Figure 1.

Single chain variable fragment (scFv) binding to programmed death-ligand 1 (PDL1) or vascular endothelial growth factor (VEGF). (A) Schematic representation of the yeast surface display (YSD) system. The scFv fused to Aga2p expressed in yeast is disulfide-bonded to Aga1p, which is covalently bound to the yeast cell wall. Expression of the scFv variant was detected by an anti-c-myc-FITC antibody, and binding was determined using either biotinylated-PDL1 or biotinylated-VEGF₁₂₁, followed by allophycocyanin (APC)-conjugated streptavidin. (B–E) Flow cytometry measurement of the expression of YSD Du scFv (B) or its binding to 25 nM soluble PDL1 (C), and expression of YSD Ran scFv (D) or its binding to 25 nM soluble VEGF₁₂₁ (E). (F, G) YSD affinity titration curves for the anti-VEGF Ran scFv (F; red dots), anti-PDL1 Du scFv (G; blue dots), At scFv (G; dark yellow squares) and Av scFv (G; orange triangles). Binding was determined by using flow cytometry analysis and different concentrations of VEGF (1 to 100 nM) or PDL1 (0.005 to 20 nM). The binding of each sample was normalized to its own expression level and then to the highest binding signal exhibited by the same scFv clone. The data was fitted to a 1:1 Langmuir kinetic binding model to yield the apparent K_D value for each scFv–target protein pair.