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.