Figure 3
Directed evolution of Fas antibody E09. (a) Analysis of the 44 highest affinity E09 variant antibodies from the six selection rounds, R1-R6. Surrogate affinity was determined using a six-point antigen titration in an ELISA-based assay. (b) Individual energy contribution of the seven mutations in the top clone EP6b_B01 to affinity improvement. Free energy of binding compared with the parent (ΔΔG0 (mut-WT)) of single-point mutants and revertants in the EP6b_B01 sequence context are shown in white and grey, respectively. Theoretical additive values of the point mutant and the revertant are shown in dashed black. Energy calculations were based on BIAcore affinity measurement of scFvs for recombinant Fas. (c) Structural basis for affinity improvement in EP6b_B01. The lower panels show close-up views from the squares in the upper panel. The left part of each panel shows the zoom from the structure FasR:E09 complex, the right part the zoom from FasR:EP6b_B01. Hydrogen bonds are indicated as black dashed lines
