Protocol for designing and evaluating antibody-drug conjugates to promote endocytosis and lysosomal trafficking

Receptor endocytosis and lysosomal delivery are critical for efficient intracellular delivery of antibody-drug conjugates (ADCs), yet many cancer targets exhibit limited internalization. Here, we present a protocol for engineering and evaluating ADCs to induce receptor ubiquitination, thereby promoting receptor uptake and transport toward lysosomal compartments. We describe steps for designing antibodies fused with E3 ligase-binding modules, drug conjugation, and cellular assays to quantify ubiquitination-dependent internalization and lysosomal accumulation in cancer cell models. For complete details on the use and execution of this protocol, please refer to Zhuang et al.¹.