Figure 2. Comparison of cytotoxic activity engaged by rituximab when targeting CHO-S cells displaying the rituximab epitope at different distances from the plasma membrane.

CHO-S cells transfected with each of the fusion proteins (Figure 1) were used as targets to investigate antibody effector mechanisms in vitro. A-B) Transfected CHO-S cells were labelled with RTX or an isotype control (ISO) and incubated with 15% human serum for 30 minutes. CDC was measured by flow cytometry using propidium iodide inclusion as a measure of cell lysis. A) shows representative data and B) results from n=3 independent experiments. C-D) CFSE labelled targets were opsonised with RTX or ISO before use as targets in an ADCP assay with murine BMDM. The percentage of BMDM which phagocytosed the target cells (defined as the F4/80⁺ CFSE⁺ population) was recorded and plotted above. C) shows representative flow cytometry data along with confocal imaging confirming phagocytosis of CFSE labelled targets. In the confocal imaging CFSE labelled targets are in green, wheat germ agglutinin (WGA) stained surface membranes are red and DAPI-stained nuclei are blue. D) results from n=3 independent experiments. E) Calcein-AM labelled targets were opsonised with RTX or ISO before acting as targets in ADCC assays with human PBMC. For all experiments the mean and SD of three independent experiments are presented. Statistical significance was assessed using a two-way ANOVA test. * p <0.05, ** p <0.005, *** p <0.001, **** p <0.0001.