Fig. 5. The mechanisms of action by which the cetuximab variants in inhibit EGFR^S492R or EGFR^G465R activation.

a The modeled structural mechanisms by which the cetuximab variants restore binding with EGFR^S492R or EGFR^G465R. The critical residues in the cetuximab variants and the EGFR mutations are highlighted as colored sticks in the interface. The structures of VH domain in the cetuximab/cetuximab variants and EGFR^S492R or EGFR^G465R ECD domain III are shown in white and gray, respectively. b The central cavities (circled in black) in the paratopes of cetuximab (PDB code: 1YY9) and its variants. c The mechanism by which the cetuximab variants inhibit the ligand-induced activation of EGFR^S492R or EGFR^G465R, as shown by western blot analysis. Stable NIH3T3 cells were stimulated with EGF (0.5 nM) and immunoblotted for total and phosphorylated EGFR, Akt, and Erk (pEGFR^Y1068, pAkt^S473, and pErk^T202/Y204). d The mechanism by which the cetuximab variants inhibit the ligand-induced receptor internalization and degradation of EGFR^S492R or EGFR^G465R, as shown by flow cytometry. The mean fluorescence intensity (MFI) of different stable HEK293T cells was determined after 48 h of preincubation with the indicated concentrations of antibodies. MFI values were normalized to the EGFR-eGFP signal in cells with no antibody treatment. All above assays were performed in triplicates. The bars indicate the mean ± SD values. Source data are provided as a Source Data file.