FIGURE 4.

Biological activity of bispecific DARPins with a flexible linker. Each symbol or bar represents the average of three data points. A, inhibition of cell viability as determined by XTT assays. Cells were treated for 72 h with different concentrations of bivalent DARPins. A 1:1 mixture of DARPins E01 and E69, E01_GS_E69, and E69_GS_E01 affected the cell proliferation similar to cetuximab (Cet) (*, p < 0.05 compared with untreated cells; Student's t test); the negative control DARPin Off7 did not. B, inhibition of cell proliferation as determined by clonogenic assays. A431 cells were treated for 7 days with 100 nm DARPin or 100 nm cetuximab, after which the medium was changed, and cells were allowed to grow for another 7 days. C, cell cycle distribution. A431 cells were treated for 24 h with 100 nm DARPin or 100 nm cetuximab, after which cells were stained with propidium iodide and measured by flow cytometry.