Binding of 17C02-based molecules to human FcγRIIIA expressed on THP-1-CD16A cells, THP-1 cells, as well as primary cells. Cells were incubated with the indicated concentration of either 17C02-based molecules, 3G8-based molecules, or albumin alone (negative control). The following were the cell types used for each panel: (A,D) THP-1-CD16A cells; (B,E,H) NK cells from healthy human donors; and (C,F,I) neutrophils from healthy human donors; as well as (G) a comparison between THP-1-CD16A and THP-1 cells. Binding of albumin fusion proteins was detected using a FITC-labeled monoclonal antihuman albumin antibody, whereas binding of deglycosylated full-length antibodies or the 1-armed antibody was detected using an APC-labeled goat F(ab’)2 antimouse IgG (Fc-specific) or an AF647-labeled donkey antihuman IgG (H + L), respectively. Stained cells were washed and analyzed by flow cytometry using the BD LSRFortessa X-20. Data analysis was performed using FlowJo v10. Data are presented as mean ± standard deviation from 3 to 5 independent experiments. The dashed line represents the mean fluorescent intensity (MFI; arbitrary units) value for the corresponding secondary antibody alone at 5 μg/mL. Statistical analysis was performed using a 2-way analysis of variance (ANOVA) and Sidak multiple comparisons test, comparing the MFI values of the 3 molecules at each antibody concentration (∗P < .05; ∗∗P < .01).
