Journal: mAbs

Article Title: Coactivation of Tie2 and Wnt signaling using an antibody-R-spondin fusion potentiates therapeutic angiogenesis and vessel stabilization in hindlimb ischemia.

doi: 10.1080/19420862.2024.2435478

Figure Lengend Snippet: Figure 2. Generation of T11-based bifunctional antibodies fused with furin-1 and −2 (Fu1/2) domains of RSPO3. (a) Effect of T11 and ang-1 on Wnt/β-catenin signaling. 293T-hTie2 cells transfected with STF and Renilla luciferase reporter plasmids were plated and treated with control IgG (10 μg/mL), T11 IgG (10 μg/mL), ang-1 (300 ng/ mL), recombinant Wnt3a (300 ng/mL) and/or recombinant RSPO3 (20 ng/mL) for 24 h. Relative luciferase values represent mean of triplicate measurements. (b) A schematic of bifunctional antibody–RSPO3 fusions. T11 was utilized as a backbone IgG, and the Fu1/2 domains of RSPO3 were appended on the C-terminus of HC or LC of T11, to generate T11–RF12 or T11–RF12-LC. (c Analysis of purified antibody fusions. T11–RF12 or T11–RF12-LC was purified and analyzed using SDS-PAGE under non-reducing and reducing conditions. (d) and (e) binding activity of antibody fusions. Binding of T11–RF12 and T11–RF12-LC to counterpart molecules was assessed using indirect ELISA with recombinant Tie2-ecd (D), LGR5, or ZNRF3 (E). One-hundred nanomolar of control IgG, T11, T11–RF12, or T11–RF12-LC was allowed to bind to each protein. Values represent mean ± SD of duplicate measurements.

Article Snippet: Antibodies (100 nM), recombinant human Angiopoietin-1 (Ang-1) (400 ng/mL; R&D Systems, Cat. # 923-AN), Wnt3a (300 ng/mL; R&D Systems, Cat. # 5036-WN), or RSPO3 (20 ng/mL; R&D Systems, Cat. # 3500-RS) were added to each well and the plate was incubated for 18 h. The wells were subsequently examined under a light microscope (Olympus), and the tube lengths and branch points were quantified using the ImageJ software (National Institutes of Health, http://rsbweb. nih.gov/ij/).

Techniques: Transfection, Luciferase, Control, Recombinant, Purification, SDS Page, Binding Assay, Activity Assay, Indirect ELISA

Journal: mAbs

Article Title: Coactivation of Tie2 and Wnt signaling using an antibody-R-spondin fusion potentiates therapeutic angiogenesis and vessel stabilization in hindlimb ischemia.

doi: 10.1080/19420862.2024.2435478

Figure Lengend Snippet: Figure 3. Enhanced activation of Wnt/β-catenin signaling and endothelial tube formation by T11–RF12. (a) and (b) activation of Wnt/β-catenin signaling by antibody fusions. 293T-hTie2 cells transfected with STF and Renilla luciferase reporter plasmids were plated and treated with 100 nM of control IgG, T11, T11–RF12, T11–RF12-LC, or recombinant RSPO3 (20 ng/mL) without (A) or with (B) recombinant Wnt3a (300 ng/mL). Relative luciferase values represent mean ± SD (n = 4). (c) and (d) induction of endothelial tube formation by T11–RF12. HUVECs were seeded on matrigel-coated plates and incubated in the presence of control IgG (ctrl IgG), T11 (100 nM), or T11–RF12 (100 nM), or RSPO3 (20 ng/mL) without (a) or with (b) recombinant Wnt3a (300 ng/mL). After 18 h, tube formation in each well was photographed (×40), and relative tube lengths or branch points were quantified by analyzing the images. Values represent mean ± SD of triplicate measurements. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Antibodies (100 nM), recombinant human Angiopoietin-1 (Ang-1) (400 ng/mL; R&D Systems, Cat. # 923-AN), Wnt3a (300 ng/mL; R&D Systems, Cat. # 5036-WN), or RSPO3 (20 ng/mL; R&D Systems, Cat. # 3500-RS) were added to each well and the plate was incubated for 18 h. The wells were subsequently examined under a light microscope (Olympus), and the tube lengths and branch points were quantified using the ImageJ software (National Institutes of Health, http://rsbweb. nih.gov/ij/).

Techniques: Activation Assay, Transfection, Luciferase, Control, Recombinant, Incubation