Figure 1. Design and validation of FLAgs as activators of the Wnt-βcatenin pathway.

(A) Surface plasmon resonance (SPR) binding kinetics of F^P Fab. Kinetics were derived from curves for soluble F^P Fab interacting with immobilized FZD CRD, and ‘x’ indicates no detectable binding. (B) Anti-LRP6 Ab inhibitory activity. Inhibition of WNT1 or WNT3A signaling by indicated LRP6 Abs in the diabody-Fc format was assessed using stimulation with purified WNT3A or upon WNT1 cDNA transfection. (C) Molecular architecture of tetravalent FLAgs. (D) Activation of βcatenin signaling by FLAgs. Dose response curves are shown for the activation of a LEF/TCF reporter gene (y-axis) in HEK293T cells by serial dilutions of pan-specific FLAg proteins (F^P+P-L6¹⁺¹, F^P+P-L6³⁺³ and F^P+P-L6¹⁺³) (x-axis). Error bars indicate SEM, n = 3. (E) Levels of βcatenin protein in RKO cells after 30 min treatment with the indicated concentrations of pan-FLAg (F^P+P-L6¹⁺³). Representative blot of three replicates. (F) Time course of βcatenin and phosphorylated Disheveled-2 (p-DVL2) protein levels in RKO cells treated with 10 nM pan-FLAg (F^P+P-L6¹⁺³). Representative blot of three replicates.

Figure 1—figure supplement 1. Development of Synthetic Modular Tetravalent FZD.

(A) SPR binding kinetics of F^P IgG. 'x’ indicates no detectable binding. (B) Epitope binning for L6¹ and L6³ IgG by competitive blocking measured using BLI. The normalized signal is shown for the binding of L6¹ or L6³ (x-axis) to immobilized LRP6-Fc pre-blocked with the indicated Ab (y-axis). (C) Schematic representation of bispecific diabody-Fc (db) and single-chain bispecific IgG (bs) modalities recognizing FZD and LRP6 receptors. (D–E) Effects of bispecific IgGs and diabody-Fcs on Wnt-βcatenin signaling, as measured using HEK29T cells stably expressing a LEF/TCF reporter and with or without WNT1 transient transfection or treatment with WNT3A (0.5 μg/ml). (F) Coomassie-stained SDS-PAGE analysis of purified FLAg F^P+P-L6¹⁺³ under oxidizing (Ox) or reducing (red) conditions. (G) βcatenin signaling stimulated by FLAg or WNT3A conditioned media, measured using LEF/TCF reporter assay in HEK293T cells. (H) Co-binding of FLAg F^P+P-L6¹⁺³ to absorbed LRP6-Fc (LRP6) and soluble concentrations of FZD4-His (FZD4) by ELISA with anti-His-HRP detection. (I) Dose response analysis of βcatenin signaling activity stimulated with mouse WNT3A (R and D systems 1324-WN/CF), human/mouse WNT5A (R and D systems, 645-WN/CF) purified proteins, FLAg F^P+P-L6¹⁺³ or CHIR99021. HEK293T cells stably expressing a LEF/TCF reporter were stimulated with the indicated doses for 16 hr. (J) The fold change of luciferase activity at maximal stimulation of Wnt reporter cells treated with 20 nM FLAg F^P+P-L6¹⁺³ , 20 nM purified WNT3A, or 12 μM CHIR99021.