3.3 Effects of avidity on yeast/mammalian cell interactions
We next sought to compare the effects of avidity on yeast/mammalian cell
interactions in the biofloating and biopanning platforms through studies
using mammalian cell lines with varying PD-L1 expression levels:
PD-L1+ CHO-K1 (dense), H2444 (medium), and MDA-MB-231
(sparse). For biofloating characterization, yeast cells expressing the
atezolizumab and nivolumab (negative control) scFvs were co-incubated
with the 3 PD-L1-expressing cell lines in suspension at a
yeast:mammalian cell ratio of 10:1. We found that yeast displaying the
atezolizumab scFv were fully bound to dense and medium PD-L1-expressing
cell lines almost immediately (Figures 4a and 4c). Minimal specific
binding of atezolizumab scFv-expressing yeast to the sparse
PD-L1-expressing cell line was observed compared to the nivolumab
scFv-expressing yeast control. Also, the percent of PD-L1-expressing
mammalian cells bound to scFv-expressing yeast declined with decreasing
antigen density. For biopanning characterization, yeast cells expressing
the atezolizumab and nivolumab scFvs were incubated with monolayers of
the 3 PD-L1-expressing mammalian cell lines. We found that atezolizumab
scFv-expressing yeast bound to the dense PD-L1-expressing cells in a
time-dependent manner, whereas almost no specific binding was detected
to the medium and sparse cell lines for all timepoints when compared to
the nivolumab scFv-expressing yeast control (Figures 4b and 4d).
Collectively, these experiments demonstrate the superior sensitivity for
the biofloating versus the biopanning platform in detecting
yeast/mammalian cell interactions at lower levels of antigen expression.
We next studied the effects of avidity on the optimal yeast:mammalian
cell co-incubation ratio in both the biofloating and biopanning systems.
Yeast displaying the atezolizumab or nivolumab (negative control) scFv
were incubated with the dense, medium, and sparse PD-L1-expressing cell
lines at various yeast:mammalian cell ratios while fixing the incubation
time at 2 hr. For biofloating experiments, atezolizumab scFv-expressing
yeast binding to the dense cell line was detected at ratios as low as
0.06:1, whereas the medium cell line required yeast:mammalian cell
ratios of >2:1 for detection (Figures 5a and 5c). Specific
binding to the sparse cell line was barely detectable by biofloating.
Notably, the maximum percentage of PD-L1-expressing mammalian cells
bound to scFv-expressing yeast declined with decreasing avidity
conditions. These results reinforce that the 10:1 yeast:mammalian cell
ratio determined from affinity titrations is optimal for biofloating
analysis. Similar to the affinity titration studies, the analogous
biopanning characterization for avidity effects demonstrate that
>10-fold higher yeast:mammalian cell ratios were required
to achieve saturation, as compared to biofloating (Figures 5b and 5d).
Almost no specific binding could be detected to the medium and sparse
cell lines using biopanning, again reiterating the higher sensitivity of
the biofloating platform.