BPS2026 – Clustering of EGFR using phase-separating ligands enhances receptor uptake and lysosomal targeting

Overexpression and sustained signaling of the epidermal growth factor receptor (EGFR) contribute to the progression of many epithelial cancers, including non-small-cell lung cancer, breast cancer, and colorectal cancer. While monoclonal antibodies have been used to trigger EGFR internalization, their therapeutic impact is often limited by receptor recycling and poor trafficking to degradative pathways. Strategies that promote sustained EGFR downregulation are therefore of growing interest. Recent studies suggest that intrinsically disordered regions (IDRs), such as the low-complexity domain of the FUS protein (FUS-LC), can drive local protein clustering. Here, we used this concept to promote receptor clustering at the plasma membrane, resulting in their targeted endocytic uptake and localization to lysosomes. Specifically, we engineered a fusion of the anti-EGFR nanobody 7D12 with FUS-LC to evaluate whether clustering-based mechanisms can enhance EGFR internalization and degradation. We used SUM159 cells expressing EGFR-GFP and quantified uptake of fluorescently labeled ligands using live-cell imaging across a range of ligand concentration levels. FUS-tagged nanobodies showed enhanced internalization relative to controls, particularly at intermediate ligand concentration, as assessed by comparing surface-bound versus internalized ligand and receptor pools. Flow cytometry corroborated this increase in uptake at the population level. To assess degradation, we measured colocalization of EGFR with the lysosomal marker LAMP1. Although internalization by FUS-tagged ligands was slower initially, by 2 hours, they promoted greater EGFR accumulation in lysosomes compared to unmodified nanobodies. These observations suggest that clustering not only enhances EGFR uptake but also favors trafficking toward degradation. Our results highlight how phase-separating IDRs can be harnessed to modulate receptor trafficking and offer a novel ligand-based platform for selective degradation of cell-surface receptors.