In vivo anti-tumor efficacy is dependent upon the ability of the targeted GSH to maintain CAR expression over time. (A) Experimental schema for in vivo assessment of tumor burden using CD19-CAR stress-test model for B-acute lymphoblastic leukemia in NSG mice. CAR⁺ cells were not purified but CAR⁺ cell numbers were calculated based on flow cytometry. (B) Kaplan Meier tumor-free survival curves for mice administered with GSH 1, 4, and 6 CARs ± insulator C1 and TRAC-CARs. Combined results from 2 experiments with 2 independent T-cell donors (n = 7-12). (C) Tumor burden curves over 90 days in the group of mice from panel B. Some mice with no tumor burden had to be euthanized owing to severe graft-versus-host disease. (D) Total CAR⁺ T-cell number in the BM of mice 10 days after infusion (results from one representative donor, n = 4-8 mice per group). Data are shown as mean ± SD. (E) Mouse cells were depleted from the BM cells of all mice illustrated in panel D, the remaining cells were pooled by group and an 18 hour cytotoxicity assay was performed with CD19-Ffluc-GFP NALM6 cells at a ratio of 3:1, T cells:NALM6 cells. Cell number calculation was done based on flow cytometry data after mouse cell depletion (supplemental Figures 6B and 8A). Data are not shown for GSH1 CARs because measurement of luciferase was skewed because of the presence of NALM6⁺ cells (not eliminated by mouse cell depletion) in BM at day 10. Data are shown as mean ± SD of 3 technical replicates from the pooled cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; Mann-Whitney U test. BM, bone marrow; BLI, bioluminescent imaging; Ffluc, firefly luciferase; ns, not significant.