Figure 3

Ex vivo B-cell depletion activity of anti-CD20 IgG1 rituximab variants in a whole blood matrix. (a) Heparinized peripheral blood from healthy donors 1 and 2 were incubated with serial dilutions of anti-CD20 IgG1 rituximab variants (fucosylated (closed circle), non-fucosylated (open circle), triple amino acid-substituted mutant S239D/S298A/I332E (closed square)). No significant difference in cytotoxicity was observed between non-fucosylated and triple amino acid-substituted mutant S239D/S298A/I332E by paired t-test. (b) Non-fucosylated anti-CD20 was added to the whole blood sample from donor 3 at 0.11 μg/mL (white column). Non-fucosylated (gray column) or fucosylated (black column) anti-CD20 was further added to the reaction at final concentrations as indicated. In both experiments, the remaining B cells after 4-h treatment were stained with anti-CD19-FITC, and the samples were analyzed on a flow cytometer to quantify the number of B cells. B-cell depletion is represented as cytotoxicity (%) as described in Materials and Methods.