Figure 5. Impairment of PMN-mediated ADCC activity by an FcγRIII-optimized anti-EGFR antibody variant is restored by additional improvement of its FcγRIIa affinity. (A, B) PMN from healthy blood donors were left untreated (A) or were stimulated with GM-CSF (B) and subsequently used as effector cells in ADCC experiments utilizing indicated antibodies at increasing concentrations and A431 cells (left panels). Results from different blood donors at highest antibody concentrations were separately depicted (right panels). Data are presented as mean ± SEM from at least three independent experiments with different blood donors. * P ≤ 0.05 for wild type antibody vs. antibody variants; # P ≤ 0.05 for S239D/I332E/G236A vs. S239D/I332E. (C) Neutrophil-enriched whole blood samples from G-CSF primed donors were used as effector source in ADCC experiments against A431 cells. Data are presented as mean ± SEM from three independent experiments with different blood donors. * P ≤ 0.05 for wild type antibody vs. antibody variants; # P ≤ 0.05 for S239D/I332E/G236A vs. S239D/I332E.