Modular Synthesis of Anti-HER2 Dual-Drug Antibody-Drug Conjugates Demonstrating Improved Toxicity

Antibodies have gained clinical success in the last two decades for the targeted delivery of highly toxic small molecule chemotherapeutics. Yet antibody-drug conjugates (ADCs) often fail in the clinic due to the development of resistance. The delivery of two mechanistically distinct small molecule drugs on one antibody is of increasing interest to overcome these challenges with single-drug ADCs. We have developed a modular synthetic strategy for the construction of a library of 19 dual-drug ADCs where drugs are conjugated through unnatural cyclopentadiene-containing amino acids and native cysteine residues on an anti-HER2 trastuzumab scaffold. Importantly, this strategy utilizes the same functional group on the linker-drug construct; this allows for the facile addition of drugs at either conjugation site and enables the evaluation of different drug-to-antibody ratios and combinations of drug pairs. We tested the library on high- and mid-HER2 expressing cell lines and observed increased toxicity in several dual-drug ADCs compared with single-drug constructs. The strategy developed herein provides a method for the facile synthesis, characterization, and evaluation of dual-payload ADCs. Simultaneous delivery of combinations of drugs with distinct mechanisms of action is critical for the next generation of targeted drug delivery.