Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody-Drug Conjugates

Two US FDA-approved antibody-drug conjugates (ADCs; Kadcyla (R) and Adcetris (R)) have accelerated clinical interest in the potential of targeted cancer therapeutics as the next generation of oncology drugs that are designed to increase efficacy while reducing overall toxicity. Thiol conjugates are produced by partial reduction of the interchain disulfides, followed by conjugation with a drug-linker, resulting in a heterogeneous mix of molecules that differ with respect to the site of conjugation and the number of drugs per antibody. ADCs that have been characterized in this class have an immunoglobulin G1 (IgG1) framework and there is little information available on IgG2 ADCs. As IgG1s and IgG2s differ in the number of disulfides and molecular conformations, each subclass could lead to unique combinations of possible conjugation sites. We conducted in-depth characterization of two ADCs, an IgG1 and an IgG2 conjugated to monomethyl auristatin E. The results demonstrate that the IgG1 monoclonal antibodies favor conjugation to the cysteines between the light and heavy chains, whereas IgG2s demonstrate preference for the hinge region cysteines. The drug-loading distribution and conjugation sites of ADCs have been reported to influence pharmacokinetics, toxicity, and clearance. Therefore, an understanding of the conjugation profiles is important for the selection and engineering of ADCs. (C) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association