Figure 3.

Binding of the mono- and bi-specific proteins to cell-expressed PDL1 and recombinant VEGF₁₂₁. (A) Binding titration curves for the Du mono-specific (blue triangles) and DuRan-Bis bi-specific (green circles) proteins to endogenous PDL1 on the surface of U87MG cells. Binding was measured using FACS at protein concentrations of 0.001–5 nM (Du) and 0.001–50 nM (DuRan-Bis). The binding was determined using an APC-conjugated anti-His tag antibody. Fluorescence signals obtained from cells treated with APC-conjugated anti-His tag antibody in the absence of Du or DuRan-Bis proteins were subtracted from all samples (containing different protein treatments), and the resulting signal intensities were then normalized to the highest binding signal exhibited by the same protein treatment. The data was fitted to a 1:1 Langmuir kinetic binding model to yield the apparent K_D values for Du and DuRan-Bis proteins. Values are the means of triplicate experiments; bars represent standard error of the mean (SEM). (B) Flow cytometry analysis showing the dual binding of the bi-specific protein DuRan-Bis in U87MG cells (that express PDL1) pre-incubated with Du or DuRan-Bis (100 nM) and then monitored to determine the enhancement in binding response upon addition of biotinylated VEGF₁₂₁ (100 nM), followed by streptavidin-conjugated APC. The following color coding is used: grey—untreated cells, black—cells treated with biotinylated VEGF₁₂₁ and then streptavidin-conjugated APC, blue—cells treated with Du followed by biotinylated VEGF₁₂₁ and then streptavidin-conjugated APC, green—cells treated with DuRan-Bis followed by VEGF₁₂₁ and then streptavidin-conjugated APC.