Amatoxin-derived payloads and their antibody-drug conjugate: unique bicyclopeptide ADCs exhibiting targeted antitumor activity.

Balancing efficacy with off-target toxicity remains a critical challenge in the development of antibody-drug conjugates (ADCs). Emerging ADC technologies, including the design of safer payloads and optimized drug-linkers, offer new avenues to improve both safety and efficacy. Amatoxins bind efficiently to eukaryotic RNA polymerase II at nanomolar concentrations, potently inhibiting transcription and subsequent protein synthesis. In this study, we designed, synthesized, and evaluated a series of novel amatoxin-based bicyclic peptide payloads. From these, three payload-linkers incorporating a Val-Ala (VA) peptide linker were generated, and two trastuzumab-based ADCs (O-3-ADC and P-3-ADC) were constructed. A novel synthetic strategy was developed in which p-aminobenzyl alcohol (PABC) was first coupled to the amatoxin-derived payload, followed by amide bond formation with the VA linker. In vitro cytotoxicity assays revealed that O-3-ADC exhibited IC₅₀ values of 8.5 ± 0.06 nM and 20 ± 0.41 nM against HeLa and SK-OV-3 cells, respectively, while P-3-ADC showed IC₅₀ values of 7.5 ± 0.04 nM and 27 ± 0.12 nM against the same cell lines. Compared to their corresponding free payloads, the antitumor activity of the two novel ADCs was enhanced by up to 255-fold. Furthermore, P-3-ADC significantly inhibited tumor growth in vivo. Collectively, these results demonstrate that the novel amatoxin-derived payloads and their corresponding ADCs possess significant antitumor potential, highlighting their promise for future targeted ADC development.