Straightforward Glycoengineering Approach to Site-Specific Antibody-Pyrrolobenzodiazepine Conjugates.

Antibody-drug conjugates (ADCs) have become a powerful platform to deliver cytotoxic agents selectively to cancer cells. ADCs have traditionally been prepared by stochastic conjugation of a cytotoxic drug using an antibody's native cysteine or lysine residues. Through strategic selection of the mammalian expression host, we were able to introduce azide-functionalized glycans onto a homogeneously glycosylated anti-EphA2 monoclonal antibody in one step. Conjugation with an alkyne-bearing pyrrolobenzodiazepine dimer payload (SG3364) using copper-catalyzed click chemistry yielded a site-specific ADC with a drug-to-antibody ratio (DAR) of four. This ADC was compared with a glycoengineered DAR two site-specific ADC, and both were found to be highly potent against EphA2-positive human prostate cancer cells in both an in vitro cytotoxicity assay and a murine tumor xenograft model.