Journal: Scientific Reports

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity

doi: 10.1038/srep07518

Figure Lengend Snippet: (a) Schematic overview of the design of the dimeric constructs. (b) Schematic overview of SPR-based off rate analysis assay. HSA was immobilized on the chip surface. A first injection of dimeric Affibody constructs resulted in a negligible off rate due to the femtomolar affinity of ABD for HSA. VEGFR2 binding was analyzed by subsequent injection of monomeric VEGFR2. The experiments were performed in duplicates. (c) Representative sensorgrams obtained from the SPR-based off-rate analysis assay, showing the injection of 40 nM monomeric human VEGFR2 over each of the four dimeric Affibody molecules. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. (d) Flow-cytometric analysis of binding of dimeric Affibody constructs to VEGFR2-expressing 293/KDR cells. Binding of the Affibody constructs is monitored by the binding of fluorescently labeled HSA to the ABD tag. A higher shift in mean log fluorescence intensity compared to the negative control construct Z Taq -ABD-Z Taq or cells labeled with secondary reagent only was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR2-expressing cells. The experiment was performed in duplicates. (e) Inhibition of VEGF-A induced phosphorylation of VEGFR2 on 293/KDR cells. Cells were pre-treated with the biparatopic binder Z VEGFR2_22 -(S 4 G) 4 -ABD 035 -(S 4 G) 4 -Z VEGFR2_40 , a combination of 30 nM or 3 nM of each of the monomeric binders Z VEGFR2_22 and Z VEGFR2_40 , 30 nM or 3 nM of the negative control construct Z Taq -ABD 035 -Z Taq , or PBS, followed by stimulation with VEGF-A. VEGFR2 phosphorylation was determined by ELISA. The biparatopic binder and the combination of monomers both resulted in a decrease in phosphorylation level compared to the controls, and a more potent inhibition was observed for the biparatopic binder. The data is presented as the OD450 for each sample normalized against the OD450 of untreated cells. The experiment was performed in duplicates.

Article Snippet: HSA (Sigma-Aldrich) was immobilized on a GLM sensor chip (Biorad Laboratories) using a ProteOn XPR36 instrument (Biorad Laboratories).

Techniques: Construct, Injection, Binding Assay, Expressing, Labeling, Fluorescence, Negative Control, Inhibition, Enzyme-linked Immunosorbent Assay