Figure 1
A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. (a) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. (b) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. (c) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. (d) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of SupT1 cells engineered to express CD19 and GFP (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) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). (f) IFN-γ or (g) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).
