(A) Schematic illustration of the BsAb in the VHS platform. The blue asterisks represent VEGFA to which the BsAb can bind, and the red asterisks represent HER2 to which the BsAb can bind. (B) Coomassie blue–stained gels of TB-VHS and its parental antibodies separated by SDS-PAGE under reducing (right) and nonreducing (left) conditions. HC, heavy chain; LC, light chain; MW, molecular weight. (C) Specific binding of TB-VHS and its parental antibodies to HER2 and human VEGFA detected by ELISA. For detecting HER2 binding, HER2 ECD recombinant protein–coated 96-well microplates were used to capture the antibodies, and antibodies were detected by HRP-labeled anti–human IgG antibody. For detecting VEGFA binding, rabbit anti–human Fc antibody–coated 96-well microplates were used to capture the antibodies, and antibodies were incubated with biotinylated human VEGFA and detected by streptavidin-HRP conjugate. (D) Thermal stability of TB-VHS. TB-VHS, bevacizumab, and trastuzumab were incubated in a water bath at 50°C for 1 hour, and then binding of these antibodies and antibodies stored at 4°C against HER2 and human VEGFA was detected by ELISA as in C. (E) Competitive binding of TB-VHS to HER2 and human VEGFA. TB-VHS (5 nM) was incubated with either 5 nM biotinylated VEGFA and increasing concentrations of HER2 ECD recombinant protein (left) or 5 nM HER2 ECD recombinant protein and increasing concentrations of VEGFA (biotinylated and unlabeled) (right) in a solution at 4°C for 1 hour. Then, separate 96-well microplates coated with rabbit anti–human Fc antibody were used to capture TB-VHS. Binding of VEGFA to TB-VHS was detected by streptavidin-HRP, and binding of HER2 ECD to TB-VHS was detected by a biotinylated anti-HER2 antibody and then streptavidin-HRP.