Figure 4.

Antibody binding to HER2 on SK-BR-3 cells, measured by immunofluorescence before and after cleavage of the linker between the mask and trastuzumab and between the two structural domains in the mask in the T-scFv40 and T-scFv69 constructs. T alone is a positive control whilst NIP228 alone is a negative control; T-scFv40 and T-scFv69, intact, digested by TEV, Factor Xa, and both enzymes; T-dAb intact and digested by TEV; T-scFvGipg013 intact. All antibodies were detected with Alexa488 anti-human secondary (green) and nuclear Hoechst stain (blue).

Figure 4 Alt text: Large figure showing microscopy images of the binding to SK-BR-3 cells of the masked antibodies, intact and cleaved, at different concentrations. The bound antibodies are observed in green, and the cell nuclei in blue. For T-scFv40: T-scFv40 + TEV and T-scFv40 + TEV+ Factor Xa bind as much as naked trastuzumab, followed by T-scFv40 + Factor Xa, followed by T-scFv40 intact. For T-scFv69: T-scFv69 + TEV + Factor Xa bind as much as naked trastuzumab, followed by T-scFv69 + TEV, followed by T-scFv69 + Factor Xa which binds as much as T-scFv69 intact. For T-dAb: very little binding to cells is observed for the intact and digested constructs.