3.2 Effects of antibody affinity on yeast/mammalian cell interactions
We sought to compare the impact of antibody binding affinity on yeast/mammalian cell interactions in the biofloating and biopanning platforms. For biofloating characterization, yeast cells expressing the atezolizumab, D12, A1, and nivolumab (an anti-PD-1 antibody serving as a negative control) scFvs were co-incubated with PD-L1+CHO-K1 cells in suspension with a yeast:mammalian cell ratio of 10:1. The co-incubation period was varied from 0 to 180 min to gauge the kinetics of this system under different affinity conditions. We found that yeast displaying the high affinity atezolizumab and medium affinity D12 scFvs were fully bound to PD-L1+ CHO-K1 cells virtually instantaneously (Figures 2a and 2c). However, the low affinity clone A1 showed a time-dependent increase in mammalian cell binding, and did not achieve the same degree of binding as the high affinity or medium affinity scFvs, even after 180 min. As anticipated, no binding of atezolizumab, D12, or A1 scFvs was detected on PD-L1-CHO-K1 cells via biofloating (Figure S2a). For biopanning characterization, scFvs were co-incubated with the PD-L1+ CHO-K1 monolayers. We found that yeast displaying the high and medium affinity clones showed detectable binding to PD-L1+ CHO-K1 cells within 60 min, whereas yeast displaying the low affinity clone did not show detectable binding to mammalian cells within 180 min (Figures 2b and 2d). As expected, no binding of atezolizumab, D12, or A1 scFvs was detected on PD-L1- CHO-K1 cells via biopanning (Figure S2b). In contrast with their instantaneous saturation using the biofloating platform, interaction between the high and medium affinity clones and the mammalian cells increased over time, demonstrating the stronger kinetic dependence of yeast/mammalian cell binding for the biopanning versus the biofloating platform. Moreover, detection of interaction between yeast displaying the low affinity clone and mammalian cells in the biofloating but not the biopanning setup indicates the superior sensitivity of the former.
We next sought to compare the impact of antibody binding affinity on the optimal yeast:mammalian cell co-incubation ratio in both the biofloating and biopanning platforms. To this end, yeast displaying atezolizumab, D12, A1, and nivolumab (negative control) scFvs were incubated with PD-L1+ CHO-K1 cells at various yeast:mammalian cell ratios while keeping the incubation time constant. For biofloating experiments, binding was detectable for ratios as low as 0.57 for the high, medium, and low affinity clones (Figure 3a). The potency of binding corresponded with scFv affinity, with the high affinity clone requiring the lowest yeast:mammalian cell ratio to achieve saturation (Figure 3c). Based on these findings, a 10:1 yeast:mammalian cell ratio was determined to be sufficient for biofloating studies. Analogous biopanning studies demonstrated that >10-fold higher yeast:mammalian cell ratios were required to achieve saturation, as compared to biofloating (Figures 3b and 3d). Binding of yeast displaying the low affinity scFv to mammalian cells was barely detectable, even at the highest yeast:mammalian cell ratio, again supporting the enhanced sensitivity of binding detection for the biofloating platform. Our results also confirm that the 40:1 yeast:mammalian cell ratio previously implemented for biopanning studies18 is sufficient to reach saturation for high affinity scFv clones.