Fig. 3: Binding of IgG-Fab regions to membrane-associated FcRn and cellular uptake of IgG1.

a Schematic overview illustrating the principle of the surface competition assay to measure membrane-associated FcRn binding. b Histograms of a representative experiment comparing the binding of AF650-labeled anti-FcRn Fab to membrane-associated FcRn expressed on the surface of HEK-FcRn-GFP cells when co-incubated with the indicated FcRn inhibitors (full-size IgG1 or Fc fragments) at pH 6.0 on ice at 4 °C. Detection (AF650-labeled anti-FcRn Fab):inhibitor molar ratio at highest inhibitor concentration = 1:25 (optimized to measure competition with MST-HN containing IgG1). c Anti-FcRn Fab-AF650 staining of cells plotted as percent gMFI in the presence of indicated inhibitors compared with untreated cells. d Anti-FcRn Fab-AF650 staining of cells plotted as in (c), but for WT IgG1 variants of different specificities with indicated numbers of Fab or H435A variant (does not bind detectably to FcRn). Control IgG1 is an antibody directed against an irrelevant and non-disclosed target. Detection (AF650-labeled anti-FcRn Fab):inhibitor molar ratio at highest inhibitor concentration = 1:250 (optimized to measure competition with WT-IgG). Data represent histograms from a representative experiment, with indicated numbers showing gMFIs averaged from measured duplicates from the same experiment in (b), with aggregated data presented as mean values from three independent experiments in (c) and five in (d) using flow cytometry. Error bars indicate standard deviations. Statistical analysis was performed using a two-way ANOVA (Tukey’s multiple-comparisons test) (c) and one-way ANOVA (Dunn’s multiple-comparisons test) comparing to Fc-WT (d) and statistically significant differences are indicated by asterisks: *<0.05, **<0.01, ****<0.0001. ns not significant.