US 11,833,214 B2
Methods of preparing cell-binding agent-drug conjugates
Scott A Hilderbrand, Swampscott, MA (US); and Daniel F. Milano, Reading, MA (US)
Assigned to IMMUNOGEN, INC., Waltham, MA (US)
Filed by IMMUNOGEN, INC., Waltham, MA (US)
Filed on Mar. 20, 2020, as Appl. No. 16/825,127.
Claims priority of provisional application 62/856,942, filed on Jun. 4, 2019.
Claims priority of provisional application 62/821,707, filed on Mar. 21, 2019.
Prior Publication US 2020/0405874 A1, Dec. 31, 2020
Int. Cl. C07K 16/40 (2006.01); A61P 35/00 (2006.01); A61K 47/68 (2017.01); A61K 31/5365 (2006.01); A61K 31/537 (2006.01); A61K 31/5513 (2006.01); A61K 31/5517 (2006.01); C07K 16/28 (2006.01)
CPC A61K 47/6803 (2017.08) [A61K 31/537 (2013.01); A61K 31/5365 (2013.01); A61K 31/5513 (2013.01); A61K 31/5517 (2013.01); A61K 47/6849 (2017.08); A61K 47/6871 (2017.08); C07K 16/2866 (2013.01); C07K 16/2896 (2013.01); C07K 16/40 (2013.01)] 9 Claims
 
1. A method of preparing a cell-binding agent-cytotoxic agent conjugate comprising a cell-binding agent (CysCBA) having one or more unpaired cysteine residues covalently linked to a cytotoxic agent, wherein the CysCBA is a cysteine-engineered antibody (CysAb) or antigen-binding fragment thereof and the method comprises the steps of:
(a) reacting a reducing agent with a capped cell-binding agent (cCysCBA) having one or more unpaired cysteine residues capped with a capping agent to form a reduced cell-binding agent, wherein the capping agent is removed to form a free thiol (—SH) group in the reduced cell-binding agent;
(b) reacting the reduced cell-binding agent with a selective oxidizing agent to form the CysCBA, wherein the free thiol group in the unpaired cysteine residue is not oxidized; and
(c) reacting the CysCBA with a compound of formula (I):

OG Complex Work Unit Chemistry
or a pharmaceutically acceptable salt thereof, thereby forming the conjugate, wherein the reduced cell-binding agent in step (a) is used in step (b) without purification; and the CysCBA in step (b) is used in step (c) without purification, wherein:
D is a cytotoxic agent represented by the following formula:

OG Complex Work Unit Chemistry
or a pharmaceutically acceptable salt thereof;
the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), trishydroxypropyl phosphine (THPP), tris (2-cyanoethyl)phosphine, dithiothreitol (DTT), (dicyclohexylphosphino)benzenesulfonic acid, bis(p-sulfonatophenyl)phenylphosphine, (diphenylphosphino)benzenesulfonic acid, (diphenylphosphino)benzoic acid, 2-(diphenylphosphino)-N,N,N-trimethylbenzylammonium triflate, (diphenylphosphino)ethylamine, 2-(diisopropylphosphino)ethylamine, or 3-(diphenylphosphino)propylamine (DPPA); and
L is a linker.