Bridging Disulfides for Stable and Defined Antibody Drug Conjugates

• DOI: 10.1021/bc500148x
• Publication Date: 2014-05-03T14:22:53Z
• Authors: George Badescu; Penny Bryant; Matthew Bird; Korinna Henseleit; Julia Swierkosz; Vimal Parekh; Rita Tommasi; Estera Pawlisz; Kosma Jurlewicz; Monika Farys; Nicolas Camper; XiaoBo Sheng; Martin Fisher; Ruslan Grygorash; Andrew Kyle; Amrita Abhilash; Mark Frigerio; Jeff Edwards; Antony Godwin
• Keywords: 0303 health sciences; Dose-Response Relationship, Drug; Molecular Structure; Cell Survival; Antineoplastic Agents; Trastuzumab; Antibodies, Monoclonal, Humanized; 3. Good health; Structure-Activity Relationship; 03 medical and health sciences; Cell Line, Tumor; MCF-7 Cells; Humans; Disulfides; Drug Screening Assays, Antitumor; Oligopeptides; Cell Proliferation

To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation.