Fig. 4: Reducing PCPs on the CAR surface mitigates T cell exhaustion induced by high CAR signaling.
From: Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness
a The sequences of optimized GD2.CAR variants. b The top three largest PCPs on optimized GD2.CAR scFv surface displayed by the BindUP web server tool. Dark blue, the first-largest PCP; medium blue, the second-largest PCP; light blue, the third-largest PCP. c The calculation of PCP scores for the optimized GD2.CAR scFvs. d The calculation of relative tonic signaling indexes for the optimized GD2.CAR scFvs. e The calculation of relative exhaustion scores for the optimized GD2.CAR scFvs. f Imaging analysis of CAR clustering on GD2WT and GD2F4.CAR-T cells. Pink, CAR; green, CAR-IRES EGFP; blue, Hoechst. Scale bars, 5 μm. g In vitro killing assay against GD2+ neuroblastoma cell line CHLA-255 of WT and mutated GD2.CAR-T cells. h, i Cytokine secretion assay of CAR-T cells after activation. WT and optimized GD2.CAR-T cells were co-incubated with their target tumor cells at the indicated E:T ratio. The levels of IL-2 and IFN-γ were determined by ELISA. j Representative bioluminescence images of tumor burden after GD2WT and GD2F4.CAR-T infusion over time. k Quantifications of luciferase intensity, which reflets tumor burden, of each mouse shown in j using IVIS system. l Survival curves for the mice shown in j. Data are presented as means ± SEM; Comparisons were determined using unpaired Student’s t-tests (d, e), two-way analysis of variance (g–i, k), and survival analysis (l); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
