Fig. 5. Activity of bnDs as bivalent molecules.

a Model of a bivalent DARPin-Fc generated by fusing the DARPin (violet with orange interaction surface) to the N-terminus of the Fc-region of human IgG1 (light grey and dark grey for the paired chains) with a preserved hinge region. b Binding of bivalent DARPin-Fc fusions to cell surface-expressed Env (JR-FL) measured by flow cytometry. DARPins-Fc fusions were titrated and incubated with cells in the presence or absence of 2 µM sCD4. Mulv Env served as a negative control. Histograms of normalized fluorescence intensities (top panel) and dose-response curves with mean fluorescence intensities (MFI, bottom panel) are depicted. See also linked experiments in Supplementary Fig. 12. c Binding of increasing concentrations of monovalent DARPins and bivalent DARPin-Fc fusions to wt (BG505-SOSIP) and V1V2 deleted Env trimer (BG505-SOSIP∆V1V2) in ELISA. Representative relative luminescent unit (RLU) data from one of two independent experiments are shown. d Comparison of monovalent and bivalent V3 inhibitors. Log IC₅₀ ratios (Fabs/mAbs and bnDs/Fc-bnDs) from a panel of 9 Tier-2 viruses (top) and corresponding V1V2-deleted viruses (bottom) are depicted. Values > 1 indicate higher potency of the bivalent inhibitor version. Individual data points are shown as circles and were calculated from geometric means of two independent experiments for wt and V1V2-deleted virus respectively (n = 2). Pairs with resistant strains were not included in the plots. Box plot limits extend from the 25th to 75th percentiles; centerline: median; whiskers indicate the minimum and maximum values. See Supplementary Data 9 and Supplementary Fig. 13 for a full data set.