Dissecting PROTAB ‐mediated degradation of cell surface proteins

Targeted protein degradation mediated by antibodies has emerged as a promising strategy for degrading extracellular or membrane‐bound proteins. Proteolysis‐Targeting Antibodies (PROTABs) are bispecific antibodies specifically designed to induce the degradation of membrane proteins by tethering them to a cell surface E3 ligase, which promotes ubiquitination and subsequent degradation. Recent studies have demonstrated the potential of PROTABs to degrade oncogenic receptors, but their underlying mechanisms remain to be fully elucidated. Here, we investigated the mechanism of action of a HER2‐targeting PROTAB comprising an anti‐Zinc and RING finger protein 3 (ZNRF3) arm and an anti‐receptor tyrosine‐protein kinase erbB‐2 (HER2) arm. We show that PROTAB induces rapid ternary complex formation, followed by receptor internalization and degradation, resulting in ~ 85% target depletion within 24 h. Mechanistically, ubiquitination enhances but is not strictly required for internalization, and degradation proceeds predominantly through the lysosomal pathway. Notably, ZNRF3 is not codegraded but instead accumulates at the cell surface, while the PROTAB antibody itself is largely recycled. Importantly, target degradation does not consistently translate into growth inhibition, highlighting the role of cellular context and target dependency. Together, these findings provide a mechanistic framework for PROTAB function and inform the rational design of next‐generation antibody‐based degraders.