Cathepsin B dependent activatable trigger fluorophore (CAT-Fluor) for in situ functional imaging of antibody-drug conjugates.

Antibody-drug conjugates (ADC) have emerged as an important class of therapeutic agents that combine the target specificity of a monoclonal antibody with the potency of a cytotoxic payload. Despite clinical success, our understanding of receptor endocytosis and ADC toxicity remains limited. Less than 1% of ADCs reach tumors, raising concerns about off-target cytotoxicity. To shed light on these issues, our study introduces a smart antibody-fluorophore conjugate (sAFC) with Cathepsin B dependent Activatable Trigger Fluorophore (CAT-Fluor) to mimic ADC behavior in situ. Using a Cathepsin B-cleavable linker, we linked a Si-rhodamine (SiR) derivative with superior near-infrared emission to antibodies, creating sAFC. Carbamoylation of the primary amino group on SiR is employed to conjugate with the linker and inhibit the electron-push-pull effect of the xanthene skeleton, thus inducing fluorescence quenching. In vitro, the anti-EGFR sAFC emulates ADC metabolism and suggests that specific proteins implicated in endocytosis, like caveolin, significantly influence ADC internalization efficacy, potentially correlating with drug resistance. In vivo studies using sAFC demonstrate that 'passenger ADCs' found in normal tissues release minimal payload, likely elucidating how ADCs mitigate dose-limiting toxicities. Therefore, our sAFC-based strategy, combining CAT-Fluor and targeted interventions, quantitatively and objectively evaluated the impact of various stages and key proteins in the physiological process, spanning from antigen recognition, endocytosis mechanism, to transport and protein hydrolysis, on ADC efficiency. This comprehensive approach lays a mechanistic foundation for advancing ADC research and development, and offers novel insights into tackling ADC efficacy, resistance and potential toxicities from the standpoint of endocytosis mechanisms.