Screening and characterization of EGFR-targeted specific nanobodies

The epidermal growth factor receptor (EGFR), also known as HER1 or ErbB1, is a member of the ErbB receptor family. It plays a critical role in epidermal growth factor (EGF)-mediated cell proliferation and signal transduction pathways. Mutations or overexpression of EGFR are closely associated with the development and progression of various solid tumors, making it an important molecular target for tumor-targeted therapy. In this study, we prepared and purified the extracellular domain of EGFR using a eukaryotic expression system with a C-terminal Avi tag, and utilized it as a biotinylated antigen for phage display screening and subsequent biochemical analyses. Using an anti-EGFR camelid immune nanobody library previously constructed in our laboratory, we performed biopanning against the purified EGFR extracellular domain via phage display technology. Through phage ELISA, sequence analysis, soluble expression screening, and further biochemical characterization, several nanobody candidates with specific binding to EGFR were identified. Among these, the representative clone NB1B3 was selected for in-depth analysis using ELISA, biolayer interferometry (BLI), and EGF competition assays. BLI-based binding kinetics revealed that the clone exhibits nanomolar affinity, while competitive ELISA demonstrated its ability to effectively compete with EGF for EGFR binding in vitro, indicating its potential inhibitory function. Taken together, these results identify NB1B3 as an EGFR-binding nanobody candidate with preliminary biochemical evidence of competition with EGF. Although its precise epitope and cellular activity remain to be determined, this study provides an experimentally supported candidate and a screening workflow for further anti-EGFR nanobody development.