Diagram of the activation of the signaling pathway by the split molecules through the binding to a bridging receptor on the targeting cell surface.

A) Efficient WNT/β-catenin signaling requires two FZD binding domains and two LRP binding domains in one WNT mimetic molecule. B) WNT mimetic molecule is split into one molecule having two FZD binding arms (2:0) and a second molecule having two LRP binding arms (0:2) and then each tethered to a different “bridging element” binding to a different epitope on the same bridging receptor (named αLRP-αBR-1 and αFZD-αBR-2) (top two panels). On targeting cells where the BR is expressed, FZD and LRP are then assembled by αLRP-αBR-1 and αFZD-αBR-2 via BR recreating the signaling competent receptor complexes (bottom panel).

Diagrams of the molecules used in the experiment to provide proof of concept for SWIFT.

A) Elements that are used in the split molecules. LRP6 binder element is in scFv format; FZD binder is in the IgG1 format; Both αGFP IgG1 and αGFP scFv are used for assembly of the negative control molecules. Two types of βKlotho binders are used. Binder#1, 39F7 IgG1, a βKlotho monoclonal antibody; Binder#2, FGF21FL and different deletion variants. B) The diagram of the assembled SWIFT molecules. C) The diagram of the assembled negative control molecules. (D-G) Bindings of various F-FGF21 fusion proteins to FZD7 and βKlotho. Bindings of FZD7 and β-Klotho to F-FGF21FL (D), F-FGF21ΔN (E), F-FGF21ΔC (F), or F-FGF21ΔNΔC (G) were determined by Octet. (H-J) Binding of LRP6 and βKlotho to L-39F7 (H), αGFP-39F7 (I), or L-αGFP (J) were determined by Octet. Mean KD values were calculated from all 7 binding curves with global fits (red dotted lines) using 1:1 Langmuir binding model. (K) Step bindings of FZD7 and βKlotho to various F-FGF21 fusion proteins. Sequential binding of F-FGF21FL (blue sensorgram), F-FGF21ΔN (red sensorgram), F-FGF21ΔC (light green sensorgram), or F-FGF21ΔNΔC (green sensorgram), followed by FZD7 CRD, then followed by addition of βKlotho on Octet shows simultaneous engagement of both FZD7 and βKlotho to the indicated F-FGF21 proteins. Sensorgrams for FZD7 and βKlotho area (red dotted box) are enlarged at the right. (L) Step binding of LRP6 and βKlotho to L-39F7 and its control proteins. Sequential binding L-39F7 (blue sensorgram), L-αGFP (light blue sensorgram), αGFP-39F7 (red sensorgram), or 39F7 IgG (green sensorgram), followed by LRP6E3E4, then followed by addition of βKlotho on Octet shows simultaneous engagement of both LRP6 and βKlotho to the indicated L-39F7 and its control proteins. Sensorgrams for LRP6 and βKlotho area (red dotted box) are enlarged on the right.

Dose dependent STF assay of SWIFT molecules in HEK293 and Huh7 cells.

A-F) Various F-FGF21 fusion proteins in the presence of L-39F7 in HEK293 STF cells (A) and Huh7 STF cells (B); various F-FGF21 fusion proteins in the presence of L-αGFP in HEK293 STF cells (C) and Huh7 STF cells (D); and various F-FGF21 fusion proteins in the presence of αGFP-39F7 in HEK293 STF cells (E) and Huh7 STF cells (F). G) Expression of bridging receptor βKlotho (KLB) in HEK293 and Huh7 cells. Data are representative of three independent experiments performed in triplicates and are shown as mean ± standard deviation (SD).

Activity of targeted molecules in primary human cells.

A) Representative images of primary human hepatocytes cultures in 2D or human small intestinal organoids. Scale bars 200 µm. B) Expression of bridging receptor βKlotho (KLB) in hepatocytes or small intestinal cells. C) WNT target gene AXIN2 expression normalized to control treatment after 24-hour treatment with 10 nM of indicated molecules in human hepatocytes. D) WNT target gene AXIN2 expression normalized to control treatment after 24-hour treatment with 10 nM of indicated molecules in human small intestinal organoids. *** = P ≤ 0.001 (one-way ANOVA), each data point represents an independent experiment performed in duplicates.