PMC

Representations of bispecific DARPins used in this work. A, model of the structure of E69_GS_E01. E69 (red) and E01 (blue) are both represented as schematic secondary structure models, whereas the flexible linker (yellow) is represented as sticks. B, stick model of the structure of E69_LZ3_E01. E69 at the N terminus is depicted in red, E01 in blue, the leucine zipper in green, and the linkers in yellow.

Epitopes of DARPins on EGFR and their spatial relation relative to bound EGF. A, comparison of the epitopes of E01 and E68 and cetuximab on domain III of EGFR. EGFR residues involved in DARPin/cetuximab binding are in red, and EGF is in green stick representation. B, epitope of E69. EGFR residues involved in E69 binding are depicted in red, and EGF is in green stick representation. C, overview of epitopes of E01 and E69. EGFR is shown in ribbon representation, with domain I in red, domain II in green, domain III in blue, and part of domain IV in gray. The EGF ligand is shown in yellow stick representation. Residues involved in E69 binding are shown in Corey-Pauling-Koltun representation in pink (domain I), and residues involved in either E01 or E68 binding are shown in Corey-Pauling-Koltun representation in cyan (domain III).

Effect of DARPin treatment on downstream signaling. A431 cells were treated for 24 h with 100 nm cetuximab (Cet) or 100 nm DARPin. Cells were then stimulated with 10 ng/ml EGF, except cells from the “None −EGF” sample. Cell lysate corresponding to 20 μg of protein was loaded onto an SDS-polyacrylamide gel; proteins were then transferred by Western blotting and detected by a fluorescently labeled secondary antibody. A digital image of the fluorescently stained Western blot is shown above, and a quantitation of the band intensity is shown below. A, detection of EGFR and phospho-EGFR (Tyr-1068). E69_LZ3_E01 dramatically reduced total EGFR. B, detection of ERK1/2 and phospho-ERK1/2 (Thr-202/Tyr-204). E01_GS_E69, E69_GS_E01, and E69_LZ3_E01 inhibited ERK1/2 phosphorylation dramatically. C, detection of Akt and phospho-Akt (Ser-473). E69_LZ3_E01 inhibited Akt phosphorylation to some extent.

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.

Epitope comparison and biological effects of monovalent DARPins binding to A431 cells. Each symbol or bar represents the average of three data points. A, epitope comparison by flow cytometry. DARPins on the x axis were genetically fused to sfGFP. A431 cells were incubated with 100 nm sfGFP-tagged DARPin and 1 μm unlabeled DARPin, cetuximab (Cet), or EGF (denoted by the differently shaded bars). E69 is the only DARPin that cannot be competed with any other DARPin, only with itself. MFI, mean fluorescence intensity. B, inhibition of cell viability as determined by XTT assays. Cells were treated for 72 h with different concentrations of DARPins. DARPins E01, E67, and E68 affected the cell proliferation (*, p < 0.05 compared with untreated cells; Student's t test), whereas E69 and negative control Off7 did not. C, 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. E01, E67, and E68 slightly inhibited cell proliferation, whereas E69 did not. D, 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. E01, E67, and E68 induced G₁ arrest (*, p < 0.05 compared with untreated cells; Student's t test), whereas E69 did not affect A431 cells.

EGFR down-regulation by E69_LZ3_E01 is caused by inhibition of receptor recycling. A, A431 cells were treated for 2 h with 100 nm DARPin_Alexa Fluor 488 at 37 °C to allow for internalization of the EGFR-DARPin complex. Residual fluorescence outside the cell was quenched by an anti-Alexa quenching antibody, whereas fluorescence from recycled EGFR-DARPin complexes was chased by unlabeled DARPin. The fluorescence signal from the internalized receptor-DARPin complex was measured and compared with that from an untreated control. E69_LZ3_E01 was able to inhibit recycling back to the surface, whereas E01 alone as well as in combination with E69 did not. B, A431 cells were treated for 20 min with the receptor-recycling inhibitor monensin or with E69_LZ3_E01. At the indicated time points, residual surface EGFR was analyzed by flow cytometry. E69_LZ3_E01 was able to inhibit receptor recycling to the same extent as monensin. C, schematic overview of the biological effect of DARPin E69_LZ3_E01 on A431 cells overexpressing EGFR. DARPin E69 is depicted in red, and E01 is depicted in blue; the DARPins are connected via a leucine zipper. Upon treatment of A431 cells with the DARPin, EGFR on the cell surface will organize in clusters. These clusters are internalized via clathrin-coated pits into the early endosome. Normally, EGFR will be recycled back to the cell surface via the recycling endosome; in the case of E69_LZ3_E01, however, recycling is inhibited, and total surface EGFR is diminished. This has a strong impact on cell proliferation and downstream signaling events.

Biological activity of bispecific DARPins connected with a leucine zipper through different linkers. Each symbol or bar represents the average of three data points. A, schematic overview of the constructs with different linker lengths. B, inhibition of A431 cell viability as determined by XTT assays. Cells were treated for 72 h with different concentrations of bispecific DARPins with a flexible Gly-Ser linker or dimerizing leucine zipper, with E01 at the N terminus and E69 at the C terminus. E01_LZ1_E69 and E01_LZ2_E69 affected cell proliferation similar to cetuximab (*, p < 0.05 compared with untreated cells; Student's t test), whereas the negative control Off7 and E01_LZ3_E69 did not. C, inhibition of A431 cell viability as determined by XTT assays. Cells were treated for 72 h with different concentrations of bispecific DARPins with a flexible Gly-Ser linker or dimerizing leucine zipper, with E69 at the N terminus and E01 at the C terminus. E69_LZ1_E01 and E69_LZ3_E01 affected cell proliferation similar to cetuximab (*, p < 0.05 compared with untreated cells; Student's t test), whereas the negative control Off7 did not. DARPin E69_LZ2_E01 affected cell proliferation, but to a lesser extent. D, inhibition of cell proliferation as determined by clonogenic assays. A431 cells were treated for 7 days with 100 nm DARPin or 100 nm cetuximab (Cet), after which the medium was changed, and cells were allowed to grow for another 7 days. E69_LZ3_E01 significantly inhibited cell proliferation (*, p < 0.02 compared with untreated cells; Student's t test), whereas the other constructs had a less pronounced effect on cell proliferation. E, inhibition of cell proliferation as determined by clonogenic assays at different concentrations. A431 cells were treated with different concentrations of DARPin or cetuximab for 7 days, after which the medium was changed, and cells were allowed to grow for another 7 days. Both cetuximab and E69_LZ3_E01 significantly inhibited cell proliferation (**, p < 0.02, and *, p < 0.01, respectively; Student's t test), whereas negative control Off7 did not show an effect. The IC₅₀ of E69_LZ3_E01 as determined from this graph is ∼100 nm. F and G, cell cycle distribution. A431 cells were treated for 24 h with 100 nm bispecific DARPin with a dimerizing leucine zipper or with 100 nm cetuximab, after which cells were stained with propidium iodide and measured by flow cytometry. The bispecific DARPins induced G₁ arrest (*, p < 0.05 compared with untreated cells; Student's t test), whereas E69_LZ3_E01 in particular significantly induced G₁ arrest (**, p < 0.02 compared with untreated cells; Student's t test).