2.3 Yeast display and PD-L1 affinity characterization
General yeast display methodologies were carried out with EBY-100 yeast cells, as previously described.7,21 The VH chain followed by the VL chain of the anti-PD-L1 antibody atezolizumab and the anti-programmed cell death protein (PD-1) antibody nivolumab were cloned into the yeast display vector pCT3CBN, separated by a (G4S)3linker (scFv format). The PD-L1-binding scFvs D12 and A1 were selected from a previously reported naïve yeast-displayed scFv library.22 These clones were displayed on yeast using the pCTCON2 expression vector. Yeast clones were grown in SD-CAA medium at 30ºC while shaking at 200 rpm. Yeast were induced in SG-CAA at an initial optical density (OD) of 1.0 (1×107 cells/mL).
To titrate PD-L1 affinity, atezolizumab, D12, and A1 scFv-displaying yeast were grown overnight, induced for 24 hr, and then aliquoted into 96-well plates at 105 cells/well. Cells were then pelleted and resuspended in PBE buffer (phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA) and 1 mM ethylenediaminetetraacetic acid (EDTA)) containing various concentrations of biotinylated PD-L1 and incubated for 2 hr at room temperature. Cells were then pelleted and washed with PBE and incubated for 15 min at 4°C with 50 nM Alexa647-conjugated streptavidin (SA-647) (Thermo Fisher Scientific) diluted in PBE. A final wash was conducted and binding was analyzed via flow cytometry using a CytoFLEX analyzer (Beckman Coulter). Data was analyzed in Prism software (GraphPad) and fitted using a single logistic model. Equilibrium dissociation constant (KD) values were determined for each clone. The experiment was performed in triplicate and repeated twice to ensure reproducibility.