Figure 3

Reconfiguration of endodomain positions enhances TetCAR function. (a) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41BB or CD28 endodomains, with a TIP-CD3ζ or TIP-41BB-CD3ζ domains. (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 of CAR expression on surface of RQR8⁺ cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 5 donors from 2 experiments. Statistical analysis was through one-way ANOVA between the groups, p values were; between FMC63-BBz and each Fab-TetCAR (****, < 0.0001). Differences between Fab-TetCARs alone were analyzed by one-way ANOVA but were not significant. (d) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; FMC-Tet-BBz (*, 0.0119). (e) IFN-γ and (f) IL-2 release after 24 h of co-culture with SupT1-CD19 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = 28-Tet-z (*, 0.0484), 28BB-Fab-Tet-z and 28BB-Fab-Tet-BBz (****, < 0.0001). P values for IL-2 = 28-Tet-z (*, 0.0150), 28BB-Fab-Tet-z (****, < 0.0001), 28-Fab-Tet-BBz (**, 0.0029) and 28BB-Fab-Tet-BBz (***, 0.0007). (g) Killing of NALM6 after 48 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; BB-Fab-Tet-z (**, 0.0083), BB-Fab-Tet-BBz (*, 0.0189) and 28-Fab-Tet-z, 28BB-Fab-Tet-z, 28-Fab-Tet-BBz and 28BB-Fab-Tet-BBz (****, < 0.0001). (h) IFN-γ and I) IL-2 release after 48 h of co-culture with NALM6 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = BB-Fab-Tet-z (*, 0.0433), 28-Fab-Tet-z (*, 0.0387), 28BB-Fab-Tet-z (****, < 0.0001) and 28BB-Fab-Tet-BBz (**, 0.0020). P values for IL-2 = 28BB-Fab-Tet-z (*, 0.0450).