976, International Publication No. 2009 / 134977, and International Publication No. 2010 / 09 Examples include those described in International Publication No. 3395, International Publication No. 2011 / 130613, International Publication No. 2011 / 130616, International Publication No. 2013 / 055993, International Publication No. 2014 / 057687, International Publication No. 2014 / 107024, International Publication No. 2014 / 134457, International Publication No. 2014 / 145090, and International Publication No. 2015 / 098099, preferably those described in International Publication No. 2014 / 057687 and International Publication No. 2015 / 098099.

[0023] There are no particular restrictions on cytotoxic drugs as long as they have antitumor effects and substituents or substructures that can bind to the linker, but examples include camptothecin, calicheamicin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin, and auristatin E. Examples of E) include maytansine, paclitaxel, pyrrolobenzodiazepine, and their derivatives, preferably camptothecin derivatives, and more preferably exatecan derivatives.

[0024] Exatecan (IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione, (also expressed as chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13(9H,15H)-dione), a topoisomerase I inhibitor, is formulated as follows:

[0025] [ka]

[0026] It is a compound represented by [formula].

[0027] In this invention, "drug linker" refers to the drug and linker portion in an antibody-drug conjugate, or in other words, the substructure other than the antibody in an antibody-drug conjugate.

[0028] In the present invention, "interchain disulfide" refers to a disulfide between two heavy chains in an antibody (heavy chain-to-heavy chain disulfide), or a disulfide between a heavy chain and a light chain (heavy chain-to-light chain disulfide).

[0029] In this invention, "interchain thiol" refers to a thiol group obtained by the reduction of an interchain disulfide of an antibody.

[0030] In the present invention, "heavy chain-inter-heavy chain thiol" refers to a thiol group obtained by the reduction of the heavy chain-inter-heavy chain disulfide of an antibody.

[0031] In the present invention, "heavy-light chain thiol" refers to a thiol group obtained by the reduction of the heavy-light chain disulfide of an antibody.

[0032] In antibody-drug conjugates, the number of drug molecules bound to a single antibody molecule is a crucial factor affecting its efficacy and safety. Antibodies have four interchain disulfides, and each disulfide is composed of two thiol groups. Therefore, the number of drug molecules bound to a single antibody molecule can be 2, 4, 6, or 8.

[0033] In an antibody-drug conjugate (hereinafter sometimes referred to as "D2") in which two drug molecules are bound to one antibody molecule, two isomers may be generated: an antibody-drug conjugate in which two drug linkers are bound to the heavy-chain-light-chain thiol (hereinafter sometimes referred to as "D2-1"), and an antibody-drug conjugate in which two drug linkers are bound to the heavy-chain-heavy-chain thiol (hereinafter sometimes referred to as "D2-2").

[0034] [ka]

[0035] (In the formula, long lines (bent lines) represent heavy chains, short lines represent light chains, and lines consisting of circles and wavy lines represent drug linkers.) As an antibody-drug conjugate (hereinafter sometimes referred to as "D4") in which four drug molecules are bound to one antibody molecule, three isomers can be generated: an antibody-drug conjugate in which four drug linkers are bound to the heavy-chain-light-chain thiol (hereinafter sometimes referred to as "D4-1"), an antibody-drug conjugate in which four drug linkers are bound to the heavy-chain-heavy-chain thiol (hereinafter sometimes referred to as "D4-2"), and an antibody-drug conjugate in which two drug linkers are bound to the heavy-chain-light-chain thiol and two drug linkers are bound to the heavy-chain-heavy-chain thiol (hereinafter sometimes referred to as "D4-3").

[0036] [ka]

[0037] (In the formula, long lines (bent lines) represent heavy chains, short lines represent light chains, and lines consisting of circles and wavy lines represent drug linkers.) As an antibody-drug conjugate (hereinafter sometimes referred to as "D6") in which six drug molecules are bound to one antibody molecule, two isomers may be generated: an antibody-drug conjugate (hereinafter sometimes referred to as "D6-1") in which four drug linkers are bound to the heavy-chain-light-chain thiol and two drug linkers are bound to the heavy-chain-heavy-chain thiol, and an antibody-drug conjugate (hereinafter sometimes referred to as "D6-2") in which two drug linkers are bound to the heavy-chain-light-chain thiol and four drug linkers are bound to the heavy-chain-heavy-chain thiol.

[0038] [ka]

[0039] (In the formula, long lines (bent lines) represent heavy chains, short lines represent light chains, and lines consisting of circles and wavy lines represent drug linkers.) In an antibody-drug conjugate (hereinafter sometimes referred to as "D8") in which eight drug molecules are bound to one antibody molecule, an antibody-drug conjugate can be formed in which four drug linkers are bound to the heavy-chain-light-chain thiol and four drug linkers are bound to the heavy-chain-heavy-chain thiol.

[0040] [ka]

[0041] (In the formula, long lines (bent lines) represent heavy chains, short lines represent light chains, and lines consisting of circles and wavy lines represent drug linkers.) The interchain thiols of the antibody bind to, for example, the 3-position of the N-substituted maleimidyl group of the drug linker intermediate by forming a thioether. That is, the binding site between the antibody and the drug linker is, for example, as shown in the following formula:

[0042] [ka]

[0043] (In the formula, "antibody-S-" is derived from the antibody.)

[0044] In the present invention, "antibody-drug conjugate composition" means a composition that may contain D2, D4, D6, D8, and an antibody without a drug linker (hereinafter sometimes referred to as "D0") in any proportion.

[0045] In the present invention, "average drug binding number" is also called the drug-to-antibody ratio (DAR), and refers to the average number of drugs (or drug linkers) that bind to one antibody molecule in an antibody-drug conjugate composition.

[0046] In the present invention, the "content" of each isomer in the antibody-drug conjugate composition is expressed in mole percent based on the antibody.

[0047] The antibody-drug conjugate preferably produced in the present invention is of the formula

[0048] [ka]

[0049] (In the formula, A indicates the binding site with the antibody.) This is an antibody-drug conjugate in which a drug linker, indicated by the symbol, and an antibody are linked by a thioether bond. This drug linker is bound to thiol groups (in other words, sulfur atoms of cysteine ​​residues) formed at disulfide bond sites between the antibody chains (two heavy chain-to-heavy chain bonds and two heavy chain-to-light chain bonds).

[0050] The antibody-drug conjugate preferred for production in the present invention can also be represented by the following formula.

[0051] [ka]

[0052] Here, the drug linker is bound to the antibody by a thioether bond. Also, n is synonymous with the so-called average drug-to-antibody ratio (DAR), and represents the average number of drug linkers bound per antibody.

[0053] The antibody-drug conjugate, which is suitably produced in the present invention, after being transferred into cancer cells,

[0054] [ka]

[0055] By releasing the compound represented by [formula], it exerts an antitumor effect.

[0056] The above-mentioned compounds are considered to be the main component of the antitumor activity of the antibody-drug conjugate, which is suitably produced in the present invention (International Publication No. 2014 / 057687 and International Publication No. 2015 / 098099).

[0057] The above compound is thought to be produced by the cleavage of the linker portion of the above antibody-drug conjugate, according to formula

[0058] [ka]

[0059] It is thought to be caused by the decomposition of the aminal structure of the compound represented by [formula].

[0060] 2. Antibodies used in the manufacture of antibody-drug conjugate compositions The antibodies used in the production of the antibody-drug conjugate compositions of the present invention may be derived from any species, but preferably from humans, rats, mice, and rabbits. If the antibodies are derived from a species other than humans, it is preferable to chimerize or humanize them using well-known techniques. The antibodies of the present invention may be polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are preferred.

[0061] The antibodies used in the production of the antibody-drug conjugate composition of the present invention preferably have properties that can target cancer cells, and are preferably those that have the ability to recognize cancer cells, the ability to bind to cancer cells, the ability to be taken up and internalized within cancer cells, and / or cytotoxic activity against cancer cells.

[0062] The binding affinity of antibodies to cancer cells can be confirmed using flow cytometry. Antibody uptake into cancer cells can be confirmed, for example, using (1) an assay that visualizes antibodies taken up into cells using a fluorescence microscope with a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay that measures the amount of fluorescence when taken up into cells using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay (Bio Techniques 28:162-165, January 2000), which uses an immunotoxin that binds to the therapeutic antibody and suppresses cell proliferation by releasing a toxin upon uptake into cells. Recombinant complex proteins of the catalytic domain of diphtheria toxin and protein G can also be used as immunotoxins.

[0063] The antitumor activity of an antibody can be confirmed in vitro by measuring its inhibitory activity on cell proliferation. For example, cancer cell lines overexpressing the antibody's target protein can be cultured, and the antibody can be added to the culture system at various concentrations to measure its inhibitory activity on focus formation, colony formation, and spheroid proliferation. In vivo, for example, antitumor activity can be confirmed by administering the antibody to nude mice transplanted with cancer cell lines highly expressing the target protein and measuring the changes in the cancer cells.

[0064] While it is desirable for the antibody itself to have antitumor effects, the antitumor effect of the antibody-drug conjugate is not essential, as it involves attaching a compound that exerts antitumor effects. For the purpose of specifically and selectively exerting the cytotoxic effects of the antitumor compound in cancer cells, it is important and desirable that the antibody has the property of migrating into (internalizing) cancer cells.

[0065] The antibodies used in the production of the antibody-drug conjugate composition of the present invention can be obtained by known means. For example, they can be obtained by immunizing animals with antigenic polypeptides using methods commonly practiced in this art, and then collecting and purifying the antibodies produced in vivo. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from non-human animals such as mice and rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity between the obtained heterologous antigen and the human antigen.

[0066] Furthermore, monoclonal antibodies can also be obtained by establishing hybridomas and fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells, according to known methods (for example, Kohler and Milstein, Nature (1975) 256, p.495-497; Kennet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)).

[0067] Antigens can be obtained by genetically modifying host cells to produce the gene encoding the antigen protein. Specifically, a vector capable of expressing the antigen gene is created, introduced into host cells to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing animals with antigen-expressing cells or cell lines expressing the antigen, which have been genetically modified as described above.

[0068] The antibodies used in the production of the antibody-drug conjugate composition of the present invention are preferably genetically modified recombinant antibodies, such as chimeric antibodies or humanized antibodies, which are artificially modified for purposes such as reducing heteroantigenicity against humans, or antibodies that have only the gene sequence of a human-derived antibody, i.e., human antibodies. These antibodies can be produced using known methods.

[0069] Chimeric antibodies are antibodies in which the variable region and constant region are heterogeneous, for example, chimeric antibodies in which the variable region of a mouse or rat-derived antibody is conjugated to the constant region of a human-derived antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0070] Examples of humanized antibodies include antibodies in which only the complementarity determining region (CDR) of a heterologous antibody is incorporated into a human-derived antibody (Nature (1986) 321, pp. 522-525), antibodies in which, in addition to the CDR sequence of a heterologous antibody, amino acid residues of a portion of the framework of the heterologous antibody are also transplanted into a human antibody using the CDR transplantation method (International Publication No. 90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (U.S. Patent No. 5821337).

[0071] Examples of human antibodies include antibodies produced using human antibody-producing mice that possess human chromosome fragments containing the genes for the heavy and light chains of human antibodies (see Tomizuka, K. et al., Nature Genetics (1997) 16, p.133-143; Kuroiwa, Y. et. al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et. al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et. al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727, etc.). Alternatively, antibodies obtained by phage display selected from a human antibody library can also be cited (see Wormstone, IM et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p.2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1(2), p.189-203; Siriwardena, D. et. al., Ophthalmology (2002) 109(3), p.427-431, etc.).

[0072] The antibodies used in the production of the antibody-drug conjugate compositions of the present invention also include modified antibodies. Such modified antibodies refer to antibodies that have been chemically or biologically modified according to the present invention. Chemically modified antibodies include those having chemical moieties attached to an amino acid backbone, or chemical moieties attached to N- or O-linked carbohydrate chains. Biologically modified antibodies include those that have undergone post-translational modifications (e.g., addition of N- or O-linked glycans, processing of the amino or carboxyl terminus, deamidation, isomerization of aspartic acid, oxidation of methionine, etc.), or those to which a methionine residue has been added to the amino terminus by expression using prokaryotic host cells. Furthermore, antibodies labeled to enable detection or isolation of the antibody or antigen according to the present invention, such as enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies, are also included in the meaning of such modified antibodies. Such modified antibodies according to the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and for detection or isolation of antibodies or antigens.

[0073] Furthermore, antibody-dependent cytotoxic activity can be enhanced by regulating the glycosylation (glycosylation, defucoseation, etc.) of the antibody bound to the antibody according to the present invention. Known techniques for regulating antibody glycosylation include, but are not limited to, International Publication No. 99 / 54342, International Publication No. 00 / 61739, and International Publication No. 02 / 31140. The antibody according to the present invention also includes antibodies in which such glycosylation has been regulated.

[0074] It is known that antibodies produced in mammalian cultured cells have a deletion of the lysine residue at the carboxyl terminus of their heavy chain (Journal of Chromatography A, 705: 129-134 (1995)), and also that two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain are deleted, and a proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector function (such as complement activation or antibody-dependent cell-mediated cytotoxicity) of the antibody. Therefore, the antibodies according to the present invention include antibodies that have undergone such modifications and functional fragments of such antibodies, as well as deletions in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and amidated deletions (for example, heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as antigen-binding ability and effector function are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody according to the present invention are not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be one of the full-length heavy chains and heavy chains selected from the group consisting of the above-mentioned deletions, or a combination of either two types. The quantity ratio of each deletion may be affected by the type of mammalian cultured cell that produces the antibody according to the present invention and the culture conditions, but the antibody according to the present invention preferably has a deletion of one amino acid residue at the carboxyl terminal in both of the two heavy chains.

[0075] Examples of antibody isotypes according to the present invention include IgG (IgG1, IgG2, IgG3, IgG4), but IgG1 or IgG2 are preferred.

[0076] The antibodies that can be used in the production of the antibody-drug conjugate composition of the present invention are not particularly limited, but examples include anti-TROP2 antibody, anti-B7-H3 antibody, anti-HER2 antibody, anti-HER3 antibody, anti-CD3 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody, anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 antibody, anti-FOLR1 antibody, anti-VEGF antibody, anti-CD20 antibody, and anti- Examples of antibodies that can be used include CD22 antibody, anti-CD70 antibody, anti-PSMA antibody, anti-CEA antibody, anti-Mesothelin antibody, anti-A33 antibody, anti-CanAg antibody, anti-Cripto antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-GPR20 antibody, and anti-CDH6 antibody. Preferably, anti-TROP2 antibody and anti-B7-H3 antibody can be used.

[0077] In the present invention, "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor-associated calcium signal transducer 2; EGP-1) and, preferably, has the activity to be internalized in TROP2-expressing cells by binding to TROP2.

[0078] Examples of anti-TROP2 antibodies include the antibody described in International Publication No. 2015 / 098099. Preferably, an antibody comprising a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 5 (the amino acid sequence described in SEQ ID NO: 1, amino acid numbers 50 to 54), CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 6 (the amino acid sequence described in SEQ ID NO: 1, amino acid numbers 69 to 85), and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 7 (the amino acid sequence described in SEQ ID NO: 1, amino acid numbers 118 to 129), and a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 8 (the amino acid sequence described in SEQ ID NO: 2, amino acid numbers 44 to 54), CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 9 (the amino acid sequence described in SEQ ID NO: 2, amino acid numbers 70 to 76), and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 10 (the amino acid sequence described in SEQ ID NO: 2, amino acid numbers 109 to 117), can be mentioned. More preferably, an antibody comprising a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 140) and a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 129) can be mentioned. More preferably, an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234), or an antibody in which a lysine residue is deleted at the carboxyl terminus of the heavy chain of the antibody.

[0079] In the present invention, "anti-B7-H3 antibody" refers to an antibody that specifically binds to B7-H3 (B cell antigen #7 homolog 3; PD-L3; CD276) and, preferably, has the activity to be internalized in B7-H3-expressing cells by binding to B7-H3.

[0080] An example of an anti-B7-H3 antibody is M30-H1-L4 (International Publication No. 2014 / 057687). Preferably, an antibody comprising a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 11 (amino acid sequences described by amino acid numbers 50 to 54 in SEQ ID NO: 3), CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 12 (amino acid sequences described by amino acid numbers 69 to 85 in SEQ ID NO: 3), and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 13 (amino acid sequences described by amino acid numbers 118 to 130 in SEQ ID NO: 3), and a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 14 (amino acid sequences described by amino acid numbers 44 to 53 in SEQ ID NO: 4), CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 15 (amino acid sequences described by amino acid numbers 69 to 75 in SEQ ID NO: 4), and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 16 (amino acid sequences described by amino acid numbers 108 to 116 in SEQ ID NO: 4), can be mentioned. More preferably, an antibody comprising a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 141) and a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 128) can be mentioned. More preferably, an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 471) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 233), or an antibody in which a lysine residue is deleted at the carboxyl terminus of the heavy chain of the antibody.

[0081] In the present invention, "anti-TROP2 antibody-drug conjugate composition" refers to an antibody-drug conjugate composition produced by the present invention in which the antibody is an anti-TROP2 antibody.

[0082] In the present invention, "anti-B7-H3 antibody-drug conjugate composition" refers to an antibody-drug conjugate composition produced by the present invention in which the antibody is an anti-B7-H3 antibody.

[0083] 3. Drug linker intermediates used in the manufacture of antibody-drug conjugate compositions The drug linker intermediate used in the production of the antibody-drug conjugate composition of the present invention is not particularly limited as long as it is a compound that reacts with the interchain thiol of the antibody, but is preferably a compound having an N-substituted maleidyl group, and more preferably a compound represented by the following formula.

[0084] [ka]

[0085] The above drug linker intermediate can be represented by the chemical name N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide, and can be manufactured by referring to the descriptions in International Publication No. 2014 / 057687, International Publication No. 2015 / 098099, and International Publication No. 2019 / 044947, etc.

[0086] 4. Conjugation of antibodies and drug linker intermediates In the present invention, the conjugation of an antibody and a drug linker intermediate is (i) A step of reacting an antibody with a reducing agent to obtain an antibody having a thiol group; then, (ii) The step of reacting the antibody having a thiol group obtained in (i) with a drug linker intermediate; Step (i) is characterized by continuing until the ratio of antibodies having four heavy-chain-light-chain thiols and the ratio of antibodies having four heavy-chain-heavy-chain thiols reach a steady state (equilibriumization of the reduction reaction). This makes it possible to stably produce antibody-drug conjugate compositions containing isomers of each antibody-drug conjugate with different drug binding sites in a fixed ratio.

[0087] In the present invention, "reaching a steady state" means that, over time, the composition ratio (%) becomes a constant value (it may fluctuate within a range that can be recognized as substantially constant, preferably fluctuating within a range of ±5%, more preferably within a range of ±4%, even more preferably within a range of ±3%, even more preferably within a range of ±2%, and even more preferably within a range of ±1%).

[0088] Furthermore, the proportion of antibodies having four heavy-light chain interchain thiols at each reaction time can be considered to be the same as the proportion of D4-1 in the antibody-drug conjugate composition produced over that reaction time (content of D4-1 / (content of D4-1 + content of D4-2) × 100).

[0089] Furthermore, the proportion of antibodies having four heavy chain-inter-heavy chain thiols at each reaction time can be considered to be the same as the proportion of D4-2 in the antibody-drug conjugate composition produced over that reaction time (content of D4-2 / ​​(content of D4-1 + content of D4-2) × 100).

[0090] Therefore, the time it takes for the composition ratio of antibodies having four heavy-chain-light-chain thiols to reach a steady state can be determined, for example, by tracking the time course of the composition ratios of D4-1 and D4-2 in the manufactured antibody-drug conjugate composition and confirming the time at which these reach a constant value (they may fluctuate within a range that can be recognized as substantially constant, preferably fluctuating within a range of ±5%, more preferably within a range of ±4%, even more preferably within a range of ±3%, even more preferably within a range of ±2%, and even more preferably within a range of ±1%).

[0091] Step (i) can preferably be carried out over 2 hours or more, more preferably over 4 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, or 31 hours or more, even more preferably over 5 hours or more, 12 hours or more, 20 hours or more, 24 hours or more, or 48 hours or more, even more preferably over 6 hours or more, 12 hours or more, 20 hours or more, 24 hours or more (or 36 hours or more), or 48 hours or more, and including the upper limit, preferably over 2 to 168 hours It can be done over a period of 4 to 50 hours, 12 to 48 hours, 16 to 48 hours, 20 to 51 hours, or 31 to 50 hours; more preferably over a period of 5 to 50 hours, 12 to 48 hours, 20 to 51 hours, 24 to 48 hours, or 48 to 50 hours; and more preferably over a period of 6 to 50 hours, 12 to 48 hours, 20 to 51 hours, 24 to 48 hours (or 36 to 48 hours), or 48 to 50 hours.

[0092] Step (i) can preferably be carried out at -5°C to 35°C, more preferably at 0°C to 20°C, even more preferably at 5°C to 20°C, and even more preferably at 5°C to 10°C, or about 10°C. In the present invention, "about 10°C" preferably means 8°C to 12°C, more preferably 9°C to 11°C, and even more preferably 10°C to 11°C, or 10°C.

[0093] If step (i) is carried out at 20°C, step (i) can preferably be carried out for 4 hours or more, more preferably for 5 hours or more, even more preferably for 6 hours or more, and including the upper limit, it can preferably be carried out for 4 to 50 hours, more preferably for 5 to 50 hours, and even more preferably for 6 to 50 hours.

[0094] If step (i) is carried out at 10°C, step (i) can preferably be carried out for 12 hours or more, or 20 hours or more, and preferably for 12 to 48 hours, or 20 to 51 hours, including the upper limit.

[0095] If step (i) is carried out at 5°C, step (i) can preferably be carried out for 16 hours or more, more preferably for 20 hours or more, even more preferably for 24 hours or more (or 36 hours or more), and including the upper limit, it can preferably be carried out for 16 to 48 hours, more preferably for 20 to 48 hours, and even more preferably for 24 to 48 hours (or 36 to 48 hours).

[0096] If step (i) is performed at 0°C, step (i) can preferably be performed for 31 hours or more, more preferably for 48 hours or more, and including the upper limit, it can preferably be performed for 31 to 50 hours, and more preferably for 48 to 50 hours.

[0097] The reaction time and reaction temperature in step (i) can be further optimized depending on the type of antibody in the antibody-drug conjugate composition being produced.

[0098] In other words, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-TROP2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234), or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), then step (i) can preferably be carried out at approximately 10°C for 20 hours or more, and, including the upper limit, can preferably be carried out at approximately 10°C for 20 to 51 hours.

[0099] Furthermore, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-B7-H3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 and amino acid sequences described in SEQ ID NO: 4 and amino acid sequence consisting of amino acid sequences described in SEQ ID NO: 4, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), then step (i) can preferably be carried out at 5-10°C (more preferably at 6°C, 7°C, 8°C, 9°C, or 10°C) for 12 hours or more, or at 5°C for 20 hours or more, and including the upper limit, it can preferably be carried out at 5-10°C (more preferably at 6°C, 7°C, 8°C, 9°C, or 10°C) for 12-48 hours, or at 5°C for 20-48 hours.

[0100] The reducing agent used in step (i) is not particularly limited as long as it can reduce the interchain disulfide of the antibody, but for example, tris(2-carboxyethyl)phosphine or its salt, dithiothreitol, or 2-mercaptoethanol can be used, preferably tris(2-carboxyethyl)phosphine or its salt can be used, and more preferably tris(2-carboxyethyl)phosphine hydrochloride can be used.

[0101] The equivalent amount of reducing agent used in step (i) per molecule of antibody (hereinafter, in this invention, "equivalent amount" means molar equivalent) can preferably be 1.9 to 2.5 equivalents per molecule of antibody, and more preferably 2 to 2.2 equivalents per molecule of antibody.

[0102] The solvent used in step (i) is not particularly limited as long as it allows the reaction to proceed, but preferably, an aqueous solution of L-histidine can be used.

[0103] The solvent used in step (i) can be further optimized depending on the type of antibody in the antibody-drug conjugate composition being produced.

[0104] In other words, if the antibody in the antibody-drug conjugate composition to be produced is an anti-TROP2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234), or an antibody in which the lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), then the solvent used in step (i) is preferably a 0.001 mol / L to 0.1 mol / L aqueous solution of L-histidine, more preferably a 0.005 mol / L to 0.02 mol / L aqueous solution of L-histidine, and even more preferably a 0.01 mol / L aqueous solution of L-histidine. The pH of the reaction solution in step (i) can preferably be adjusted using an aqueous solution of disodium hydrogen phosphate, and the pH is preferably 6.4 to 7.4, more preferably 6.7 to 7.1, and even more preferably 6.9.

[0105] Furthermore, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-B7-H3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 and amino acid sequences described in SEQ ID NO: 4 and amino acid sequence consisting of amino acid sequences described in SEQ ID NO: 4, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), then the solvent used in step (i) is preferably a 0.005 mol / L to 0.3 mol / L aqueous solution of L-histidine, more preferably a 0.025 mol / L to 0.1 mol / L aqueous solution of L-histidine, and even more preferably a 0.05 mol / L aqueous solution of L-histidine.

[0106] The reaction solution in step (i) may contain a buffer derived from antibody production.

[0107] Step (i) is preferably carried out in the presence of a chelating agent. The chelating agent is not particularly limited as long as it can be used in the reduction of interchain disulfides of the antibody, but for example, ethylenediaminetetraacetic acid (hereinafter also referred to as "EDTA"), diethylenetriaminepentaacetic acid, or glycol etherdiaminetetraacetic acid can be used, and ethylenediaminetetraacetic acid can preferably be used.

[0108] The chelating agent can preferably be used in an amount of 1 to 20 equivalents per antibody molecule, more preferably in an amount of 3 to 8 equivalents per antibody molecule, even more preferably in an amount of 4 to 6 equivalents per antibody molecule, and even more preferably in an amount of 5 or 6 equivalents per antibody molecule.

[0109] The buffer used in step (i) may contain a surfactant. In the present invention, "surfactant" refers to a substance having hydrophilic and hydrophobic groups that can be used as one of the components of a pharmaceutical preparation. Examples of such surfactants include polysorbate (including polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and polysorbate 60 (Tween 60)), polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene castor oil, or sodium lauryl sulfate, and more preferably polysorbate 20 or polysorbate 80.

[0110] The type of surfactant used in step (i) can be appropriately selected depending on the type of antibody in the antibody-drug conjugate composition to be manufactured.

[0111] For example, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-TROP2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234), or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), then the buffer used in step (i) may preferably contain polysorbate 80.

[0112] Furthermore, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-B7-H3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 to 21 to 233, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), then the buffer used in step (i) may preferably contain polysorbate 20.

[0113] The drug linker intermediate used in step (ii) can preferably be used in an amount of 4 to 5 equivalents per antibody molecule, more preferably in an amount of 4.2 to 4.6 equivalents per antibody molecule, and even more preferably in an amount of 4.4 equivalents per antibody molecule.

[0114] The drug linker intermediate used in step (ii) can preferably be added to the reaction solution obtained in step (i) in a state of being dissolved in a solvent. The solvent is not particularly limited as long as it can be used in the binding reaction with the antibody, but preferably dimethyl sulfoxide, aqueous dimethyl sulfoxide, acetone, or aqueous acetone can be used, more preferably aqueous dimethyl sulfoxide can be used, and even more preferably 80% aqueous dimethyl sulfoxide can be used. Furthermore, these solvents can preferably be used in a state containing acetic acid, and more preferably in a state containing aqueous acetic acid.

[0115] Step (ii) can preferably be carried out at 0 to 20°C, more preferably at 5 to 20°C, and even more preferably at 5 to 10°C, or about 10°C.

[0116] The reaction time for step (ii) is preferably 20 minutes to 4 hours, and more Preferably, the duration is 0.5 to 2 hours.

[0117] After step (ii), a reagent having a thiol group may be used to quench the excess drug linker intermediate.

[0118] The reagent having a thiol group is not particularly limited as long as it can react with the maleidyl group of the drug linker intermediate, but for example, N-acetylcysteine ​​and cysteine ​​can be used, and preferably N-acetylcysteine ​​can be used.

[0119] The reagent containing the thiol group can preferably be used in amounts of 2 to 8 equivalents, more preferably in amounts of 3 to 7 equivalents, and even more preferably in amounts of 3 to 5 equivalents.

[0120] The step of adding a reagent having a thiol group can preferably be carried out at 0 to 20°C, more preferably at 5 to 20°C, and even more preferably at 5 to 10°C, or about 10°C.

[0121] The resulting antibody-drug conjugate composition can be purified by ultrafiltration to remove impurities derived from the drug linker intermediate.

[0122] In the present invention, "ultrafiltration" refers to a purification method that separates large solute molecules from small solute molecules, or solute molecules from solvent molecules, by filtering through a membrane (ultrafiltration membrane) having a pore size in the range of approximately 0.001 μm to approximately 0.05 μm. Ultrafiltration membranes generally have a molecular weight cutoff (MWCO) in the range of 1 kDa to 1000 kDa. MWCO is generally defined as the molecular weight of spherical solutes that are retained by 90% of the membrane. The ultrafiltration of the present invention can preferably be performed using an ultrafiltration membrane with an MWCO of 1 kDa to 100 kDa, and more preferably using an ultrafiltration membrane with an MWCO of 30 kDa. Examples of materials for the ultrafiltration membrane include regenerated cellulose, cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyethersulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, nylon, and ceramic. The ultrafiltration of the present invention can preferably be performed using an ultrafiltration membrane made of regenerated cellulose, but is not limited thereto. Examples of ultrafiltration membranes used in the present invention include Pellicon® XL Cassette Ultracel® (manufactured by Merck), Pellicon® 2 Ultracel® (manufactured by Merck), and Pellicon® 3 Ultracel® (manufactured by Merck). Ultrafiltration specifically refers to a method of forced filtration by pressure or centrifugal operation, while a method of filtration by passive diffusion is sometimes called "dialysis filtration." However, any method using an ultrafiltration membrane is included in "ultrafiltration" in the present invention.

[0123] The average number of drug-bound antibodies in the antibody-drug conjugate composition produced by the present invention is preferably 2 to 6, more preferably 3 to 5, even more preferably 3.5 to 4.5, and even more preferably about 4. In the present invention, "about 4" is preferably 3.8 to 4.2, more preferably 3.9 to 4.1, and even more preferably 4.

[0124] Furthermore, the content of the antibody-drug conjugate (D4) with four drug linkers attached in the antibody-drug conjugate composition produced by the present invention is preferably 50% or more, and including the upper limit, it is preferably 50-59%, 50-60%, 50-70%, 50-80%, 50-90%, or 50-100%.

[0125] Furthermore, in the antibody-drug conjugate composition produced by the present invention, the proportion of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain interchain thiol is preferably 1.5 to 2.5 times, and more preferably 1.5 to 2.33 times, the proportion of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain interchain thiol.

[0126] In other words, the ratio of the content of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain-light-chain thiol to the content of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain-heavy-chain thiol in the antibody-drug conjugate composition produced by the present invention is preferably 60:40 to 71:29, and more preferably 60:40 to 70:30. 5. Characterization of Antibody-Drug Conjugate Compositions The average number of drug-bound molecules per antibody in a manufactured antibody-drug conjugate composition can be calculated, for example, by measuring the UV absorbance of the antibody-drug conjugate and its conjugation precursor at two wavelengths, 280 nm and 370 nm (UV method), or by treating the antibody-drug conjugate with a reducing agent and quantifying each resulting fragment by HPLC measurement (HPLC method) (see International Publication Nos. 2014 / 057687, 2015 / 098099, and 2017 / 002776, etc.). The measurement conditions for the HPLC method can be optimized depending on the type of antibody in the manufactured antibody-drug conjugate composition. (1-1) Preparation of samples for HPLC analysis (reduction of antibody-drug conjugates) A diluted solution (60 μL) of the antibody-drug conjugate composition is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture is incubated at 37°C for 20 or 30 minutes to cleave the disulfide between the antibody-drug conjugate chains. The resulting sample is then used for HPLC analysis.

[0127] (1-2) HPLC analysis If the antibody in the antibody-drug conjugate composition to be manufactured is an anti-TROP2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234), or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the process can be carried out, for example, as follows.

[0128] Measurement device: High-performance liquid chromatograph Detector: UV absorbance meter (measurement wavelength: 280 nm) Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Agilent Technologies) Column temperature: 80℃ Mobile phase A: 0.05% trifluoroacetic acid (TFA) aqueous solution Mobile phase B: Acetonitrile solution containing 0.04% TFA Gradient program (mobile phase B): 27%-36% (0 min-12.5 min), 36%-42% (12.5 min-15 min), 42%-27% (15 min-15.1 min), 27%-27% (15.1 min-25 min)

[0129] Furthermore, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-B7-H3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 to 21 to 233, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the process can be carried out, for example, as follows.

[0130] Measurement device: High-performance liquid chromatograph Detector: UV absorbance meter (measurement wavelength: 280 nm) Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 Å; Agilent Technologies) Column temperature: 80℃ Mobile phase A: 0.05% trifluoroacetic acid (TFA) aqueous solution Mobile phase B: Acetonitrile solution containing 0.04% TFA Gradient program (mobile phase B): 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)

[0131] (1-3) Data Analysis Compared to the light chain (L0) and heavy chain (H0) of an antibody without a bound drug, the light chain (L1) and heavy chain (H1) bound to a single drug, the heavy chain (H2) bound to two drugs, and H3) bound to three drugs become more hydrophobic and retain longer in proportion to the number of bound drugs. Therefore, they elute in the order of L0, L1, H0, H1, H2, and H3. By comparing the retention times of L0 and H0, the detection peak can be assigned to one of L0, L1, H0, H1, H2, or H3.

[0132] Because drug linkers absorb UV light, the peak area value is corrected according to the following formula using the molar extinction coefficients of the light chain, heavy chain, and drug linkers, depending on the number of drug linker bonds.

[0133]

number

[0134]

number

[0135] Here, the molar extinction coefficients (280 nm) of the light and heavy chains in each antibody can be estimated from the amino acid sequences of the light and heavy chains of each antibody using a known calculation method (Protein Science, 1995, vol.4, 2411-2423).

[0136] For example, if the antibody is an anti-TROP2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234)), then 27640 can be used as the estimated molar extinction coefficient of the light chain and 83810 as the estimated molar extinction coefficient of the heavy chain.

[0137] Furthermore, if the antibody is an anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 471) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 233)), then 30160 can be used as the estimated molar extinction coefficient of the light chain and 87250 as the estimated molar extinction coefficient of the heavy chain.

[0138] The molar extinction coefficient (280 nm) of the drug linker can be obtained by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine ​​to convert the maleimidyl group to succinimidothioether, and then using the measured molar extinction coefficient (280 nm) of the compound obtained.

[0139] The ratio (%) of each chain's peak area to the total peak area correction value is calculated according to the following formula.

[0140]

number

[0141] The average number of drug conjugates in an antibody-drug conjugate composition is calculated according to the following formula.

[0142] Average number of drug-bound cells = (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

[0143] The content of each isomer with different drug binding numbers and positions in the manufactured antibody-drug conjugate composition can be calculated, for example, by separating the elution times of each isomer using HPLC and measuring the area value of each isomer's content (see International Publication No. 2017 / 002776, etc.). The HPLC method can be optimized depending on the type of antibody in the manufactured antibody-drug conjugate composition.

[0144] (2-1)HPLC analysis A diluted solution of the antibody-drug conjugate composition is used for HPLC analysis. Alternatively, a sample prepared by incubating the diluted solution at 37°C for 60 minutes is used for HPLC analysis.

[0145] If the antibody in the antibody-drug conjugate composition to be manufactured is an anti-TROP2 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234), or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the process can be carried out, for example, as follows.

[0146] Measurement device: High-performance liquid chromatograph Detector: UV absorbance meter (measurement wavelength: 280 nm) Column: TSK-gel Butyl-NPR (4.6 x 35 mm, 2.5 μm; Tosoh Corporation) Column temperature: 25℃ Mobile phase A: 30 mM phosphate buffer (pH 7.0) containing 1.5 M ammonium sulfate. Mobile phase B: A mixed solution containing 90% 30 mM phosphate buffer (pH 7.0) and 10% isopropyl alcohol. Gradient program (mobile phase B): 25%-65% (0 min-15 min), 65%-95% (15-15.1 min), 95% (15.1-18 min), 95%-25% (18 min-18.1 min), 25% (18.1 min-25 min)

[0147] Furthermore, if the antibody in the antibody-drug conjugate composition to be manufactured is an anti-B7-H3 antibody (preferably an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 to 21 to 233, or an antibody in which a lysine residue at the carboxyl terminus of the heavy chain of the antibody is deleted), the process can be carried out, for example, as follows.

[0148] Measurement device: High-performance liquid chromatograph Detector: UV absorbance meter (measurement wavelength: 280 nm) Column: TSK-gel Butyl-NPR (4.6 x 35 mm, 2.5 μm; Tosoh Corporation) Column temperature: 25℃ Mobile phase A: 30 mM phosphate buffer (pH 7.0) containing 1.5 M ammonium sulfate. Mobile phase B: A mixed solution containing 90% 30 mM phosphate buffer (pH 7.0) and 10% isopropyl alcohol. Gradient program (mobile phase B): 20%-64% (0 min-15 min), 64%-95% (15-15.1 min), 95% (15.1-18 min), 95%-20% (18 min-18.1 min), 20% (18.1 min-25 min)

[0149] (2-2) Data Analysis Under the HPLC separation conditions of (2-1), elution occurs in the order of D0, D2, D4-1, D4-2, D6, and D8, depending on the difference in salt concentration. Since the drug linker has UV absorption, the peak area value is corrected according to the following formula using the molar extinction coefficients of the antibody and drug linker, depending on the number of drug linkers bound.

[0150]

number

[0151] Here, the molar extinction coefficients (280 nm) of the light and heavy chains in each antibody can be estimated from the amino acid sequence of each antibody using a known calculation method (Protein Science, 1995, vol.4, 2411-2423).

[0152] For example, if the antibody is an anti-TROP2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234)), then 223400 can be used as an estimated value for the molar extinction coefficient of the antibody.

[0153] Furthermore, if the antibody is an anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 471) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 233)), then 235320 can be used as an estimated value for the molar extinction coefficient of the antibody.

[0154] The molar extinction coefficient (280 nm) of the drug linker can be obtained by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine ​​to convert the maleimidyl group to succinimidothioether, and then using the measured molar extinction coefficient (280 nm) of the compound obtained.

[0155] The content of each isomer can be calculated by calculating the ratio (%) of each peak area to the total peak area correction value according to the following formula.

[0156]

number

[0157] 6. Pharmaceutical Compositions The antibody-drug conjugate composition produced by the present invention may be administered containing one or more pharmaceutically compatible components. The pharmaceutically compatible components can be appropriately selected from pharmaceutical additives and other substances commonly used in this field, depending on the dosage and concentration of the antibody-drug conjugate composition produced by the present invention. For example, the antibody-drug conjugate composition produced by the present invention may be administered as a pharmaceutical composition containing a buffer such as a histidine buffer, an excipient such as sucrose or trehalose, and a surfactant such as polysorbate 80 or polysorbate 20.

[0158] The pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention can be applied to patients as systemic therapy, as well as applied topically to cancer tissue to produce therapeutic effects.

[0159] A pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention can be suitably used in mammals, and more preferably in humans.

[0160] A pharmaceutical composition comprising the antibody-drug conjugate composition produced by the present invention can preferably be used as an injectable preparation, more preferably as an aqueous injectable preparation or a lyophilized injectable preparation, and even more preferably as a lyophilized injectable preparation.

[0161] When the pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention is an aqueous injection, it can preferably be administered intravenously by drip infusion after being diluted with an appropriate diluent. Examples of diluents include glucose solution (preferably 5% glucose solution) and physiological saline solution.

[0162] When the pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention is a lyophilized injectable preparation, it can preferably be dissolved in sterile water for injection, diluted in an appropriate diluent as needed, and then administered intravenously by drip infusion. Examples of diluents include glucose solution (preferably 5% glucose solution) and physiological saline solution.

[0163] Examples of routes of delivery that can be used to administer a pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention include intravenous, intradermal, subcutaneous, intramuscular, and intraperitoneal routes, with intravenous routes being preferred.

[0164] The antibody-drug conjugate composition produced by the present invention can be administered to humans at intervals of 1 to 180 days, preferably at intervals of 1, 2, 3, or 4 weeks, and more preferably at intervals of 3 weeks. Furthermore, the antibody-drug conjugate composition produced by the present invention can be administered in doses of approximately 0.001 to 100 mg / kg per dose, preferably at doses of 0.8 to 12.4 mg / kg per dose. When the antibody-drug conjugate composition used in the present invention is an anti-TROP2 antibody-drug conjugate composition, doses of 0.27 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6.0 mg / kg, or 8.0 mg / kg per dose can be administered at intervals of 3 weeks. When the antibody-drug conjugate composition used in the present invention is an anti-B7-H3 antibody-drug conjugate composition, preferably, a dose of 0.8 mg / kg, 1.6 mg / kg, 3.2 mg / kg, 4.8 mg / kg, 6.4 mg / kg, or 8.0 mg / kg can be administered at intervals of once every three weeks.

[0165] Pharmaceutical compositions comprising antibody-drug conjugate compositions produced by the present invention can be used for the treatment of cancer, preferably breast cancer (including triple-negative breast cancer and luminal breast cancer), gastric cancer (sometimes called gastric adenocarcinoma), colorectal cancer (sometimes called colorectal cancer, including colon cancer and rectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), esophageal cancer, head and neck cancer (including salivary gland cancer and pharyngeal cancer), and gastroesophageal junction cancer. It can be used for the treatment of at least one cancer selected from the group consisting of biliary tract cancer (including bile duct cancer), Paget's disease, pancreatic cancer, ovarian cancer, uterine carcinosarcoma, urothelial carcinoma, prostate cancer, bladder cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, endometrial cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, glioblastoma pleomorphoni, sarcoma, osteosarcoma, and melanoma.

[0166] The pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention can be selectively used as an agent for drug therapy, a major treatment for cancer, and as a result can slow the growth of cancer cells, suppress proliferation, and even destroy cancer cells. Through these actions, cancer patients can achieve relief from cancer-related symptoms and an improvement in quality of life (QOL), thus achieving therapeutic effects while preserving the lives of cancer patients. Even if the destruction of cancer cells is not achieved, the suppression and control of cancer cell proliferation can lead to longer survival while achieving a higher QOL in cancer patients.

[0167] In addition to the use of drugs alone in such drug therapies, pharmaceutical compositions containing antibody-drug conjugate compositions manufactured according to the present invention can also be used in combination with other therapies in adjuvant therapy, such as surgery, radiation therapy, and hormone therapy. Furthermore, they can also be used as drug therapies in neoadjuvant therapy.

[0168] In addition to the therapeutic uses described above, pharmaceutical compositions containing the antibody-drug conjugate composition produced by the present invention can also be expected to have preventive effects, such as suppressing and even destroying the proliferation of minute metastatic cancer cells. For example, it can be expected to have an effect of suppressing and destroying cancer cells in bodily fluids during the metastatic process, or suppressing and destroying minute cancer cells immediately after implantation in any tissue. Therefore, it can be expected to have an inhibitory and preventive effect on cancer metastasis, especially after surgical removal of cancer.

[0169] The pharmaceutical composition containing the antibody-drug conjugate composition produced by the present invention can be administered in combination with other cancer treatment agents, thereby enhancing the antitumor effect. Other cancer treatment agents used for this purpose include irinotecan (CPT-11), cisplatin, carboplatin, oxaliplatin, fluorouracil (5-FU), gemcitabine, capecitabine, paclitaxel, docetaxel, doxorubicin, epirubicin, cyclophosphamide, and mitomycin C. C) Tegafur / Gimeracil / Oteracil combination drug, Cetuximab, Panitumumab, Bevacizumab, Ramucirumab, Regorafenib, Trifluridine / Tipiracil combination drug, Gefitinib, Erlotinib, Afatinib Afatinib, Osimertinib, Methotrexate, Pemetrexed, Tamoxifen, Toremifene, Fulvestrant, Leuprorelin, Goserelin, Letrozole, Anastrozole, Progesterone formulation, Trastuzumab emtansineExamples include emtansine, trastuzumab, pertuzumab, lapatinib, nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, ipilimumab, tremelimumab, olaparib, rucaparib, niraparib, talazoparib, and veliparib, but the list is not limited to drugs with antitumor activity. [Examples]

[0170] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0171] [Example 1] Tracking the time course of the composition ratios of D4-1 and D4-2 in the production of an anti-TROP2 antibody-drug conjugate composition. A solution containing anti-TROP2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234)) (equivalent to 500 mg of antibody) was placed in a polypropylene tube. Then, 0.1 g / g polysorbate 80 aqueous solution (50 μL), 0.5 mol / L EDTA aqueous solution (5 equivalents relative to the antibody), and 0.01 mol / L L-histidine aqueous solution (38 mL) were added, and then 0.3 mol / L disodium hydrogen phosphate aqueous solution was added to adjust the pH to 6.9. Next, an aqueous solution containing 1 mg / g tris(2-carboxyethyl)phosphine hydrochloride (2.12 g; 2.15 equivalents per molecule of antibody) was added at 0°C, 10°C, or 20°C under stirring, and the mixture was stirred at the same temperature to produce an antibody containing thiol groups. After 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 20 hours, 22 hours, 24 hours, 25 hours, 28 hours, 31 hours, 44 hours, 48 ​​hours, 50 hours, or 51 hours, a portion of the reaction solution is taken and the formula is applied to it.

[0172] [ka]

[0173] The compound shown in the formula was added and reacted with an antibody having a thiol group.

[0174] [ka]

[0175] (In the formula, A indicates the binding site with the antibody.) An anti-TROP2 antibody-drug conjugate composition was obtained in which a drug linker, indicated by [the symbol], and an anti-TROP2 antibody were linked by a thioether bond.

[0176] The time course of the reaction was tracked by measuring the content of D4 in D0-D8 and the composition ratio of D4-1 and D4-2 within D4 in the obtained anti-TROP2 antibody-drug conjugate composition.

[0177] The results at a reaction temperature of 0°C are shown in Table 1.

[0178] [Table 1]

[0179] The proportion of D4-1 decreased with increasing reaction time, while the proportion of D4-2 increased with increasing reaction time. It was confirmed that the proportions of D4-1 and D4-2 reached a constant value after a reaction time of 31 hours or more (preferably 48 hours or more). From this, it was confirmed that at a reaction temperature of 0°C, the proportion of antibodies having four heavy-chain interchain thiols and the proportion of antibodies having four heavy-chain interchain thiols reach steady-state values ​​after 31 hours or more (preferably 48 hours or more). The results at a reaction temperature of 10°C are shown in Table 2.

[0180] [Table 2]

[0181] The proportion of D4-1 decreased with increasing reaction time, while the proportion of D4-2 increased with increasing reaction time. It was confirmed that the proportions of D4-1 and D4-2 reached constant values ​​after 20 hours of reaction time. From this, it was confirmed that at a reaction temperature of 10°C, the proportion of antibodies with four heavy-chain interchain thiols and the proportion of antibodies with four heavy-chain interchain thiols reach steady-state values ​​after 20 hours. The results at a reaction temperature of 20°C are shown in Table 3.

[0182] [Table 3]

[0183] The D4 content was slightly lower at 0°C and 10°C than at 20°C, and showed a slight decreasing trend as the reaction time increased. The proportion of D4-1 decreased with increasing reaction time, while the proportion of D4-2 increased with increasing reaction time. It was confirmed that the proportions of D4-1 and D4-2 reached constant values ​​after a reaction time of 4 hours or more (preferably 5 hours or more, more preferably 6 hours or more). From this, it was confirmed that at a reaction temperature of 20°C, the proportion of antibodies with four heavy-chain interchain thiols and the proportion of antibodies with four heavy-chain interchain thiols reached steady-state values ​​after 4 hours or more (preferably 5 hours or more, more preferably 6 hours or more). Figure 5 shows the change over time in the content of D4 in D0 to D8 as a result of reaction temperatures of 0°C, 10°C, and 20°C, and Figure 6 shows the change over time in the composition ratio of D4-1 and D4-2 in D4.

[0184] [Example 2] Tracking the time course of the composition ratios of D4-1 and D4-2 in the production of an anti-B7-H3 antibody-drug conjugate composition.

[0185] A solution containing anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 471) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 233)) (equivalent to 250 mg of antibody) was placed in a polypropylene tube, and then polysorbate 20 (2.9 mg), 0.5 mol / L EDTA aqueous solution (6 equivalents relative to the antibody), and 0.05 mol / L L-histidine aqueous solution (12.5 mL) were added. Next, under stirring at 5°C or 10°C, an aqueous solution containing tris(2-carboxyethyl)phosphine hydrochloride (1.03 mg; 2.1 equivalents per antibody molecule) was added, and the mixture was shaken at the same temperature to produce an antibody containing a thiol group. After 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, or 48 hours, a portion of the reaction solution was taken and the formula was applied.

[0186] [ka]

[0187] A compound represented by was added and reacted with an antibody having a thiol group. Accordingly, the formula

[0188]

Chemical Formula

[0189] (wherein, A represents the binding position to the antibody) An anti-B7-H3 antibody-drug conjugate composition, in which the drug linker represented by and the anti-B7-H3 antibody are bound via a thioether bond, was obtained.

[0190] Changes in the reaction over time were tracked by measuring the content of D4 among D0 to D8 and the constituent ratio of D4-1 and D4-2 in D4 in the obtained anti-B7-H3 antibody-drug conjugate composition. The results at a reaction temperature of 5°C are shown in Table 4.

[0191]

Table 4

[0192] The constituent ratio of D4-1 decreased as the reaction time increased, and the constituent ratio of D4-2 increased as the reaction time increased. It was confirmed that when the reaction time is 16 hours or longer (preferably 20 hours or longer, more preferably 24 hours or longer (or 36 hours or longer)), the constituent ratios of D4-1 and D4-2 come to exhibit constant values. From this, it was confirmed that when the reaction temperature is 5°C, after 16 hours or longer (preferably 20 hours or longer, more preferably 24 hours or longer (or 36 hours or longer)), the constituent ratio of antibodies having four inter-heavy chain-light chain thiols and the constituent ratio of antibodies having four inter-heavy chain thiols reach steady-state values. The results at a reaction temperature of 10°C are shown in Table 5.

[0193]

Table 5

[0194] The proportion of D4-1 decreased with increasing reaction time, while the proportion of D4-2 increased with increasing reaction time. It was confirmed that the proportions of D4-1 and D4-2 reached constant values ​​after 12 hours of reaction time. From this, it was confirmed that at a reaction temperature of 10°C, the proportion of antibodies with four heavy-chain interchain thiols and the proportion of antibodies with four heavy-chain interchain thiols reached steady-state values ​​after 12 hours.

[0195] Figure 7 shows the change over time in the content of D4 in D0 to D8 as a result of reactions at reaction temperatures of 5°C and 10°C, and Figure 8 shows the change over time in the composition ratio of D4-1 and D4-2 in D4.

[0196] [Example 3] Production of an anti-TROP2 antibody-drug conjugate composition by the method of the present invention A solution containing anti-TROP2 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 20 to 470 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 20 to 234) (10.4 kg of solution weight: equivalent to 400 g of antibody) was placed in a single-use reactor. Then, 0.1 g / g polysorbate 80 aqueous solution (40 mL), 0.5 mol / L EDTA aqueous solution (28 mL; 5 equivalents relative to the antibody), and 0.01 mol / L L-histidine aqueous solution (30 kg) were added, and the pH was adjusted to 6.9 by adding 0.3 mol / L disodium hydrogen phosphate aqueous solution. Under stirring at 10-11°C, an aqueous solution containing 1 mg / g tris(2-carboxyethyl)phosphine hydrochloride (1.70 kg; 2.15 equivalents per molecule of antibody) was added and reduced. The mixture was then stirred at an internal temperature of 10-11°C for 30 hours until the composition ratio of antibodies having four heavy-chain interchain thiols reached a steady state.

[0197] The resulting reaction solution was then subjected to stirring at an internal temperature of 10°C, and the formula was applied.

[0198] [ka]

[0199] The compound indicated by (13.4 g; 4.4 equivalents per antibody molecule) was dissolved in a mixture of 10% aqueous acetic acid (7.4 mL) and 80% aqueous dimethyl sulfoxide (1.5 L), and this mixture was added over 26 minutes. The mixture was stirred at the same temperature for 50 minutes to bind to the antibody containing the thiol group. Next, 0.17 L of aqueous 0.05 M N-acetylcysteine ​​(3 equivalents per antibody molecule) was added, and the mixture was stirred at the same temperature for 1 hour. Finally, the pH was adjusted to 5 using 10% aqueous acetic acid.

[0200] The obtained solution was ultrafiltration using a Pellicon® 2 Ultracel® (Merck) roller pump while adding 0.01 mol / L histidine buffer (pH 5) to remove compound-derived impurities. The solution was concentrated and adjusted to pH 6, and the formula was obtained.

[0201] [ka]

[0202] (In the formula, A indicates the binding site with the antibody.) A solution containing an anti-TROP2 antibody-drug conjugate composition was obtained, in which a drug linker represented by [the formula shown] and an anti-TROP2 antibody were linked by a thioether bond.

[0203] Further, to the above solution, there were added a pH 6 histidine buffer in which sucrose (1.6 kg) was dissolved, a pH 6 histidine buffer, and a 0.1 g / L polysorbate 80 aqueous solution (28 g), followed by addition of a histidine buffer (pH 6) containing 9% sucrose to adjust the protein concentration to 20 g / L, thereby obtaining a pharmaceutical composition (19.0 kg) comprising an anti-TROP2 antibody-drug conjugate composition. The obtained pharmaceutical composition had a protein concentration of 20.3 g / L, a protein yield of 374 g, and an average number of drug conjugations per antibody molecule of 4.0. The content of each isomer was D0: 3%, D2: 18%, D4: 53% (of which D4-1: 60%, D4-2: 40%), D6: 20%, and D8: 4%.

[0204] [Example 4] Production of anti-B7-H3 antibody-drug conjugate composition by the method of the present invention A solution containing an anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence set forth at amino acid positions 20 to 471 of SEQ ID NO: 3 and a light chain consisting of the amino acid sequence set forth at amino acid positions 21 to 233 of SEQ ID NO: 4) (solution weight: 10.2 kg, equivalent to 200 g of antibody) was placed in a single-use reactor, and polysorbate 20 (4.6 g), 0.5 mol / L aqueous EDTA solution (18 g; 6 equivalents relative to the antibody), and 0.05 mol / L aqueous L-histidine solution (10 kg) were further added. Under stirring at 5°C, an aqueous solution containing tris(2-carboxyethyl)phosphine hydrochloride (0.831 g; 2.1 equivalents per antibody molecule) was added for reduction, followed by stirring at an internal temperature of 5°C for 4 hours and at 10°C for 15 hours, so that the constitution ratio of an antibody having four inter heavy chain-light chain thiols and the constitution ratio of an antibody having four inter heavy chain-heavy chain thiols reached a steady-state value.

[0205] To the obtained reaction solution, under stirring at an internal temperature of 9 to 11°C, a compound of formula

[0206]

Chemical Formula

[0207] The compound indicated by (6.64 g; 4.4 equivalents per antibody molecule) was dissolved in 80% dimethyl sulfoxide aqueous solution (1.1 kg) containing 10% acetic acid aqueous solution (3.7 mL), and this solution was added over 50 minutes. The mixture was stirred at the same temperature for 24 minutes to bind to the antibody containing the thiol group. Next, 0.05 M N-acetylcysteine ​​aqueous solution (0.14 kg; 5 equivalents per antibody molecule) was added, and the mixture was stirred at the same temperature for 1 hour. The pH was then adjusted to 5 using 10% acetic acid aqueous solution.

[0208] The obtained solution was ultrafiltration using a Pellicon® 2 Ultracel® (Merck) roller pump, while adding 0.01 mol / L histidine buffer (pH 5) to remove compound-derived impurities. The solution was concentrated and adjusted to pH 5.9, and the formula was obtained.

[0209] [ka]

[0210] (In the formula, A indicates the binding site with the antibody.) A solution containing an anti-B7-H3 antibody-drug conjugate composition was obtained, in which a drug linker indicated by [the formula] and an anti-B7-H3 antibody were linked by a thioether bond.

[0211] Furthermore, to the above solution, a pH 5.9 histidine buffer containing sucrose (0.83 kg) and another pH 5.9 histidine buffer were added, and then a 9% sucrose-containing histidine buffer (pH 5.9) was added to adjust the protein concentration to 20 g / L, obtaining a pharmaceutical composition (10.1 kg) containing an anti-B7-H3 antibody-drug conjugate composition. The obtained pharmaceutical composition had a protein concentration of 20.4 g / L, a protein yield of 198 g, and an average number of drug bindings per antibody molecule of 4.0. The content of each isomer was D0: 3%, D2: 20%, D4: 54% (of which D4-1: 64%, D4-2: 36%), D6: 18%, and D8: 5%.

[0212] [Example 5] Production of anti-B7-H3 antibody-drug conjugate composition by the method of the present invention A solution containing anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 20 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 21 to 233) (51.0 g liquid weight: equivalent to 1.0 g antibody) was mixed with 100 mg / g aqueous solution of polysorbate 20 (0.23 mL), 0.5 mol / L aqueous solution of EDTA (6 equivalents relative to the antibody), and 0.05 mol / L aqueous solution of L-histidine (50 mL). Under stirring at 5°C, an aqueous solution containing tris(2-carboxyethyl)phosphine hydrochloride (4.50 mg; 2.3 equivalents per antibody molecule) was added to reduce the solution, and the mixture was stirred at an internal temperature of 5°C for 24 hours until the composition ratio of antibody with 4 interchain thiols and the composition ratio of antibody with 4 interchain thiols reached steady-state values.

[0213] The resulting reaction solution is then stirred at an internal temperature of 4-6°C, and the formula is...

[0214] [ka]

[0215] The compound shown (33 mg; 4.4 equivalents per antibody molecule) was dissolved in 80% dimethyl sulfoxide aqueous solution (5.5 g) containing 10% acetic acid aqueous solution (0.018 mL), and this solution was added over 20 minutes, and the mixture was stirred at the same temperature for 60 minutes to bind to the antibody containing the thiol group. Next, 0.05 M N-acetylcysteine ​​aqueous solution (0.68 g; 5 equivalents per antibody molecule) was added, and the mixture was stirred at the same temperature for 1 hour. Then, the pH was adjusted to 5 using 10% acetic acid aqueous solution, and the formula was used.

[0216] [ka]

[0217] (In the formula, A indicates the binding site with the antibody.) A solution was obtained containing an anti-B7-H3 antibody-drug conjugate composition in which a drug linker represented by and an anti-B7-H3 antibody were linked by a thioether bond. The content of each isomer in the solution was D0: 4%, D2: 15%, D4: 54% (of which D4-1: 65%, D4-2: 35%), D6: 21%, and D8: 5%.

[0218] [Example 6] Production of an anti-B7-H3 antibody-drug conjugate composition by the method of the present invention A solution containing anti-B7-H3 antibody (an antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 20 to 471 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 21 to 233) (30.6 g by liquid weight: equivalent to 600 mg of antibody) was mixed with 100 mg / g aqueous solution of polysorbate 20 (0.14 mL), 0.5 mol / L aqueous solution of EDTA (6 equivalents relative to the antibody), and 0.05 mol / L aqueous solution of L-histidine (30 mL). Under stirring at 10°C, an aqueous solution containing tris(2-carboxyethyl)phosphine hydrochloride (2.47 mg; 2.1 equivalents per antibody molecule) was added to reduce the solution, and the mixture was stirred at an internal temperature of 10°C for 24 hours until the composition ratio of antibody with 4 interchain thiols and the composition ratio of antibody with 4 interchain thiols reached steady-state values.

[0219] The resulting reaction solution is then stirred at an internal temperature of 9-11°C, and the formula is...

[0220] [ka]

[0221] The compound shown (20 mg; 4.4 equivalents per antibody molecule) was dissolved in 3.3 g of 80% dimethyl sulfoxide aqueous solution containing 0.011 mL of 10% acetic acid aqueous solution, and this solution was added over 20 minutes. The mixture was stirred at the same temperature for 45 minutes to bind to the antibody containing the thiol group. Next, 0.41 g of 0.05 M N-acetylcysteine ​​aqueous solution (5 equivalents per antibody molecule) was added, and the mixture was stirred at the same temperature for another 45 minutes. Then, the pH was adjusted to 5 using 10% acetic acid aqueous solution, and the formula was used.

[0222] [ka]

[0223] (In the formula, A indicates the binding site with the antibody.) A solution was obtained containing an anti-B7-H3 antibody-drug conjugate composition in which a drug linker represented by and an anti-B7-H3 antibody were linked by a thioether bond. The content of each isomer in the solution was D0: 4%, D2: 22%, D4: 54% (of which D4-1: 64%, D4-2: 36%), D6: 17%, and D8: 3%. [Sequence Listing Free Text]

[0224] SEQ ID NO: 1: Amino acid sequence of the heavy chain of the anti-TROP2 antibody Sequence ID 2: Amino acid sequence of the light chain of the anti-TROP2 antibody SEQ ID NO: 3: Amino acid sequence of the heavy chain of the anti-B7-H3 antibody SEQ ID NO: 4: Amino acid sequence of the light chain of the anti-B7-H3 antibody Sequence ID 5: Amino acid sequence of CDRH1 of the anti-TROP2 antibody Sequence ID 6: Amino acid sequence of CDRH2, an anti-TROP2 antibody SEQ ID NO: 7: Amino acid sequence of CDRH3 of the anti-TROP2 antibody Sequence ID 8: Amino acid sequence of CDRL1 in the anti-TROP2 antibody Sequence ID 9: Amino acid sequence of CDRL2 in the anti-TROP2 antibody Sequence ID 10: Amino acid sequence of CDRL3 in the anti-TROP2 antibody Sequence ID 11: Amino acid sequence of CDRH1, an anti-B7-H3 antibody Sequence ID 12: Amino acid sequence of CDRH2, an anti-B7-H3 antibody SEQ ID NO: 13: Amino acid sequence of CDRH3 of the anti-B7-H3 antibody Sequence ID No. 14: Amino acid sequence of CDRL1 in anti-B7-H3 antibody Sequence ID 15: Amino acid sequence of CDRL2 in the anti-B7-H3 antibody Sequence ID 16: Amino acid sequence of CDRL3 in anti-B7-H3 antibody

Claims

1. (i) A step of reacting an antibody with a reducing agent to obtain an antibody having a thiol group; then, A method for producing an antibody-drug conjugate composition, comprising the step of reacting the antibody having a thiol group obtained in (i) with a drug linker intermediate, A manufacturing method characterized in that step (i) is carried out until the composition ratio of the antibody having four heavy-chain-light-chain thiols and the composition ratio of the antibody having four heavy-chain-heavy-chain thiols reach a steady state.

2. The manufacturing method according to claim 1, wherein step (i) is carried out over a period of four hours or more.

3. The manufacturing method according to claim 1, wherein step (i) is carried out over a period of 12 hours or more.

4. The manufacturing method according to claim 1, wherein step (i) is carried out over a period of 16 hours or more.

5. The manufacturing method according to claim 1, wherein step (i) is carried out over a period of 20 hours or more.

6. The manufacturing method according to claim 1, wherein step (i) is carried out over a period of 31 hours or more.

7. The manufacturing method according to any one of claims 1 to 6, wherein step (i) is performed at 0 to 20°C.

8. The manufacturing method according to any one of claims 1 to 6, wherein step (i) is performed at 5 to 20°C.

9. The manufacturing method according to any one of claims 1 to 6, wherein step (i) is performed at 5 to 10°C.

10. The manufacturing method according to any one of claims 1 to 6, wherein step (i) is performed at approximately 10°C.

11. The manufacturing method according to any one of claims 1 to 10, wherein the average number of drug-bound antibodies in the antibody-drug conjugate composition produced is 3.5 to 4.

5.

12. The manufacturing method according to any one of claims 1 to 11, wherein the antibody-drug conjugate composition produced contains 50% or more of an antibody-drug conjugate to which four drug linkers are bound.

13. The manufacturing method according to any one of claims 1 to 12, wherein the proportion of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain inter-light-chain thiols in the manufactured antibody-drug conjugate composition is 1.5 to 2.5 times the proportion of antibody-drug conjugates in which four drug linkers are bound to the heavy-chain inter-heavy-chain thiols.

14. The manufacturing method according to any one of claims 1 to 13, wherein the reducing agent is used in an amount of 1.9 to 2.5 equivalents per molecule of antibody.

15. The method for producing a product according to any one of claims 1 to 14, wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof.

16. The method for producing a drug linker intermediate having an N-substituted maleidyl group, according to any one of claims 1 to 15.

17. The drug linker intermediate is, 【Chemistry 1】 A method for producing a compound represented by any one of claims 1 to 15.

18. The drug linker of the antibody-drug conjugate composition to be manufactured is of the formula 【Chemistry 2】 (In the formula, A indicates the binding site with the antibody, and the drug linker is bound to the antibody by a thioether bond.) The manufacturing method according to claim 17, as shown in [reference].

19. The method for producing an antibody according to any one of claims 1 to 18, wherein the antibody is an anti-TROP2 antibody or an anti-B7-H3 antibody.

20. The method for producing an antibody according to claim 19, wherein the antibody is an anti-TROP2 antibody.

21. The method for producing the anti-TROP2 antibody according to claim 20, wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 5, CDRH2 having the amino acid sequence described in SEQ ID NO: 6, and CDRH3 having the amino acid sequence described in SEQ ID NO: 7, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 8, CDRL2 having the amino acid sequence described in SEQ ID NO: 9, and CDRL3 having the amino acid sequence described in SEQ ID NO:

10.

22. The method for producing anti-TROP2 antibodies according to claim 20, wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 1, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 2,

23. The method for producing anti-TROP2 antibodies according to claim 20, wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234).

24. The method for producing the anti-TROP2 antibody according to claim 23, wherein the lysine residue at the heavy chain carboxyl terminus is deleted.

25. The method for producing an antibody according to claim 19, wherein the antibody is an anti-B7-H3 antibody.

26. The method for producing an anti-B7-H3 antibody according to claim 25, wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 11, CDRH2 having the amino acid sequence described in SEQ ID NO: 12, and CDRH3 having the amino acid sequence described in SEQ ID NO: 13, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 14, CDRL2 having the amino acid sequence described in SEQ ID NO: 15, and CDRL3 having the amino acid sequence described in SEQ ID NO:

16.

27. The method for producing anti-B7-H3 antibody according to claim 25, wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 3, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 4, consisting of the amino acid sequence described in SEQ ID NO:

4.

28. The method for producing anti-B7-H3 antibody according to claim 25, wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 471) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 233).

29. The method for producing the anti-B7-H3 antibody according to claim 28, wherein the lysine residue at the heavy chain carboxyl terminus is deleted.

30. An antibody-drug conjugate composition produced by the manufacturing method described in any one of claims 1 to 29.

31. (i) A step of reacting an antibody with a reducing agent to obtain an antibody having a thiol group; then, A method for producing an antibody-drug conjugate composition, comprising the step of reacting the antibody having a thiol group obtained in (i) with a drug linker intermediate, (i) The process is carried out at 0-20°C for 4 hours or more. A method for producing an antibody-drug conjugate composition characterized in that the content of antibody-drug conjugates with four drug linkers attached is 50% or more, and the proportion of antibody-drug conjugates with four drug linkers attached to the heavy-chain interchain thiol is 1.5 to 2.5 times the proportion of antibody-drug conjugates with four drug linkers attached to the heavy-chain interchain thiol.

32. The manufacturing method according to claim 31, wherein step (i) is carried out over a period of 12 hours or more.

33. The manufacturing method according to claim 31, wherein step (i) is carried out over a period of 16 hours or more.

34. The manufacturing method according to claim 31, wherein step (i) is carried out over a period of 20 hours or more.

35. The manufacturing method according to claim 31, wherein step (i) is carried out over a period of 31 hours or more.

36. The manufacturing method according to any one of claims 31 to 35, wherein step (i) is performed at 5 to 20°C.

37. The manufacturing method according to any one of claims 31 to 35, wherein step (i) is performed at 5 to 10°C.

38. The manufacturing method according to any one of claims 31 to 35, wherein step (i) is performed at approximately 10°C.

39. The manufacturing method according to any one of claims 31 to 38, wherein the average number of drug-bound antibodies in the antibody-drug conjugate composition produced is 3.5 to 4.

5.

40. The manufacturing method according to any one of claims 31 to 39, wherein the reducing agent is used in an amount of 1.9 to 2.5 equivalents per molecule of antibody.

41. The method for producing a product according to any one of claims 31 to 40, wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof.

42. The method for producing a drug linker intermediate having an N-substituted maleidyl group, according to any one of claims 31 to 41.

43. The drug linker intermediate is, 【Transformation 3】 A method for producing a compound represented by any one of claims 31 to 41.

44. The drug linker of the antibody-drug conjugate composition to be manufactured is of the formula 【Chemistry 4】 (In the formula, A indicates the binding site with the antibody, and the drug linker is bound to the antibody by a thioether bond.) The manufacturing method according to claim 43, as shown in [reference].

45. The method for producing an antibody according to any one of claims 31 to 44, wherein the antibody is an anti-TROP2 antibody or an anti-B7-H3 antibody.

46. The method for producing an antibody according to claim 45, wherein the antibody is an anti-TROP2 antibody.

47. The method for producing the anti-TROP2 antibody according to claim 46, wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 5, CDRH2 having the amino acid sequence described in SEQ ID NO: 6, and CDRH3 having the amino acid sequence described in SEQ ID NO: 7, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 8, CDRL2 having the amino acid sequence described in SEQ ID NO: 9, and CDRL3 having the amino acid sequence described in SEQ ID NO:

10.

48. The method for producing an anti-TROP2 antibody according to claim 46, wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 1, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 2,

49. The method for producing anti-TROP2 antibodies according to claim 46, wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 1 (amino acid numbers 20 to 470) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 2 (amino acid numbers 21 to 234).

50. The method for producing the anti-TROP2 antibody according to claim 49, wherein the lysine residue at the heavy chain carboxyl terminus is deleted.

51. The manufacturing method according to claim 45, wherein the antibody is an anti-B7-H3 antibody.

52. The method for producing an anti-B7-H3 antibody according to claim 51, wherein the antibody comprises a heavy chain containing CDRH1 having the amino acid sequence described in SEQ ID NO: 11, CDRH2 having the amino acid sequence described in SEQ ID NO: 12, and CDRH3 having the amino acid sequence described in SEQ ID NO: 13, and a light chain containing CDRL1 having the amino acid sequence described in SEQ ID NO: 14, CDRL2 having the amino acid sequence described in SEQ ID NO: 15, and CDRL3 having the amino acid sequence described in SEQ ID NO:

16.

53. The method for producing anti-B7-H3 antibody according to claim 51, wherein the antibody comprises a heavy chain including a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 3, and a light chain including a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 4, consisting of the amino acid sequence described in SEQ ID NO:

4.

54. The method for producing anti-B7-H3 antibody according to claim 51, wherein the antibody comprises a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 3 (amino acid numbers 20 to 471) and a light chain consisting of the amino acid sequence described in SEQ ID NO: 4 (amino acid numbers 21 to 233).

55. The method for producing the anti-B7-H3 antibody according to claim 54, wherein the lysine residue at the heavy chain carboxyl terminus is deleted.

56. An antibody-drug conjugate composition produced by the manufacturing method described in any one of claims 31 to 55.

Images