Method for selectively manufacturing antibody-drug conjugate
A method for producing an antibody-drug conjugate composition, comprising: (i) a step of reacting an antibody with a reducing agent in a buffer to reduce interchain disulfides, and (ii) a step of reacting drug linker intermediates with the antibody having thiol groups obtained in the step (i), wherein the reaction temperature in the step (i) is −10° C. to 10° C., and the average number of bound drugs in the produced antibody-drug conjugate composition is 3.5 to 4.5, and the content of antibody-drug conjugates in which four drug linkers are bound to heavy-light interchain thiols, in the produced antibody-drug conjugate composition is 50% or more; and an antibody-drug conjugate composition, wherein the content of antibody-drug conjugates wherein the average number of bound drugs is 3.5 to 4.5, and the content of antibody-drug conjugates in which four drug linkers are bound to heavy-light interchain thiols, is 50% or more.
1. A method for producing an antibody-drug conjugate composition, comprising:
(i) a step of reacting an antibody with a reducing agent in a buffer to reduce interchain disulfides, wherein the buffer comprises a chelating agent; and
(ii) a step of reacting drug linker intermediates with the antibody having thiol groups obtained in the step (i), wherein
the reaction temperature in the step (i) is −10° C. to 10° C., and
the average number of bound drugs in the produced antibody-drug conjugate composition is 3.5 to 4.5, and the content of the antibody-drug conjugates in which four drug linkers are bound to heavy-light interchain thiols, in the produced antibody-drug conjugate composition is 50% or more.
2. The production method according to claim 1 , wherein the average number of bound drugs in the produced antibody-drug conjugate composition is 4.0 to 4.1.
3. The production method according to claim 1 , wherein the reaction temperature in the step (i) is −5° C. to 5° C.
4. The production method according to claim 3 , wherein the reaction temperature in the step (i) is −3° C. to 3° C.
5. The production method according to claim 4 , wherein the reaction temperature in the step (i) is 0° C. to 2° C.
6. The production method according to claim 5 , wherein the reaction temperature in the step (i) is 0° C. to 1° C.
7. The production method according to claim 1 , wherein the reducing agent is used in an amount of 2 to 3 molar equivalents per molecule of the antibody.
8. The production method according to claim 1 , wherein the reducing agent is tris(2-carboxyethyl)phosphine or a salt thereof.
9. The production method according to claim 8 , wherein the salt of tris(2-carboxyethyl)phosphine is tris(2-carboxyethyl)phosphine hydrochloride.
10. The production method according to claim 1 , wherein the buffer is a histidine buffer.
11. The production method according to claim 1 , wherein the chelating agent is ethylenediaminetetraacetic acid.
12. The production method according to claim 1 , wherein the antibody is an anti-TROP2 antibody, an anti-CD98 antibody, an anti-B7-H3 antibody, or an anti-HER2 antibody.
13. The production method according to claim 1 , wherein the drug linker intermediate has an N-substituted maleimidyl group.
14. The production method according to claim 13 , wherein
the drug linker intermediate is
wherein -GGFG- (SEQ ID NO: 35) represents a tetrapeptide residue consisting of glycine-glycine-phenylalanine-glycine.
15. The production method according to claim 12 , wherein the antibody is an anti-TROP2 antibody.
16. The production method according to claim 15 , wherein the anti-TROP2 antibody retains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 5 (TAGMQ), CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 6 (WINTHSGVPKYAEDFKG), CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 7 (SGFGSSYWYFDV), CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 8 (KASQDVSTAVA), CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 9 (SASYRYT), and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 10 (QQHYITPLT).
17. The production method according to claim 15 , wherein the anti-TROP2 antibody consists of a heavy chain consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 470 of SEQ ID NO: 2, and a light chain consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 234 of SEQ ID NO: 4.
18. The production method according to claim 15 , wherein the anti-TROP2 antibody consists of a heavy chain consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 469 of SEQ ID NO: 2, and a light chain consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 234 of SEQ ID NO: 4.
19. The production method according to claim 12 , wherein the antibody is an anti-B7-H3 antibody.
20. The production method according to claim 19 , wherein the anti-B7-H3 antibody retains CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 27 (NYVMH), CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 28 (YINPYNDDVKYNEKFKG), CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 29 (WGYYGSPLYYFDY), CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 30 (RASSRLIYMH), CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 31 (ATSNLAS), and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 32 (QQWNSNPPT).
21. The production method according to claim 19 , wherein the anti-B7-H3 antibody consists of a heavy chain consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 471 of SEQ ID NO: 25, and a light chain consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 233 of SEQ ID NO: 26.
22. The production method according to claim 19 , wherein the anti-B7-H3 antibody consists of a heavy chain consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 470 of SEQ ID NO: 25, and a light chain consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 233 of SEQ ID NO: 26.
23. The production method according to claim 12 , wherein the antibody is an anti-HER2 antibody.
24. The production method according to claim 23 , wherein the anti-HER2 antibody consists of a heavy chain consisting of an amino acid sequence consisting of amino acid residues at positions 1 to 449 of SEQ ID NO: 33, and a light chain consisting of an amino acid sequence consisting of amino acid residues at positions 1 to 214 of SEQ ID NO: 34.
25. The production method according to claim 23 , wherein the anti-HER2 antibody consists of a heavy chain consisting of an amino acid sequence shown in SEQ ID NO: 33, and a light chain consisting of an amino acid sequence shown in SEQ ID NO: 34.
26. The production method according to claim 14 , wherein the drug linker intermediate is
wherein -GGFG- (SEQ ID NO: 35) represents a tetrapeptide residue consisting of glycine-glycine-phenylalanine-glycine.