Figure 4.
S-2HG–treated CAR-T cells show enhanced antitumor activity. (A) Schematic representation of the in vivo model and the different CAR-T populations used. NSG mice were injected IV with 1 × 106 Raji-GFP-LUC cells/mouse and 7 days after mice were treated with either anti-CD19-CAR-T cells or tEGFR (vector-control) T cells. Naive or total CD8 T cells were isolated from the same donor, activated, and transduced with the appropriate lentiviral construct. The anti-CD19-CAR (4-1BB) was used for CAR-T cells. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15. The NSG mice received 2.5 × 104 CAR+ cells, or tEGFR cells or PBS. (B) Bioluminescence imaging of tumor growth of mice treated with the following conditions: CAR-Vehicle (naive); CAR-S-2HG (naive); CAR-Vehicle (total); CAR-S-2HG (total); tEGFR and PBS. The geometric mean is shown for each condition; n = 11 mice per condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice/condition for CAR-Vehicle (total) and CAR-S-2HG (total); n = 6 mice for tEGFR and n = 5 mice for PBS. Two independent experiments using CD8 T cells from 2 different donors. On the second experiment, the IVIS imaging on day 11 and day 32 was performed 1 day later. Statistical analysis was performed for the indicated groups by the Wilcoxon test in the log10 values. (C) Bioluminescence imaging of tumor growth for the individual mice per condition. Conditions shown: PBS; CAR-Vehicle (naive); CAR-S-2HG (naive); tEGFR; CAR-Vehicle (total); and CAR-S-2HG (total). Two independent experiments using CD8 T cells from 2 different donors. On the second experiment the IVIS imaging on day 11 and day 32 was performed 1 day later; n = 11 mice/condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice/condition for CAR-Vehicle (total) and CAR-S-2HG (total); n = 6 mice for tEGFR and n = 5 mice for PBS. (D-E) Percentage of human live CD8 CAR+ TCM (CCR7+/CD45RO+) cells in peripheral blood of mice 12/13 days post adoptive cell transfer (D), and 20/21 days post adoptive cell transfer (E). All conditions were compared with CAR-S-2HG (naive) with the unpaired Student t test; n = 10 to 11 mice/condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice/condition for CAR-Vehicle (total) and CAR-S-2HG (total), 2 independent experiments using CD8 T cells from 2 different donors. (F) Kaplan-Meier survival curve of Raji-GFP-LUC-bearing mice treated as in (A) and using a 1 × 109 total flux cutoff; n = 11 mice per condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice per condition for CAR-Vehicle (total) and CAR-S-2HG (total); n = 6 mice for tEGFR and n = 5 mice for PBS; 2 independent experiments using CD8 T cells from 2 different donors. Statistical analysis was performed using a log-rank Mantel-Cox test. For all panels: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

S-2HG–treated CAR-T cells show enhanced antitumor activity. (A) Schematic representation of the in vivo model and the different CAR-T populations used. NSG mice were injected IV with 1 × 106 Raji-GFP-LUC cells/mouse and 7 days after mice were treated with either anti-CD19-CAR-T cells or tEGFR (vector-control) T cells. Naive or total CD8 T cells were isolated from the same donor, activated, and transduced with the appropriate lentiviral construct. The anti-CD19-CAR (4-1BB) was used for CAR-T cells. After activation, cells were treated with vehicle (H2O) or S-2HG (0.4 mM) from days 4 to 15. The NSG mice received 2.5 × 104 CAR+ cells, or tEGFR cells or PBS. (B) Bioluminescence imaging of tumor growth of mice treated with the following conditions: CAR-Vehicle (naive); CAR-S-2HG (naive); CAR-Vehicle (total); CAR-S-2HG (total); tEGFR and PBS. The geometric mean is shown for each condition; n = 11 mice per condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice/condition for CAR-Vehicle (total) and CAR-S-2HG (total); n = 6 mice for tEGFR and n = 5 mice for PBS. Two independent experiments using CD8 T cells from 2 different donors. On the second experiment, the IVIS imaging on day 11 and day 32 was performed 1 day later. Statistical analysis was performed for the indicated groups by the Wilcoxon test in the log10 values. (C) Bioluminescence imaging of tumor growth for the individual mice per condition. Conditions shown: PBS; CAR-Vehicle (naive); CAR-S-2HG (naive); tEGFR; CAR-Vehicle (total); and CAR-S-2HG (total). Two independent experiments using CD8 T cells from 2 different donors. On the second experiment the IVIS imaging on day 11 and day 32 was performed 1 day later; n = 11 mice/condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice/condition for CAR-Vehicle (total) and CAR-S-2HG (total); n = 6 mice for tEGFR and n = 5 mice for PBS. (D-E) Percentage of human live CD8 CAR+ TCM (CCR7+/CD45RO+) cells in peripheral blood of mice 12/13 days post adoptive cell transfer (D), and 20/21 days post adoptive cell transfer (E). All conditions were compared with CAR-S-2HG (naive) with the unpaired Student t test; n = 10 to 11 mice/condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice/condition for CAR-Vehicle (total) and CAR-S-2HG (total), 2 independent experiments using CD8 T cells from 2 different donors. (F) Kaplan-Meier survival curve of Raji-GFP-LUC-bearing mice treated as in (A) and using a 1 × 109 total flux cutoff; n = 11 mice per condition for CAR-Vehicle (naive) and CAR-S-2HG (naive); n = 10 mice per condition for CAR-Vehicle (total) and CAR-S-2HG (total); n = 6 mice for tEGFR and n = 5 mice for PBS; 2 independent experiments using CD8 T cells from 2 different donors. Statistical analysis was performed using a log-rank Mantel-Cox test. For all panels: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

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