Activity of EpoRm-CART cells in xenograft models of ALL. (A) NOD-scid-IL2RGnull mice were injected IV with 5 to 20 × 106 T cells transduced with CAR, EpoRm-CAR, or GFP only; one group of mice received no T cells. Two weeks later, mice were injected IV with 2.5 × 105 Nalm6 cells expressing luciferase. Nalm6 cell engraftment as measured by luminescence after IP injection of aqueous d-luciferin potassium salt (150 µg/g body weight) is shown. The gray area corresponds to signals <108 photons/second. On day 56, none of the mice that received no T cells, or T cells transduced with GFP only at 5 × 106 (n = 2), 10 × 106 (n = 4), or 20 × 106 (n = 2), had signals <108 photons/second. Likewise, none of the mice that received CAR T cells at 5 × 106 (n = 4) or 10 × 106 (n = 5) remained in remission. By contrast, 2 of the 4 mice that received 5 × 106 EpoRm-CAR T cells and all 5 who received 10 × 106 remained in remission, as did all mice that received 20 × 106 CAR (n = 6) and EpoRm-CAR T cells (n = 6). (B) Ventral images for the mice that received no T cells or 10 × 106 T cells are shown. The full set of images is show in supplemental Figure 8. (C) Aggregate overall survival for mice shown in panels A and B. Survival group according to different levels of cells received is shown in supplemental Figure 8. (D) Presence of T cells in the mice shown in panels A to C 13 days after infusion, before injection of Nalm6 cells. Mouse blood was collected by cheek prick, and cells were stained with APC (2D1; BioLegend) or PE/Cy7-conjugated anti-human CD45 (HI30; BD Pharmingen), and anti-mouse CD45-PE (30-F11; BD Pharmingen), as well as with anti-human CD3-APC (SK7; BD Biosciences) and anti-human CD19-PE (4G7; BD Biosciences). Percentage of human T cells (hCD45+ hCD3+ hCD19– GFP+) among all CD45+ (human and mouse) lymphoid cells is shown. **P < .01. Results of sequential measurements are shown in supplemental Figure 9A. (E) Nalm6 cells transduced with luciferase were injected IV in NOD-scid-IL2RGnull mice (5 × 105 cells per mouse). Four days later, tumor engraftment was assessed, and 10 × 106 T cells were injected IV. Ventral images illustrate Nalm6 cell engraftment as measured by luminescence after IP injection of aqueous d-luciferin potassium salt (150 µg/g body weight). Mice images on day 4 were taken with enhanced sensitivity to illustrate tumor engraftment. (F) Luminescence measurements in the groups of mice shown in panel E. The number of mice in each group with signals <108 photons/second on day 70 is shown. (G) Overall survival of mice shown in panels E and F. (H) Presence of T cells (hCD45+ hCD3+ hCD19– GFP+) in the mice shown in panels E to G 13 days after T-cell infusion. **P < .01. Results of sequential measurements are shown in supplemental Figure 9B.