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).