Figure 6
Phylomer CPP delivery is compatible with cell specific targeting and half-life extension approaches. (a) CHO-K1 cells stably expressing EGFR receptor or (b) CHO-K1 cells were treated with 1746del_SpyT conjugated to EGFRAffibody_Bouganin_SpyC (EGFRAffbd_Boug_SpyC) toxin. After 48 h incubation, the cell viability was assessed according to the resazurin reduction potential. Comparison of 100 nM immunotoxin treatment in CHO-K1_EGFR (a, right) vs CHO-K1 (b, right) cells shows that conjugation to 1746del improved delivery compared to the EGFRAffibody alone, and that the 1746del-conjugate retains EGFRAffibody-encoded specificity. Results are representative of 3 independent experiments. Error bars represent standard deviation from the mean of duplicate samples. Significance was assessed by one-way ANOVA with Dunnett’s multiple comparison test (**p < 0.01; ***p < 0.001). (c) T47D cells were treated with 1746c27_PAP_linker_SpyT with and without conjugation to the PAS_SpyC fusion protein. 1746c27-dependent PAP-induced cytotoxicity was detected for all PAS conjugates in comparison to the buffer control (Tris). The Furin-cleavable conjugate exhibited the greatest potency, but all PAS conjugates showed dose-dependent cell toxicity in a comparable concentration range. Linkers are Cathepsin B FKFL cleavage motif (BF), Cathepsin B Valine-Citrulline cleavage motif (Ba) and Furin RKKR cleavage motif (Fur). Results are representative of two independent experiments. Error bars represent standard deviation from the mean of duplicate samples.
