Figure Legends
Figure 1: Yeast/mammalian cell-cell selection platform layouts. (a) Schematic of yeast surface display of an scFv (left) and mammalian cell expression of a target MP (right). (b) In the biofloating platform, yeast and mammalian cells are co-incubated in suspension. (c) In the biopanning platform, yeast are incubated on top of mammalian cell monolayers.
Figure 2: Effects of scFv affinity on kinetics of yeast/mammalian cell interactions for biofloating versus biopanning platforms. Yeast expressing the anti-PD-L1 atezolizumab (High), D12 (Medium), or A1 (Low) scFvs or the anti-PD-1 (control) scFv were co-incubated with PD-L1+ CHO-K1 cells for varying lengths of time. (a) Flow cytometry histogram overlays depicting yeast binding (as measured by cmyc detection) within the mammalian cell population via biofloating for various incubation timepoints. (b) Phase contrast microscopy images of yeast binding to mammalian cells via biopanning for various incubation timepoints. (c) Quantification of biofloating results shown in (a), displaying the percentage of mammalian cells that are bound to yeast cells at various timepoints. (d) Quantification of biopanning results shown in (b), presenting the number of bound yeast cells per image at various timepoints.
Figure 3: Effects of scFv affinity on equilibrium yeast/mammalian cell interactions for biofloating versus biopanning platforms. Yeast expressing the anti-PD-L1 atezolizumab (High), D12 (Medium), or A1 (Low) scFvs or the anti-PD-1 (control) scFv were co-incubated with PD-L1+ CHO-K1 cells over a range of yeast:mammalian cell ratios. (a) Flow cytometry histogram overlays showing yeast binding (as measured by cmyc detection) within the mammalian cell population via biofloating for various yeast:mammalian cell ratios. (b) Phase contrast microscopy images of yeast binding to mammalian cells via biopanning for various yeast:mammalian cell ratios. (c) Quantification of the data shown in (a), displaying the percentage of mammalian cells that are bound to yeast cells at various yeast:mammalian cell ratios. (d) Quantification of the results shown (b), displaying the number of bound yeast cells per image at various yeast:mammalian cell ratios.
Figure 4: Effects of avidity on kinetics of yeast/mammalian cell interactions for biofloating versus biopanning platforms. Atezolizumab (PD-L1-specific) or nivolumab (control) scFv-expressing yeast were co-incubated with PD-L1+ CHO-K1 (Dense), H2444 (Medium), or MDA-MB-231 (Sparse) cells for varying lengths of time. (a) Flow cytometry histogram overlays depicting yeast binding (as measured by cmyc detection) within the mammalian cell population via biofloating for various incubation timepoints. (b) Phase contrast microscopy images of yeast binding to mammalian cells via biopanning for various incubation timepoints. (c) Quantification of biofloating results shown in (a), displaying the percentage of mammalian cells that are bound to yeast cells at various timepoints. (d) Quantification of biopanning results shown in (b), presenting the number of bound yeast cells per image at various timepoints.
Figure 5: Effects of avidity on equilibrium yeast/mammalian cell interactions for biofloating versus biopanning platforms. Atezolizumab (PD-L1-specific) or nivolumab (control) scFv-expressing yeast were co-incubated with PD-L1+ CHO-K1 (Dense), H2444 (Medium), or MDA-MB-231 (Sparse) cells over a range of yeast:mammalian cell ratios. (a) Flow cytometry histogram overlays showing yeast binding (as measured by cmyc detection) within the mammalian cell population via biofloating for various yeast:mammalian cell ratios. (b) Phase contrast microscopy images of yeast binding to mammalian cells via biopanning for various yeast:mammalian cell ratios. (c) Quantification of the data shown in (a), displaying the percentage of mammalian cells that are bound to yeast cells at various yeast:mammalian cell ratios. (d) Quantification of the results shown (b), displaying the number of bound yeast cells per image at various yeast:mammalian cell ratios.
Figure 6: Enrichment of a specific clone spiked into a naïve yeast-displayed scFv library using suspension cell-based versus adherent cell-based selections. (a) Suspension cell-based selection scheme using MACS wherein biotinylated target antigen-expressing mammalian cells are incubated with a yeast-displayed scFv library and immobilized within a magnetic column via streptavidin-coated magnetic beads to allow for enrichment of target antigen-specific yeast. (b) Analysis of the yeast population following each round of evolution performed using the suspension cell-based selection scheme shown in (a). A naïve yeast-displayed scFv library doped with 1:1000 PD-L1-specific atezolizumab scFv-displaying yeast was evolved against PD-L1+ CHO-K1 cells. Enrichment of atezolizumab scFv-expressing yeast (red) and depletion of other scFvs (blue) was monitored by flow cytometry. (c) Adherent cell-based selection scheme wherein a yeast library is incubated over mammalian cell monolayers and washed to allow for enrichment of target antigen-specific yeast. (d) Analysis of the yeast population following each round of evolution performed using the adherent cell-based selection paradigm shown in (c). A naïve yeast-displayed scFv library doped with 1:1000 PD-L1-specific atezolizumab scFv-displaying yeast was evolved against PD-L1+ CHO-K1 cells. Enrichment of atezolizumab scFv-expressing yeast (red) and depletion of other scFvs (blue) were monitored by flow cytometry.
Figure S1: Expression of PD-L1 on various mammalian cell lines. Flow cytometry histograms depicting PD-L1 levels on the PD-L1+ CHO-K1 (left), H2444 (middle), and MDA-MB-231 (right) cell lines. Staining with fluorescently labeled isotype control antibody (red) and anti-PD-L1 antibody (blue) are presented. Note that antigen expression is uniform on each cell line.
Figure S2: Selective binding of anti-PD-L1 scFv-expressing yeast clones to PD-L1+ but not PD-L1- CHO-K1 cells on biofloating and biopanning platforms. Yeast expressing the anti-PD-L1 atezolizumab (High), D12 (Medium), or A1 (Low) scFvs were co-incubated with PD-L1- CHO-K1 or PD-L1+ CHO-K1 cells for 180 min. (a) Flow cytometry histogram overlays depicting yeast binding (as measured by cmyc detection) within the mammalian cell population via biofloating. (b) Phase contrast microscopy images of yeast binding to mammalian cells via biopanning.