An enhanced variant of the antimitotic toxin cryptophycin was conjugated to the anti‐Her2 monoclonal antibody (mAb) Trastuzumab upon Michael addition. Either antibodies with freed hinge‐region cysteines or THIOMAB formats with engineered cysteines in the mAbs light chain were added to a maleimide derivative of cryptophycin. These Antibody‐Drug Conjugates (ADCs) showed retained binding to Her2 positive tumor cells and highly efficient cell killing in double‐digit pM range on high Her2‐expressing SK‐BR‐3 cells. Two ADCs (DAR 6, DAR 3) showed superior cell killing of the cell lines JIMT‐1 and RT112 with medium receptor expression level in comparison with a DAR 6 MMAE ADC serving as reference. The observed cell cytotoxicity is target‐dependent since no impact on cell viability was observed for low Her2‐expressing MDA‐MB468 cells. Particularly the DAR 3 ADC in THIOMAB format exhibiting desirable biophysical properties and high potency emerged as a promising candidate for further in vivo investigations.
