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, and Antony Godwin. Bioconjugate Chem., 2014, 25 (6), pp 1124–1136
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.
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