Fig. 4

Amino acid positions that control the divergent cellular responses to IgG1 and IgG4 Fc hexamers can be identified using statistically designed mutagenesis. The DoE panel of 16 Fc hexamers were tested in efficacy (phagocytosis) and safety (WB cytokine release, platelet activation, C1q binding) assays. Results are displayed as effects plots including all seven IgG1/4 divergent CH2 residues. Data represent the difference in arithmetic means for graphs a, c and d  and the ratio of geometric means for graph b as compared to IgG1 WT. Error bars represent 95% confidence intervals. a Analysis of phagocytosis inhibition was performed using the mean target cell remaining values for DoE Fc hexamers at 0.8 and 4 μg/ml. n = 1 donor. b Analysis of WB cytokine release was performed using the geometric means of cytokine release (IFN-γ, pg/ml) for DoE Fc hexamers at 33 μg/ml. A log-transformation (base 10) was applied to the data before analysis. n = 3 donors. c Analysis of platelet activation was performed using the mean % of platelets CD62p⁺. The overall mean across donors was taken for DoE Fc hexamers at 100, 50, and 25 μg/ml. n = 3 donors. d Analysis of C1q binding was performed on the mean of Abs 450 nm values for DoE Fc hexamers at 500 and 100 µg/ml