In Vitro and In Vivo Evaluation of Cysteine Rebridged Trastuzumab-MMAE Antibody Drug Conjugates with Defined Drug-to-Antibody Ratios.

The conjugation of monomethyl auristatin E (MMAE) to trastuzumab using a reduction bis-alkylation approach that is capable of rebridging reduced (native) antibody interchain disulfide bonds has been previously shown to produce a homogeneous and stable conjugate with a drug-to-antibody ratio (DAR) of 4 as the major product. Here, we further investigate the potency of the DAR 4 conjugates prepared by bis-alkylation by comparing to lower drug loaded variants to maleimide linker based conjugates possessing typical mixed DAR profiles. Serum stability, HER2 receptor binding, internalization, in vitro potency, and in vivo efficacy were all evaluated. Greater stability compared with maleimide conjugation was observed with no significant decrease in receptor/FcRn binding. A clear dose-response was obtained based on drug loading (DAR) with the DAR 4 conjugate showing the highest potency in vitro and a much higher efficacy in vivo compared with the lower DAR conjugates. Finally, the DAR 4 conjugate demonstrated superior efficacy compared to trastuzumab-DM1 (T-DM1, Kadcyla), as evaluated in a low HER2 expressing JIMT-1 xenograft model.

Bridging disulfides for stable and defined antibody drug conjugates.

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.