Preliminary Evaluation of Synthetic Amanitin Analogs as Novel Payloads for Potent Antibody−Drug Conjugates

Alpha-amanitinthe principal toxin of Amanita phalloides, infamous for its hepatotoxicity and lethalityhas been receiving considerable attention as a candidate payload in antibody−drug conjugates (ADCs). Previously, we identified three synthetic amatoxins with heightened cytotoxicity against CHO, HEK 293, and HeLa cells. In this work, these analogs were advanced as ADC payloads using the well-established anti-HER2 antibody trastuzumab to provide a head-to-head comparative study against α-amanitin, where all payloads (synthetic or natural) are linked via a glutaric linker; esterification to the dihydroxy-isoleucine on the toxin and via an amide to an antibody lysine. Tested as ADC payloads, one synthetic amatoxin outperformed the natural toxin across various drug−antibody ratios (DARs) and across a panel of HER2-overexpressing cell lines; against SK-BR-3 cells, this ADC shows a 25-fold greater potency at DAR 2.1 compared to the same ADC linked to α-amanitin at DAR 2.0. At DAR values of 5−6, this toxin confers low-femtomolar IC50 values on the corresponding trastuzumab conjugate when assayed against SK-BR-3 cells and shows exalted cytotoxicity against a panel of HER2-overexpressing cell lines compared to the ADC linked to α-amanitin.