Figure 2.
CD7-CARCD7− T cells have potent anti–T-ALL activity. (A-B) Serial stimulation assay with CD7-CARCD7−, HER2-CARBulk, or HER2-CARCD7− T cells and CCRF target cells. (A) CCRF cells at a 1:1 E:T ratio and fresh target cells were added every 72 hours if a luciferase-based cytotoxicity assay demonstrated >50% killing (N = 3, at stimulation 1, P < .001, 1-way ANOVA, CD7-CARCD7− between stimulation 1 and 5, ns, unpaired t test). Double-gradient heat map (purple, 100% target cell lysis; white, 0% target cell lysis). (B) Multiplex analysis of cytokine production by CD7-CARCD7− T cells or HER2-CARBulk and HER2-CARCD7− against CCRF cells at a 1:1 E:T ratio. At stimulation 1 by 2-way ANOVA: CD7-CARCD7− vs HER2-CARBulk, P < .05 for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, IL-4, IL-5, IL-8, MCP-1, Regulated on Activation, Normal T cell Expressed and Secreted (RANTES), tumor necrosis factor alpha (TNF-α) ; P < .01 for IL-17A, IP-10; P < .0001 for IFN-γ. CD7-CARCD7− vs HER2-CARCD7−P < .05 for IFN-γ, IL-4, and tumor necrosis factor α (TNF-α) P < .05. Double-gradient heat map (blue, 100 pg/mL; white, 102 pg/mL; yellow, 106 pg/mL). (C-I) In vivo testing of CD7-CARCD7− T cells. (C) Schematic of xenograft experiments: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected IV via tail vein with 1 × 104 CCRF cells on day 0 and a single IV dose of 3 × 106 (N = 16) or 10 × 106 (N = 20) CD7-CARCD7− T cells on day 7; mice injected with tumor-only served as a control (N = 20). Mice were monitored via IVIS imaging and tracked for (D) bioluminescence (total flux photons per second) and (E) survival (P < .0001, Mantel-Cox log-rank, P = ns between dose levels). (F) Schematic of tumor rechallenge experiments. (G) Mice surviving and having no detectable tumor burden after 125 days of treatment with CD7-CARCD7− T cells, received a second dose of 1 × 104 CCRF cells (N = 5 for 3 × 106 group and N = 4 for 10 × 106 group); naïve age-matched mice served as control (N = 5) (day 146, P = .007, 1-way ANOVA). (H-I) Peripheral blood, spleen, and bone marrow (N = 9) was collected from mice on days 212 to 225 (∼100 days postrechallenge). All surviving animals had <0.1% residual disease in the marrow. Flow cytometry analysis of CD3, CD4, CD8, and F(ab’)2 fragments to determine the presence of CAR T cells and their immunophenotype. Bulk T cells and CD7-CARCD7− T cells served as an immunophenotype reference (CAR+ vs CAR− T cells, P < .0001, 2-Way ANOVA, CD4 vs CD8 P < .01, 2-way ANOVA). ☩, one mouse that died of ischemia-associated tubular necrosis and one that died due to unknown cause; ns, not significant.

CD7-CARCD7− T cells have potent anti–T-ALL activity. (A-B) Serial stimulation assay with CD7-CARCD7−, HER2-CARBulk, or HER2-CARCD7− T cells and CCRF target cells. (A) CCRF cells at a 1:1 E:T ratio and fresh target cells were added every 72 hours if a luciferase-based cytotoxicity assay demonstrated >50% killing (N = 3, at stimulation 1, P < .001, 1-way ANOVA, CD7-CARCD7− between stimulation 1 and 5, ns, unpaired t test). Double-gradient heat map (purple, 100% target cell lysis; white, 0% target cell lysis). (B) Multiplex analysis of cytokine production by CD7-CARCD7− T cells or HER2-CARBulk and HER2-CARCD7− against CCRF cells at a 1:1 E:T ratio. At stimulation 1 by 2-way ANOVA: CD7-CARCD7− vs HER2-CARBulk, P < .05 for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, IL-4, IL-5, IL-8, MCP-1, Regulated on Activation, Normal T cell Expressed and Secreted (RANTES), tumor necrosis factor alpha (TNF-α) ; P < .01 for IL-17A, IP-10; P < .0001 for IFN-γ. CD7-CARCD7− vs HER2-CARCD7−P < .05 for IFN-γ, IL-4, and tumor necrosis factor α (TNF-α) P < .05. Double-gradient heat map (blue, 100 pg/mL; white, 102 pg/mL; yellow, 106 pg/mL). (C-I) In vivo testing of CD7-CARCD7− T cells. (C) Schematic of xenograft experiments: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected IV via tail vein with 1 × 104 CCRF cells on day 0 and a single IV dose of 3 × 106 (N = 16) or 10 × 106 (N = 20) CD7-CARCD7− T cells on day 7; mice injected with tumor-only served as a control (N = 20). Mice were monitored via IVIS imaging and tracked for (D) bioluminescence (total flux photons per second) and (E) survival (P < .0001, Mantel-Cox log-rank, P = ns between dose levels). (F) Schematic of tumor rechallenge experiments. (G) Mice surviving and having no detectable tumor burden after 125 days of treatment with CD7-CARCD7− T cells, received a second dose of 1 × 104 CCRF cells (N = 5 for 3 × 106 group and N = 4 for 10 × 106 group); naïve age-matched mice served as control (N = 5) (day 146, P = .007, 1-way ANOVA). (H-I) Peripheral blood, spleen, and bone marrow (N = 9) was collected from mice on days 212 to 225 (∼100 days postrechallenge). All surviving animals had <0.1% residual disease in the marrow. Flow cytometry analysis of CD3, CD4, CD8, and F(ab’)2 fragments to determine the presence of CAR T cells and their immunophenotype. Bulk T cells and CD7-CARCD7− T cells served as an immunophenotype reference (CAR+ vs CAR− T cells, P < .0001, 2-Way ANOVA, CD4 vs CD8 P < .01, 2-way ANOVA). ☩, one mouse that died of ischemia-associated tubular necrosis and one that died due to unknown cause; ns, not significant.

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