Fig. 4: Dual targeting BiCisCAR with two different CSDs shows enhanced expansion, tumor-infiltrating and limited exhaustion.

A Schema of testing 1E + 6 CAR T-cells in an RMS559 I.M. mouse model. Tumor size (B), bioluminescence images (C), and total bioluminescence flux (D) were monitored for RMS559_Luc orthotopic xenograft. *p = 0.05, **p = 0.0042, by mixed-effects analysis. (E) Kaplan–Meier survival analysis (n = 5 per group). **p < 0.01, ns, not significant, by Log-rank test. F Mean counts ± SEM of CAR⁺ T-cells (gating from CD45⁺CD3⁺ T-cells) in blood 21 days after CAR T-cell infusion. n  =  5 per group. One-way ANOVA with Holm-Sidak’s multiple comparison tests was performed for Log10(CAR⁺ T-cell counts). *p < 0.05, **p < 0.01, ***p < 0.001. G Schema of testing CAR T-cells in a JR I.M xenograft NSG mouse model. n = 5 per treated group, except for CD276.8HTM.BBz CAR and FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T-cells group (n = 6) in figures H–O. Tumor size (H) or bioluminescence kinetics (I) for JR_Luc orthotopic xenografts. **p = 0.0063, ****p < 0.0001, by mixed-effects analysis. CAR T-cells (CD45⁺CD3⁺ CAR⁺) in blood (J) or tumor (K) 16 or 21 days after CAR T-cell treatment. Data are shown as individual values (shapes) and mean ± SEM (bar graphs). ***p = 0.0002, ****p < 0.0001, by ordinary one-way ANOVA with Tukey’s multiple comparisons tests. Data from day 16 are shown as colored shapes; while data from day 21 are shown as black shapes in Figures K, M–O. L A representative flow cytometry experiment detects CD39 and Tim-3 expression on CAR⁺ T-cells from JR xenografts at day 21 post-infusion. Percentages of CD39⁺ (M), Tim-3⁺ (N), and CD39⁺ Tim-3⁺ (O) CAR⁺ T-cells in tumors from mice 16 or 21 days after CAR T-cells infusion. Values are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, by one-way ANOVA with Tukey’s multiple comparisons tests. The full lists of p values in E, F, M, N, and O can be found in the Source Data file. All data is provided in the Source Data file.