Figure 1
Generation and validation of WT1-28z CAR T cells. (a) Schematic representation of the WT1-28z constructs. WT1/HLA-A*02:01-specific scFv derived from the 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; black box: κ leader sequence; gray box: (Gly4Ser)3 linker. (b) Flow cytometry histograms depicting the expression of WT1-28z in retrovirally transduced into primary human T cells, as detected by binding to a fluorescently-labeled WT1/HLA-A*02:01 Tetramer (representative figure of n>10). (c) WT1-28z CAR T cells are more toxic than control irrelevant antigen-specific control CAR T cells 4H11-28z or 19-28z CAR T cells against AML-14, BV173 and OVCAR3 in standard 51Cr release assays (representative figures, n=3 for each cell line). (d) WT1-28z CAR T cells when co-cultured with AML-14, BV173, or OVCAR3 cell lines for 24 h have significantly enhanced release of IFN-γ (*P=<0.0001, 0.03, or 0.0013 respectively) and IL-2 (*P⩽0.0001, 0.013, or <0.0001 respectively), as compared with control 4H11-28z or 19-28z CAR T cells (n=3 for each cell line). (e) Standard 51Cr release assays with antigen-specific WT1-28z CAR T cells showed enhanced cytotoxicity against primary AML samples as compared with a control irrelevant antigen-specific 4H11-28z CAR T cells (n=5) (*P=0.029). (f) WT1-28z CAR T cells have significantly enhanced release of IFN-γ and IL-2 (*P=0.04) when co-cultured with AML patient samples for 24 h, as compared with 4H11-28z CAR T cells (n=3).
