Extended Data Fig. 2. Epitope variant receptors show preserved ligand-mediated activation.
a. FLT3 epitope engineered variants preserve kinase activation. Western blot of proteins extracts from K562 cells expressing FLT3 variants by Sleeping Beauty transposase. Cells were serum-starved overnight and stimulated with different concentrations of human FLT3L for 10 min at 37 °C. pFLT3 Y589-591, total FLT3 and Actin were probed on the same lysates. Total FLT3 was probed after stripping of the pFLT3 membrane. Normalized pFLT3 signal intensity (on actin) is reported on the right. Two-way ANOVA, the p-value of the editing effect is reported. Uncropped blots are reported in Supplementary Fig. 2. b. KIT epitope engineered variant preserves kinase activation. Western blot of proteins extracts from NIH-3T3 cells expressing KIT variants by Sleeping Beauty transposase. Cells were serum-starved overnight and stimulated with different concentrations of human SCF for 10 min at 37 °C. pFLT3 Y719, total KIT and Actin were probed on the same lysates. Normalized pKIT signal intensity (on total KIT) is reported in the right plot. Two-way ANOVA, the p-value of the editing effect is reported. Uncropped blots are reported in Supplementary Fig. 2. c. CD123 epitope engineered variants preserve STAT5 activation. BaF3 cells expressing CD123 variants by Sleeping Beauty transposase were starved for murine IL-3 and stimulated with different concentrations of human IL-3. Cells were evaluated for STAT5 phosphorylation by intracellular flow cytometry after 48h (left, representative FACS plots show the CD123 S59P condition at different hIL-3 doses; right, pSTAT5 PE MFI). Two-way ANOVA, the p-value of the editing effect is reported. d. FLT3, KIT, CD123 epitope engineered variants induce proliferative responses similar to WT receptors. BaF3 cells expressing FLT3, KIT and CD123 variants by Sleeping Beauty transposase were starved for murine IL-3 overnight and stimulated with different concentrations of human FLT3, SCF and IL-3, respectively. Cells were cultured for 5 days and analysed by flow cytometry to obtain absolute counts (CountBeads). Plots report absolute counts normalized to the unstimulated condition. N = 4. e. Top: Bidirectional lentiviral vector expressing a 2nd generation CAR and a truncated Epidermal Growth Factor Receptor (EGFRt). f. Percentage of EGFRt+ (left) and fold expansion (right) of T cells after transduction with 4G8-CAR at different multiplicity of infection (MOI). Days, D.