FIG. 1.

Two-color imaging shows tumor response and T-cell distribution with a single injection of mRNA-engineered anti-CD19 CAR T cells. (A) Time course of tumor response. NSG mice were given 1×10⁶ CBG Nalm-6 on day 0, followed by 1×10⁷ CBR CAR T cells targeting CD19 (top row) or mesothelin via the SS1 scFv (bottom row). T cells are widely distributed and the leukemia signal undetectable after 2 days (day 7). T cells with no target for the CAR are less widely distributed, and leukemia remains present. Over time, the leukemia can be seen to relapse in the skull base, jaw, and abdomen (day 13, middle panel) followed by disseminated progression. Leukemia progresses unchecked in the ss1-BBz-treated mice. (B) Single-color components of day 13 overlays from part A show that T cells are widely distributed in both CD19 and mesothelin-directed groups by this time point. Despite the co-localization in the mesothelin CAR T cells, they have no effect on leukemia progression. (C) Spectra acquired for use in unmixing from mice in the prone position. (D) CTX (60 mg/kg IP) effectively reduces transferred T cells. Mice were treated as in (A), with 1×10⁶ CBG Nalm-6 on day 0 followed by 1×10⁷ CBR CAR T cells targeting CD19 on day 5. About 24 hr after T-cell injection, leukemia is still visible in the femurs. After 48 hr, the red T-cell signal has increased and the green leukemia signal has diminished. About 24 hr before day 11, a single dose of IP CTX is given, and imaging shows a reduction in the red signal consistent with reduction in T-cell number. There are no detectable T cells or leukemia cells in the peripheral blood at these time points. CAR, chimeric antigen receptor; CBG, click beetle green; CTX, cyclophosphamide; NSG, NOD-SCID-γc^−/−; scFv, single-chain Fv.