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.