Extended Data Fig. 3: P2G3 LS shows strong Fc-mediated functional activity in ADCC cell killing and ADCP assays.
a) Antibody dependent cellular cytotoxicity assay (ADCC) performed with CEM NKR Luciferase cells stably expressing cell surface 2019-nCoV Spike. P2G3 exhibits potent ADCC activity in killing Spike positive cells. ADCC experiments performed with five replicates per condition using effector cells from five different healthy donors with each donor evaluated in one to three different experiments (n = 50 tests per condition). CEM NKR Spike cells were incubated with 0.30 µg/ml of the indicated human IgG mAbs. Violin plots are shown with median values as thick middle line and quariles for upper and lower lines. Statistical difference evaluated by two-way ANOVA with p-values presented as p = 0.0096 (**), p = 0.0001 and 0.0002 (from left to right ***) and p < 0.0001 (****). b) Flow cytometry gating strategy for the selection of U937 cells, the removal of cell doublets and the evaluation of cells for which Spike-coated fluorescent beads have undergone phagocytosis. The threshold gate for Spike-specific phagocytosis of beads was set using and isotope control antibody and representative dot plots for P2G3 mediated ADCP of 2019-nCoV Spike-coated beads by U937 are shown with >4000 cells analysed per condition. c, d) Antibody dependent cellular phagocytosis assay performed ancestral 2019-nCoV and with Omicron BA.1 variant Spike protein biotinylated and bound to streptavidin coated fluorescent beads. Beads mixed with the indicated antibody concentrations were incubated with the U937 monocyte effector cell line and antibody dependent cellular phagocytosis of the Spike coated beads was evaluated by flow cytometry. Dashed lines correspond to individual antibodies and solid lines indicate combinations of P2G3 and P5C3. Results shown are representative data for three separate experiments with each concentration response tested in duplicates or triplicates. Mean values ± SEM are shown.
