CAR-iNKT cells are more potent than CAR T cells. (A) Cytotoxic activity at 24 hours of untransduced T cells and iNKT cells, and of their bispecific CAR-transduced counterparts against parental SEM cells and their single or double CD19/CD133 gene-edited sublines. (B) Avidity assay comparing bispecific CAR T cells vs CAR-iNKT cells against SEM cells. Data are presented as the mean ± SEM of n = 3 experiments. ∗P < .05, by 1-way ANOVA. (C) Expression level of indicated cytokines by bispecific CAR T cells and CAR-iNKT cells after 4 and 24 hours coculture with SEM leukemia cells; 1-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (D) Schematic of experiment for panels E-G and H-I. (E-F) Leukemia burden by BLI and overall survival of SEM leukemia-bearing mice treated with indicated dose of bispecific CAR T cells and CAR-iNKT cells (n = 7 mice per group). Log-rank test, ∗∗∗P < .001. (G) Overall survival of CRISPRKMT2A-AFF1 leukemia-bearing mice treated with indicated dose of bispecific CAR T cells and CAR-iNKT cells (n = 7 mice per group). Log-rank test, ∗∗P < .01. (H) Absolute numbers of CAR T cells and CAR-iNKT cells in BM and spleen at euthanasia of mice treated as shown in panels E-F. One-way ANOVA; ∗∗P < .01. (I) Absolute numbers of CAR T cells and CAR-iNKT cells in BM and spleen at euthanasia of mice treated as shown in panel D, 1-way ANOVA; ∗∗∗∗P < .0001. (J-K) Schematic of the experiment (J) and leukemia burden assessed by BLI of SEM leukemia-bearing mice treated with 5 × 106 CAR-iNKT cells or CAR T cells (K). (L-N) Absolute numbers of leukemia cells in spleen (L), BM (M), and meningeal space (N) at euthanasia of mice treated as shown in panel K; ∗∗P < .01. (O) Leukemia-free survival of mice treated with 5 × 106 CAR-iNKT cells or CAR T cells. Log-rank test, ∗∗∗∗P < .0001. E:T, effector to target.
