3.1 Development and characterization of the biofloating platform
We designed a new platform known as biofloating for quantitative analysis of the interaction between yeast displayed scFvs and target MPs on mammalian cells (Figure 1a). Biofloating entails the co-incubation of yeast and mammalian cells in suspension to enable ready compatibility with flow cytometry analysis (Figure 1b). A system was previously reported that detects interactions between scFv-expressing yeast and MP-expressing mammalian cells by incorporating fluorescent proteins into both cell types.19 In contrast, our approach detects yeast/mammalian cell interactions via staining of yeast with fluorescently-labeled anti-cmyc antibody and intracellular staining of mammalian cells.
We compare our new platform to biopanning, an established technique for analysis of yeast/mammalian cell interactions in which yeast are incubated with mammalian cells adhered to a plate and binding is observed using phase contrast microscopy (Figure 1c). Here, we interrogate differences between the biofloating and biopanning platforms under varying affinity and avidity conditions. We use PD-L1 as our target MP for comparison of these platforms. To evaluate the effects of affinity on specific yeast/mammalian cell interactions, we use 3 scFvs with varying affinities to PD-L1: the scFv format of the FDA-approved anti-PD-L1 antibody drug atezolizumab and 2 anti-PD-L1 scFvs our lab discovered from a previously reported synthetic library,22 denoted D12 and A1. We conducted yeast surface binding titrations with these clones against soluble PD-L1. Atezolizumab, D12, and A1 scFvs showed yeast surface KDvalues of 1.1 nM (high), 60 nM (medium), and 650 nM (low), respectively (Table 1). We also interrogated the kinetic profiles of the soluble anti-PD-L1 scFvs binding to immobilized PD-L1 using bio-layer interferometry (BLI). Association rate constants (kon) and dissociation rate constants (koff) are reported in Table 2. The effects of avidity on binding were investigated by using 3 cell lines with varying PD-L1 expression. The PD-L1+CHO-K1, H2444, and MDA-MB-231 cell lines were found to express PD-L1 at 2.0×106 molecules/cell (dense), 1.3×106 molecules/cell (medium), and 3×105 molecules/cell (sparse), respectively (Table 3). In each PD-L1+ cell line, cells uniformly expressed the antigen (Figure S1).