A TIM-3–Fc decoy secreted by primary T cells enhances CAR19 T-cell efficacy and persistence in vivo. (A) Cartoon depicting the experimental strategy to block galectin-9 binding to TIM-3 on CAR19 T cells through a TIM-3–Fc decoy secreted by coadministered non-CAR T cells. (B) Representative fluorescence microscopy image (left) and transduction levels (tdTo+) measured by flow cytometry (right) of activated UT and TIM-3–Fc–transduced (tdTo+) primary T cells. (C) TIM-3 expression, quantified by flow cytometry, on UT and TIM-3–Fc T cells 7 days after transduction. (D) Proliferation of UT and TIM-3–Fc T cells. These experiments were performed with T cells from 3 different donors. (E) Experimental design to compare CAR19 and CAR19 + TIM-3–Fc T-cell persistence in vivo. NSG mice were injected IV with 1 × 106 B-ALL PDX cells (galectin-9 positive), and 4 days later, a single IV injection of 2 × 106 T cells was administered in mice (UT, TIM3Fc, CAR19, and CAR19 + TIM-3–Fc; n = 6 mice per group); 0.5 × 106 CAR19+ or/and 0.5 × 106 TIM-3–Fc+ cells were administered. Leukemia and T-cell expansion were monitored over time. In vivo CAR19 and CAR19 + TIM-3–Fc T cells were rechallenged with fresh B-ALL cells on day 25. (F) Follow-up of leukemic and T-cell persistence in PB of treated mice over time. (G) End point total and CAR+ (GFP+) T-cell quantification in PB, spleen, and BM in the different groups of treated mice. (H) Experimental design to compare CAR19 and CAR19 + TIM-3–Fc T-cell efficacy and persistence in vivo under stress conditions (1 × 106 T cells comprising 0.175 × 106 CAR19+ or/and 0.325 × 106 TIM-3–Fc+; n = 6 mice per group). Mice were followed up for 3 weeks. (I) End point quantification of blasts (J) and T cells (total and CAR+(GFP+) in PB, spleen, and BM in the indicated groups of treated mice. All data are expressed as mean ± standard error of the mean. ∗P ≤ .05; ∗∗P ≤ .01. MFI, mean fluorescence intensity; MHC, major histocompatibility complex; PE-Cy7, phycoerythrin-cyanine 7; TCR, T-cell receptor; tdTo, tdTomato; W, week.

A TIM-3–Fc decoy secreted by primary T cells enhances CAR19 T-cell efficacy and persistence in vivo. (A) Cartoon depicting the experimental strategy to block galectin-9 binding to TIM-3 on CAR19 T cells through a TIM-3–Fc decoy secreted by coadministered non-CAR T cells. (B) Representative fluorescence microscopy image (left) and transduction levels (tdTo+) measured by flow cytometry (right) of activated UT and TIM-3–Fc–transduced (tdTo+) primary T cells. (C) TIM-3 expression, quantified by flow cytometry, on UT and TIM-3–Fc T cells 7 days after transduction. (D) Proliferation of UT and TIM-3–Fc T cells. These experiments were performed with T cells from 3 different donors. (E) Experimental design to compare CAR19 and CAR19 + TIM-3–Fc T-cell persistence in vivo. NSG mice were injected IV with 1 × 106 B-ALL PDX cells (galectin-9 positive), and 4 days later, a single IV injection of 2 × 106 T cells was administered in mice (UT, TIM3Fc, CAR19, and CAR19 + TIM-3–Fc; n = 6 mice per group); 0.5 × 106 CAR19+ or/and 0.5 × 106 TIM-3–Fc+ cells were administered. Leukemia and T-cell expansion were monitored over time. In vivo CAR19 and CAR19 + TIM-3–Fc T cells were rechallenged with fresh B-ALL cells on day 25. (F) Follow-up of leukemic and T-cell persistence in PB of treated mice over time. (G) End point total and CAR+ (GFP+) T-cell quantification in PB, spleen, and BM in the different groups of treated mice. (H) Experimental design to compare CAR19 and CAR19 + TIM-3–Fc T-cell efficacy and persistence in vivo under stress conditions (1 × 106 T cells comprising 0.175 × 106 CAR19+ or/and 0.325 × 106 TIM-3–Fc+; n = 6 mice per group). Mice were followed up for 3 weeks. (I) End point quantification of blasts (J) and T cells (total and CAR+(GFP+) in PB, spleen, and BM in the indicated groups of treated mice. All data are expressed as mean ± standard error of the mean. ∗P ≤ .05; ∗∗P ≤ .01. MFI, mean fluorescence intensity; MHC, major histocompatibility complex; PE-Cy7, phycoerythrin-cyanine 7; TCR, T-cell receptor; tdTo, tdTomato; W, week.

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