Figure 2

Generation and validation of WT1-28z/IL-12 CAR T cells. (a) Schematic representation of the WT1-28z/IL-12 CAR constructs. WT1/HLA-A*02:01-specific scFv derived from heavy (VH) and light (VL) chain variable regions of the ESK1 antibody; CD28: human CD28 transmembrane and cytoplasmic signaling domains; z-chain: human TCR zeta chain cytoplasmic signaling domain; flexi human IL-12 (hIL-12 f); LTR: 50 and 30 long terminal repeat; black box: κ leader sequence; gray box: (Gly4Ser)3 linker. (b) IL-12 expression by viral producer cell lines stably transduced with CAR constructs show that only 293Galv9 cells transduced with WT1-28z/IL-12 express IL-12 (n=3 independent experiments) (P=0.01). (c) The IL-12 produced by the WT1-28z/IL-12 CAR in 293Galv9 is functional, as determined by increases in levels of IFN-γ produced by peripheral blood mononuclear cell cultured in supernatant (n=3 independent experiments) (P=0.03). (d) Flow cytometry histograms depicting the expression of WT1-28z/IL-12 CARs in retrovirally transduced primary human T cells, as detected by binding to a fluorescently-labeled WT1/HLA-A*02:01 tetramer (representative figure of n>10). (e) WT1-28z/IL-12 CAR T cells have significantly enhanced release of IFN-γ (*P=0.008, 0.01, respectively) and IL-2 (*P= 0.002, 0.002, respectively), when co-cultured with Set2 cells for 24 hours, as compared to 19-28z or 19-28z/IL-12 CAR T cells (n=4). (f) WT1-28z/IL-12 CAR T cells proliferate after co-culture with Set2 cells (n=4).