Figure 2.

YSD library in vitro affinity maturation. (a) First cycle of FACS sort panning and selection. The top 7% clone-expressing yeast cells were sorted (marked), then further panned by two additional cycles. (b,c) Library panning results demonstrate improved binding (at 0.05 μM nanoparticle polymer SLe^a-PAA-Biotin) with increased panning cycles. Unlabeled Pan3 negative control (Control) cells were stained only with secondary antibodies and compared to native YSD clone, mutated-RA9-scFv YSD library (Lib) and the three library panning cycles outputs (Pan 1, 2, 3). (d) Five single colonies from panning cycle 3 show high antigen binding (FACS against 0.05 μM SLe^a-PAA-Biotin) compared to the native and unlabeled Pan3 control. (e) Apparent K_D was evaluated by FACS, binding of native and selected RA9-mutants clones expressed on yeast cells was examined at 10 serial dilutions of nanoparticle polymer SLe^a-PAA-Biotin (3333–0.16 nM). In (c–e) cells were gated on single chain fragment variable (scFv) expressing cells and geometric mean fluorescence intensity of antigen binding measured. K_D was calculated according to non-linear fit with one-site specific binding using GraphPad Prism 8.0. Representative of two independent experiments.