Figure 5.

Screening of Fc libraries for variants with enhanced FcγRIIb binding. (A) Enrichment of cells with the highest binding to FcγRIIb. HEK293T cells were transduced with lentiviral Fc variant libraries and stained by FITC conjugated anti-FLAG antibody, biotinylated FcγRIIb and streptavidin-PE. Three rounds of selection were performed. (B) The amino acid distribution at each mutation region of the original libraries and the enriched pools after the 2^nd or 3^rd round of selection. The relative frequency of each amino acid at each position is represented by the height of the corresponding symbol. (C, D) Binding of Fc variants with the selected substitutions (C) or the combination of substitutions (D) to FcγRs was measured by surface plasmon resonance (SPR). ΔRU is equal to the response unit of Fc variant subtracted by the response unit of wild-type Fc.