[Experimental and computational analyses of altered cetuximab binding induced by EGFR mutations]

Acquired mutations in the extracellular domain of epidermal growth factor receptor (EGFR) are major causes of cetuximab resistance. However, the structural mechanisms by which different mutations impair antibody binding remain to be clarified. In this study, four clinically reported EGFR mutations, S492R, G465R, S464L, and I491M, were selected for investigation. Corresponding mutant EGFR fusion proteins were constructed, expressed, and purified. Enzyme-linked immunosorbent assay was performed to evaluate their binding ability to cetuximab. In parallel, SAAMBE-3D, FoldX, and Rosetta were employed to analyze mutation-induced changes in binding free energy, interfacial geometric complementarity, and local interaction patterns. The results showed that the half-maximal effective concentration (EC₅₀) of cetuximab binding to wild-type EGFR was 0.05 nmol/L. S492R, G465R, and S464L completely abolished cetuximab binding, whereas I491M only increased the EC₅₀ to 0.23 nmol/L, showing a weaker effect on antibody binding. Binding free energy analysis showed that all the four mutations reduced the stability of the EGFR-cetuximab complex, with I491M exerting a comparatively mild effect. Further analyses of interfacial geometric complementarity and structural features revealed that S492R and G465R mainly impaired interfacial matching by introducing marked steric hindrance and unfavorable repulsive effects, and S464L weakened antigen-antibody interactions by disrupting a critical hydrogen-bond network. I491M caused only mild perturbations in local hydrophobic interactions and conformational stability. In summary, by integrating in vitro binding assays with multiple structure-based computational methods, this study demonstrates that extracellular domain mutations of EGFR can attenuate cetuximab binding through enhanced steric hindrance, reduced interfacial complementarity, and disruption of key interaction networks. These findings provide experimental and theoretical evidence for understanding the structural basis of acquired cetuximab resistance and offer a reference for the rational design of antibodies targeting mutant EGFR.