Fig 1: F4L5.13 specifically binds FZD4/LRP5 co-receptors F4L5.13 is specific for FZD4, as determined by biolayer interferometry (BLI). F4L5.13 or the isotype control molecule (100 nM) was tested for binding to immobilized recombinant FZD CRDs or the ectodomain of the unrelated receptor Her2. BLI sensors coated with Fc were used to determine the baseline response.Binding kinetics, as determined by BLI, for the binding of immobilized F4L5.13 to FZD4 CRD or LRP5. Experiments were performed in triplicate, and data are presented as the mean ± SD.F4L5.13 selectively binds to LRP5 over LRP6. LRP5/6 proteins coated on a maxisorp plate are recognized in a concentration-responsive manner by F4L5.13 but not by a non-targeting IgG control (4275). F4L5.13 binds to LRP5 with a low nanomolar EC50 value and is > 50-fold selective for LRP5 over LRP6. Data are presented as the mean ± SD from three independent experiments.Activation of ßcatenin signaling by WNT3A, F4L5.13, or recombinant NDP (30 nM each) in HEK293T cells transfected with plasmids encoding FZD4 and/or LRP5. Values represent fold activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n = 3.Binding of F4L5.13 to the cell surface of HEK293T cells overexpressing FZD4 and LRP5 by flow cytometry. Data are representative of two experiments.
Fig 2: F4L5.13 treatment activates the Wnt-ßcatenin pathway in LEF/TCF reporter cell lines and cultured endothelial cells Top: Molecular architecture of tetravalent F4L5.13. Bottom: Schematic for activation of Wnt/ßcatenin signaling by F4L5.13.Activation of ßcatenin signaling by F4L5.13 or recombinant NDP (30 nM each) in HEK293T cells transfected with FZD4 and/or LRP5. Values represent fold activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n = 3.Dose–response curves for the activation of a LEF/TCF reporter gene in HEK293T cells transfected with plasmids encoding FZD4, LRP5 and with either GFP or TSPAN12 by serial dilutions of F4L5.13 or NDP proteins (x-axis). Data are presented as mean ± SEM, n = 3.RT–qPCR of Axin2 expression in bEnd.3 cells treated with serial dilutions of F4L5.13, NDP, or isotype control for 24 h. Data are presented as mean ± SEM, n = 3.RT–qPCR of Axin2 in bEnd.3 cells treated with NDP (200 ng/ml), isotype control or F4L5.13 (1,200 ng/ml) and transfected with control, Tspan12 or Fzd4 siRNAs. Data are presented as mean ± SD, n = 3 technical replicates. Data are representative of two independent experiments.Time course of phosphorylated Dishevelled-3 (p-DVL3) and ßcatenin protein levels in bEnd.3 cells treated with 30 nM of F4L5.13 or NDP. Histogram represents the ratio of the DVL3 phosphorylation levels over total DVL3 protein and ßcatenin levels over ß-Tubulin measured by densitometry of independent experiments. Data are presented as mean ± SEM, n = 4–6 (*P < 0.05 as compared with NT). Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (*P < 0.05 as compared with NT).
Fig 3: Downregulation of Tspan12, Fzd4, and Lrp5 in bEnd.3 cells A, BRT–qPCR of Tspan12 (A) and Fzd4 (B) mRNA expression in bEnd.3 cells transfected with scrambled, Fzd4- or Tspan12-targeting siRNA and treated or not with isotype control, F4L5.13 or NDP for 24 h. Data are presented as mean ± SD, n = 2 technical replicates. Data are representative of two independent experiments.CRT–qPCR of Axin2 in bEnd.3 cells transfected with control or Lrp5-targeting siRNAs and treated or not with isotype control, F4L5.13 or NDP for 24 h. Data are presented as mean ± SD, n = 3 technical replicates. Data are representative of two independent experiments.DRT–qPCR of Lrp5 mRNA expression in bEnd.3 cells transfected with scrambled or Lrp5 siRNA and treated or not with isotype control, F4L5.13 or NDP for 24 h. Data are presented as mean ± SD, n = 3 technical replicates. Data are representative of two independent experiments.
Fig 4: A FZD4:LRP5 antibody agonist reduces pathological neovascularization in the OIR model Schematic diagram of the OIR model (top) and representative images of P17 OIR retinas are shown (bottom). Neonatal mice were exposed to 75% oxygen from P7 to P12 to induce vessel loss. Then mice received intravitreal injections of PBS vehicle, mouse-specific anti-VEGF (as a positive control) delivered at 0.1 µg/µl, or F4L5.13v2 (a second-generation F4L5.13 modality, in which the N-terminal FZD4-specific diabody was replaced with a FZD4-specific Fab) at 50 nM or 500 nM target vitreous concentrations. Mice were returned to room air from P12 to P17 to induce maximum pathologic neovascularization at P17. At P17, retinas from injected mice were collected, dissected as flat-mounts, and stained with Isolectin B4. Scale bar = 500 µm.Quantification of the ratio of neovascular and avascular area on total area of vascularization. Data are presented as mean ± SEM, n = 11–13 retinas per group. Significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test (indicated P-values are in comparison to Vehicle).
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