Fig. 5: Functional characterization of selected CAR variants confirms their diverse phenotypes and potential enhanced properties.

a Ten CAR signaling domain variants were selected for individual characterization along with the clinically used 28z and BBz CARs. b Proportion of T cell differentiation subsets observed in CAR or TCR-negative (TCR-) T cells following a 4 day co-culture with SKBR3 cells (4:1 E:T ratio) as measured by CD62L and CD45RA surface expression (naive T cells (Tn), stem central memory T cells (Tscm), central memory T cells (Tcm), Effector Memory T cells (TEM), Effector Memory RA-positive T cells (TEMRA)). c Proportion of CAR or TCR- T cells simultaneously expressing different number of exhaustion markers (PD1, TIM3, TIGIT, LAG3, and CTLA4) following the co-culture conditions described in b. d, e CAR T cell-mediated cytotoxicity of HER2+ /GFP+ tumor cells quantified over time by fluorescence microscopy. The fluorescence change values represent the difference in GFP intensity compared to time point 0, and a dashed line represents the baseline where no GFP+ cells are left. CAR T cells were co-cultured at different E: T ratios with either SKBR3 adherent cells in a sparse 2D culture in d or with a single tumor spheroid of MCF-7 cells in e. f, g Heat maps comparing the area under the curve (AUC) of the killing curves in d, e across different CAR variants. h Cytokine secretion profile of a selection of 8 cytokines following a 4 day co-culture of CAR T cells and SKBR3 cells at a 4:1 E: T ratio. The levels of cytokines in the co-culture medium were quantified by fluorescence-encoded multiplex bead assays and are shown as fold change compared to the benchmark 28z CAR. To assess significant differences between each variant and 28z, a one-way ANOVA and Dunnett’s multiple comparisons test was used with the following significance indicators: *p-value <0.05, **p-value <0.01, ***p-value <0.001 and ****p-value <0.0001. In all panels, TCR- refers to T cells without a TCR and error bars represent the S.D. (n = 2 independent technical replicates for variants A-J and n = 2 technical replicates from two independent experiments for control groups). Source and statistical data are provided as a Source Data file.