Efficacy of a site-specific anti-Nectin-4 antibody-drug conjugate against Nectin-4-positive triple-negative breast cancer models.

Maleimide-linked antibody-drug conjugates (ADC) like Enfortumab Vedotin (EV) have limited in vivo drug-linker stability. "PODS" is a phenyloxadiazolyl methylsulfone derivatized linker that has been shown to link radioactive payloads to biologics with superior in vivo stability, compared with maleimide. For the first time, we have synthesized and characterized a novel PODS-based, cathepsin-B cleavable, Nectin-4-targeting auristatin-E (MMAE) loaded ADC, Cys2-N4MU01-PODS-PEG8-VC-PAB-MMAE (Cys2-N4MU01-PMMAE) and evaluated its anti-tumor efficacy against triple-negative breast cancer (TNBC) models. The ADC was constructed via attachment of payload to engineered cysteine on N4MU01. In vitro characterization and cytotoxicity of Cys2-N4MU01-PMMAE was assessed on Nectin-4-positive TNBC cell lines MDA-MB-468, E0771nectin-4, and 4T1nectin-4. Nectin-4-negative 4T1 cells and EV were used as control cell line and ADC, respectively. The in vivo anti-tumor efficacy of Cys2-N4MU01-PMMAE compared with EV, was evaluated in MDA-MB-468 and 4T1nectin-4 mouse xenograft models. Cys2-N4MU01-PMMAE was constructed with a drug-to-antibody ratio (DAR) of 2.2. The binding affinity of Cys2-N4MU01-PMMAE on Nectin-4+ TNBC cell was slightly lower than that of the naked antibody, but this did not affect ADC internalization. The in vitro 50% inhibition concentration (IC50) of Cys2-N4MU01-PMMAE on MDA-MB-468, E0771nectin-4, and 4T1nectin-4 were <57 nM, but inferior to that of EV. Both ADCs had no dose-dependent cytotoxicity on Nectin-4-negative 4T1 cells. In vivo, Cys2-N4MU01-PMMAE showed anti-tumor effects against 4T1nectin-4 xenograft but not MDA-MB-468 and was slightly superior to EV. Cys2-N4MU01-PMMAE warrants further optimization to enhance its effectiveness against TNBC models. This study also re-enforces the rationale for the development of novel moderate affinity and highly stable targeted therapeutics against other cancers.