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. 2022 Mar 3;298(4):101798. doi: 10.1016/j.jbc.2022.101798

Figure 6.

Figure 6

In vitro ADCP and ADCC mediated by Fc variant antibodies.A, flow cytometric ADCP assay with human THP-1 monocytic cells at pH 7.4. Her2-coated fluorescent beads were labeled with an intracellular pH fluorogenic probe before incubation with THP-1 cells and hu4D5 antibodies bearing wild-type (WT) Fc, acid-Fc, or an isotype control with wild-type Fc and an irrelevant Fab domain. After incubation, the cells were scanned by flow cytometry and the phagocytosis score calculated as %(bead internalization) ∗ GMFI(bead association). B, antibody variants (50 ng/ml) were added to calcein-loaded Her2-positive SKOV3 target cells in the presence of NK92 cells stably expressing FcγRIIIa (V158) for 4 h at an E:T ratio of 10:1. The data shown are representative of replicate experiments. C, dose-dependent ADCC assay with hu4D5 antibodies and calcein-loaded Her2-positive SKBR3 target cells in the presence of NK92 (V158) cells. The data shown are pooled from two experimental replicates, each performed with two technical replicates. Mean and SD are shown for each data point (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 determined by t test in GraphPad), as well as the fitted four-parameter logistic curves (smooth line). ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis.