Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

Figure 2

Strategy for the isolation of high affinity scFvs from the yeast libraries. (a) The graph reports the binding curve of the wild type yeast to recombinant human PD-L1, a dimer of two identical molecules, each containing the PD-L1 extracellular region linked to the Fc tag (rhPD-L1-Fc). The yeast clone expressing the wild type scFv was incubated with increasing rhPD-L1-Fc concentrations and analyzed by flow cytometry. The mean fluorescence intensity of the anti-Fc antibody, used to detect the yeast-PD-L1 complexes, was plotted as a function of rhPD-L1-Fc concentration. (b and c) The panels represent different cell sorting strategies used for the selection of high affinity clones from library 3 (b) and library 10 (c). Yeast cells were stained with two fluorescent antibodies, detecting the scFvs and the rhPD-L1-Fc antigen, respectively. At each sorting cycle, unstained samples (left columns) were used to determine fluorescence thresholds, while samples stained in absence of the target antigen (middle columns) were used to exclude any non‐specific signal from the sorting gate. The sorted yeasts are shown in the polygonal gate (right plots), and they are expressed as percentage of PD-L1-binding cells (present in the upper right quadrant). rhPD-L1-Fc concentrations used for each selection cycle are indicated on the right.

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