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

Figure 3

Validation of affinity improvement of the selected yeast clones. (a) The dot plots show the binding of all different mutated clones to dimeric rhPD-L1-Fc, in comparison with the wild type clone. As preliminary affinity screening, only the PD-L1 concentration used in the last sorting (10 pM) was tested. For most clones (whose name is indicated in the plots), a population of yeasts bound to rhPD-L1-Fc appeared at this concentration (blue population in the upper right quadrant), which was almost undetectable in the wild type clone. The fluorescence thresholds were fixed using an unstained control (not shown). (b) The chart represents the titration curves of the yeast-scFv clones to dimeric rhPD-L1-Fc, performed by flow cytometry. The mean fluorescence intensity of PD-L1 binding was normalized on the scFv expression, and this ratio was plotted against rhPD-L1-Fc concentration. For the two clones in common to both libraries, only the copy from library 3 is shown (clones 3_3 and 3_14). (c) The table reports the half-saturating antigen concentrations (K_D) of the clones and their relative binding improvement, expressed as fold change compared with the wild type yeast.

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