Design and in vitro validation of bispecific CD19-CD133 CAR-iNKT cells. (A) Design of CD19 and CD133 monospecific and CD19-CD133 bispecific CARs. (B) Flow cytometric analysis of highly pure, expanded iNKT cells, gated on CD3 and coexpressing invariant TCRVα24 (identified with the 6B11 mAb) and TCRVβ11, expressing the indicated CARs, as assessed by L-protein staining. (C) Cytotoxicity assay of indicated mono- and bispecific CAR-iNKT cells against SEM cells at 4 and 24 hours. Error bars represent standard deviation of triplicate assays. (D) Flow cytometric analysis of CD19 and CD133 expression in parental and gene-edited SEM leukemia cells. (E) Cytotoxicity assay of mono- and bispecific CAR-iNKT cells against the target cells shown in panel D. (F) Flow chamber avidity assay of indicated CAR-iNKT cells over a range of pressures (left), and representative images of CAR-iNKT cells bound to SEM cells at the beginning and end of the assay (right). Data are presented as the mean ± SEM of n = 3 experiments. ∗∗P < .01; ∗∗∗∗P < .0001, by 1-way analysis of variance (ANOVA).