Skip to main content
. 2018 Nov 20;78(2):228–237. doi: 10.1136/annrheumdis-2018-213523

Figure 1.

Figure 1

VIB9600 specifically binds FcγRIIA, competes with IgG for binding to FcγRIIA, causes receptor internalisation but fails to induce effector mechanisms. (A) Epitope competition data with IV.3 (mouse IgG 2b Ab), CamIV3 (humanised framework regions with IV.3 CDRs) and VIB9600 (humanised and optimised IV.3) on both human FcγRIIA 131 H (left) and FcγRIIA 131R (right). Representative data from two independent experiments are shown. (B) VIB9600 binding to human FcγRs in an ELISA-based binding assay. Plots represent the mean±SD. A representative plot of two independent experiments is shown. (C) In ADCC and CDC assays, the effects of VIB9600 were compared with wild type control 9600 IgG1 and isotype control IgG (R347-Tm) as indicated. in the ADCC assay, primary NK cells (effectors) were incubated with adherent FcγRIIA-expressing HEK-293 cells (targets) for 5 hour, and % cytotoxicity was determined. For CDC assays, baby rabbit complement was incubated with adherent FcγRIIA-expressing HEK-293 cells (targets), and % cytotoxicity was determined after 1 hour. Plots represent the mean±SD. Representative plots of three independent experiments are shown. (D) Binding of VIB9600 and control Ab (R347-Tm) to human FcγRIIA-expressing neutrophils in the presence and absence of 10 mg/mL IVIg (as indicated) was determined by flow cytometry (M.F.I). Representative data from two independent experiments. (E) Human monocytes were stained with CD14-Alexa 488 (green) and VIB9600-Alex 647 (red), and internalisation of FcγRIIA on human monocytes was visualised by confocal microscopy at time 0 and after culturing at room temperature for 1 hour. A representative image of three independent experiments is shown. (F) Available cell surface FcγRIIA on human monocytes and neutrophils in whole blood from healthy donors with either a 131 H/H or 131 R/R genotype (as indicated) was examined following a 2-hour incubation with VIB9600 or control Ab (R347-TM) by flow cytometry (M.F.I). (G) Similarly, cell surface FcγRIIA on cynomolgus monkey monocytes and neutrophils in whole blood was examined following a 12-hour incubation with VIB9600 or control Ab (R347-TM) by flow cytometry (M.F.I). Representative data from three independent humans and cynomolgus monkey experiments are shown. Ab, antibody; ADCC, antibody-dependent cell-mediated cytotoxicity; CDC, complement-dependent cytotoxicity; TM, triple mutation.