Fig. 2. Schematic diagram of the SGAPAE approach for directed evolution of cetuximab.

The structure-guided and phage-assisted evolution (SGAPAE) approach was applied to develop cetuximab variants for reversing S492R- or G465R-mediated resistance. First, the crystal structure of the wild-type EGFR/cetuximab Fab complex was obtained from the Protein Data Bank (PDB code: 1YY9). Second, the critical residues in the interface were identified by evaluating the interfacial free energy of the known structure of the EGFR/cetuximab Fab complex and determining the energy difference between the bound and unbound states of the interface with the Rosetta platform. Third, an epitope-restricted cetuximab mutant phage-display library with a size of 10⁶–10⁷ phages was generated by randomization of identified critical residues (n = 6). Finally, the cetuximab variants that bound to both EGFR^WT and EGFR^Mut were selected after panning the cetuximab mutant phage-display library.