Calicheamicin derivative-carrier conjugates

摘要

Methods for preparing monomeric cytotoxic drug/carrier conjugates with a drug loading significantly higher than in previously reported procedures and with decreased aggregation and low conjugate fraction (LCF) are described. Cytotoxic drug derivative/antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described. Monomeric calicheamicin derivative/anti-CD22 antibody conjugates, compositions comprising the conjugates and uses of the conjugates are also described.

主权项

1. A method for preparing a composition comprising monomeric calicheamicin derivative/antibody conjugates with reduced low conjugated fraction (LCF) having the formula, Pr(—X—W)m, wherein:
Pr is an anti-CD22 antibody comprising SEQ ID NO: 1 for CDR-H1, SEQ ID NO: 2 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 16 or residues 50-66 of SEQ ID NO: 23 or residues 50-66 of SEQ ID NO: 27 for CDR-H2, SEQ ID NO: 3 for CDR-H3, SEQ ID NO: 4 for CDR-L1, SEQ ID NO: 5 for CDR-L2 and SEQ ID NO: 6 for CDR-L3; W is a calicheamicin derivative comprising a calicheamicin; X is a hydrolyzable linker that links the antibody to the calicheamicin derivative and that is capable of releasing the calicheamicin derivative from the calicheamicin derivative/antibody conjugate after binding and entry into target cells; m is the average loading for a purified conjugation product such that the calicheamicin constitutes 4-10% of the calicheamicin derivative/antibody conjugate by weight; the method comprising the steps of: (1) adding the calicheamicin derivative to the antibody wherein the calicheamicin derivative is 4.5-11% by weight of the antibody; (2) incubating calicheamicin derivatives and antibodies in a non-nucleophilic, protein-compatible, buffered solution having a pH in the range from about 7 to 9 to produce a composition comprising the monomeric calicheamicin derivative/antibody conjugates, wherein the solution further comprises (a) an organic cosolvent, and (b) an additive comprising at least one C6-C18 carboxylic acid or its salt, and wherein the incubation is conducted at a temperature ranging from about 30° C. to about 35° C. for a period of time ranging from about 15 minutes to 24 hours; and (3) subjecting the produced in step (2) to a chromatographic separation process to separate monomeric calicheamicin derivative/antibody conjugates with a loading in the range of 4-10% by weight calicheamicin and with low conjugated fraction (LCF) below 10 percent from unconjugated antibody, calicheamicin derivative, and aggregated conjugates.