Pharmacologic control of CAR-T cell function using dasatinib

Abstract

1. Dasatinib potently and reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation.

2. Dasatinib could be repurposed as a safety switch to mitigate CAR-mediated toxicity in patients.

Graphical abstract

Figure 1.

Dasatinib is a potent, rapid, and reversible inhibitor of CAR-T cell function. Mock (untransduced), CD19.28ζ, and CD19.BBζ CAR-T cells were cocultured with CD19⁺ Nalm6-GL at 1:1 and/or 1:8 effector:target (E:T) ratio in the presence of the concentrations of dasatinib noted. (A) CD69 and CD107a expression following 6 hours of coculture at 1:1 E:T (representative histograms, similar results observed using n = 3 donors). (B) Cocultures at a 1:1 and 1:8 E:T were analyzed in an IncuCyte to assess CAR-T cell cytotoxicity. Tumor GFP fluorescence intensity was normalized to the first time point (triplicate wells; representative donor, top and middle). The fold change in tumor fluorescence intensity from t = 0 to t = 72 hours for 1:8 E:T cultures across 3 donors is shown with each dot representing results from 1 donor (bottom). (C) IL-2 and interferon-γ secretion after a 24-hour coculture (top, triplicate wells from 1 representative donor; bottom, normalized data from 3 independent donors, with each dot representing results from 1 donor). (D) CAR-T cells were preloaded with Cell Trace Violet (CTV) cytosolic dye and cocultured at a 1:1 E:T in 1 μM dasatinib or vehicle for 72 hours. CAR-T cell proliferation was assessed via flow cytometry (representative histograms of n = 3 donors). (E) CAR-T cells were cocultured in 1 μM dasatinib or vehicle and analyzed in an IncuCyte as described previously. At t = 20 hours, dasatinib was either removed (top) or added (bottom) (triplicate wells; result is representative of n = 3 donors). (F) CAR-T cells cultured in the presence of 1 μM dasatinib or vehicle were stimulated with 5 μg/mL anti-FMC63 idiotype + 5 μg/mL goat anti-mouse secondary for 5 minutes. Cells were lysed; phosphoprotein and total protein levels were subsequently assessed via western blot (representative blots, n = 3 donors). Representative plots display the mean ± standard deviation of 3 technical replicates; dot plots display the mean ± standard error of the mean of 3 independent donors. *P < .05; **P < .01; ****P < .0001; not significant (ns), P > .05. IFN-γ, interferon-γ; ND, not detectable.

Figure 2.

Dasatinib suppresses CAR-T cell expansion, cytokine secretion, and tumor control in vivo. (A) 1 × 10⁶ CD19⁺ Nalm6-GL, which stably express GFP and luciferase, were engrafted into 6- to 8-week-old NSG mice via IV injection (n = 5 mice/group). At 4 days postengraftment, 1 × 10⁶ mock (untransduced) or CD19.BBζ CAR-T cells were infused via IV injection. Mice were subsequently dosed with 50 mg/kg dasatinib or vehicle on the day of infusion and everyday thereafter either twice daily (shown) or daily (replicate experiment). (B) Tumor growth was monitored via bioluminescence imaging as quantified in (C) (representative plot of n = 2 independent experiments). (D) At day 8 after CAR-T infusion (day 12 postengraftment), where indicated, mice that had received vehicle were switched to 50 mg/kg dasatinib twice daily for 7 days (representative plot, n = 2 independent experiments). (E) Blood samples were collected retroorbitally on day 8 after CAR-T infusion (day 12 postengraftment), and circulating CAR-T cells were quantified via flow cytometry (n = 5 mice from n = 1 experiment). (F-G) Blood samples were collected retroorbitally on day 3 after CAR-T infusion (day 7 postengraftment), and plasma was isolated after a brief centrifugation. Circulating concentrations of cytokines, chemokines, and growth factors were measured via Luminex (mock n = 3 mice, vehicle and dasatinib n = 5 mice from n = 1 experiment). (G) Heat map values were generated by normalizing to the sum of the mean concentrations of the 3 experimental groups. Representative plots display mean ± standard error of the mean of replicate mice within 1 experiment (n = 2 independent experiments). **P < .01; ***P < .001; ****P < .0001; ns, P > .05.