Figure 2
Optimization of TetCAR surface expression and signaling. (a) Schematic overview of TetCAR constructs containing 41BB-ζ or CD28-ζ endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. (b) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. (c) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis. P values = FMC63-BBz versus Fab-Tet-BBz (***, 0.0003) or Fab-Tet-28z (***, 0.0002), FMC63-Tet-BBz versus Fab-Tet-BBz (ns, 0.089), FMC63-Tet-28z versus Fab-Tet-28z (*, 0.045). (d) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) control, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Šidák’s multiple comparisons within each group ± minocycline. P values for each construct + /- minocycline were: FMC63-Tet-BBz (**, 0.0023), FMC63-Tet-28z (***, 0.0003) and Fab-Tet-28z (*, 0.0279). (e) IFN-γ and (f) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the TetCAR groups at 0 nM minocycline (with Tukey’s multiple comparisons) or within these groups ± minocycline (with Šidák’s multiple comparisons). P values were; between FMC63-Tet-BBz and Fab-Tet-BBz (**, 0.0097, IFN-γ) and between FMC63-Tet-28z and Fab-Tet-28z (****, < 0.0001, IFN-γ and **, 0.0017, IL-2). P values between the TetCAR constructs ± minocycline were: FMC63-Tet-BBz (***, 0.0007, IFN-γ), FMC63-Tet-28z (*, 0.0409, IFN-γ), Fab-Tet-BBz (****, < 0.0001, IFN-γ and *, 0.0152, IL-2) and Fab-Tet-28z (****, < 0.0001 both IFN-γ and IL-2).
