Extended Data Fig. 3: CAR T cell culture with NAC or dasatinib and ITAM attenuation enhances the antileukaemia activity of dual-CAR T cells.
From: Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors
a,b. BM185-CD19/BM185-CD20 antigen-loss escape model. BALB/c mice were treated with Zip-sorted dual-CAR BALB/c T cells cultured with 1 μM dasatinib (2 days), 10 mM NAC (3 days), or DMSO (3 days). a, Leukaemia BLI from two combined experiments. Log-transformed BLI AUC values were compared using a Vardi test with FDR correction. b, Survival. c, NFAT, AP-1, or NFκB EGFP reporter analysis of unstimulated BALB/c T cells gated on dual-CAR positive or CAR-negative population following 24 h culture with 1 μM dasatinib, 10 mM NAC, or DMSO. Representative of n = 2 donor experiments, with mean ± SEM of triplicate wells. Statistical comparison via two-way ANOVA, with Tukey’s test. d,e, PD-1 and TOX expression in Zip-sorted dual-CAR or delta/delta BALB/c T cells following 24 h co-culture with BM185-CD19 or no targets. Mean ± SEM of triplicate samples were compared with a two-way ANOVA, with Tukey’s test. f, Intracellular flow cytometry analysis of TCF1 and TOX expression in Zip-sorted dual-CAR or delta/delta BALB/c T cells cultured for 2 days with 1 μM dasatinib or DMSO. Representative experiments from n = 2 donors. g, Diagram depicting ITAM mutations in 1XX CAR. h, Survival of BALB/c mice injected with BM185-CD19/BM185-CD20 (1:1) and treated with Zip-sorted dual-CAR WT CD3ζ or 1XX ITAM mutant dual-CAR T cells, from three combined experiments. i, Survival of BALB/c mice injected with dual-target-antigen expressing BM185-CD19-CD20 leukaemia and treated with WT CD3ζ or 1XX dual-CAR T cells, from three combined experiments. Survival curves were compared via log-rank tests or pairwise log-rank test, with FDR correction.
