Figure 2. Impaired ADCC activity triggered by Fc-engineered antibody is restored using FcγRIII negative granulocytes. (A) Fc receptor expression was analyzed by indirect immunofluorescence. Unfractionated PMN from healthy donors (upper panel), patients with PNH (middle panel) or eosinophils (lower panel) were collected from freshly drawn peripheral blood. PMN from patients with PNH expressed low levels of the GPI-linked FcγRIII (CD16), while FcγRII (CD32) expression was similar to PMN from healthy donors. Unfractionated PMN from healthy donors could be divided into FcγRIII negative eosinophils and FcγRIII positive neutrophils (upper panel). FcγRIII negative eosinophils were isolated from the PMN fraction from healthy donors with eosinophilia and analyzed for FcR expression (lower panel). Eosinophils did express FcγRII (CD32) and FcαRI (CD89). (B) In ADCC experiments against A431 cells, PMN from healthy donors were effective with wild type but not with S239/I332E mutated anti-EGFR antibodies (left panel; all 10 µg/ml). PMN from PNH patients (middle panel) and eosinophils from healthy donors with eosinophilia (right panel) mediated similar ADCC with the S239D/I332E antibody variant and wild type antibody. For the three blood donor types, MNC-mediated killing was enhanced with Fc-engineered antibody (lower panel). In ADCC experiments against A431 cells, MNC-mediated killing was enhanced with Fc-engineered S239D/I332E antibody compared with wild type antibody (left panel). Data are presented as mean ± SEM from three independent experiments. * P ≤ 0.05 EGFR-targeted antibody vs. control antibody.